Comparative Analysis of Antibodies and Heavily Glycosylated Macromolecular Immune Complexes by Size-Exclusion Chromatography Multi-Angle Light Scattering, Native Charge Detection Mass Spectrometry, and Mass PhotometryClick to copy article linkArticle link copied!
- Maurits A. den BoerMaurits A. den BoerBiomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The NetherlandsNetherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The NetherlandsMore by Maurits A. den Boer
- Szu-Hsueh LaiSzu-Hsueh LaiBiomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The NetherlandsNetherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The NetherlandsMore by Szu-Hsueh Lai
- Xiaoguang Xue
- Muriel D. van KampenMuriel D. van KampenGenmab, Uppsalalaan 15, 3584 CT Utrecht, The NetherlandsMore by Muriel D. van Kampen
- Boris Bleijlevens
- Albert J. R. Heck*Albert J. R. Heck*Email: [email protected]Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The NetherlandsNetherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The NetherlandsMore by Albert J. R. Heck
Abstract
Qualitative and quantitative mass analysis of antibodies and related macromolecular immune complexes is a prerequisite for determining their identity, binding partners, stoichiometries, and affinities. A plethora of bioanalytical technologies exist to determine such characteristics, typically based on size, interaction with functionalized surfaces, light scattering, or direct mass measurements. While these methods are highly complementary, they also exhibit unique strengths and weaknesses. Here, we benchmark mass photometry (MP), a recently introduced technology for mass measurement, against native mass spectrometry (MS) and size exclusion chromatography multi-angle light scattering (SEC-MALS). We examine samples of variable complexity, namely, IgG4Δhinge dimerizing half-bodies, IgG-RGY hexamers, heterogeneously glycosylated IgG:sEGFR antibody–antigen complexes, and finally megadalton assemblies involved in complement activation. We thereby assess the ability to determine (1) binding affinities and stoichiometries, (2) accurate masses, for extensively glycosylated species, and (3) assembly pathways of large heterogeneous immune complexes. We find that MP provides a sensitive approach for characterizing antibodies and stable assemblies, with dissociation correction enabling us to expand the measurable affinity range. In terms of mass resolution and accuracy, native MS performs the best but is occasionally hampered by artifacts induced by electrospray ionization, and its resolving power diminishes when analyzing extensively glycosylated proteins. In the latter cases, MP performs well, but single-particle charge detection MS can also be useful in this respect, measuring masses of heterogeneous assemblies even more accurately. Both methods perform well compared to SEC-MALS, still being the most established method in biopharma. Together, our data highlight the complementarity of these approaches, each having its unique strengths and weaknesses.
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Introduction
Materials and Methods
Protein Samples
Native MS and CD-MS
SEC-MALS
Mass Photometry
Results and Discussion
Jump-Diluted IgG4Δhinge Dimers Dissociate during MP Analysis
Figure 1
Figure 1. Qualitative and quantitative characterization of IgG4Δhinge mutants by MP. (A) Mass histogram showing particle counts of “wt” IgG4Δhinge in PBS, jump-diluted from 16 μM and measured at 4 nM, with normal distributions fitted for HL (bright red, 26%) and (HL)2 (dark red, 74%). This histogram corresponds to the first 80 s after jump dilution. Masses are indicated as the mean of a fitted normal distribution. (B) Monomer–dimer distribution during an extended experiment in triplicate (shades of red) revealed that the abundance of “wt” (HL)2 decreased during the analysis time window. Data were split into bins of 100 events, and an exponential decay function was fitted to the dimer abundance within the bin to determine the koff. (C) Determined koff was used to estimate the ratio between HL and (HL)2 at the instant of jump dilution for a dilution series of the “wt” measured in triplicate, revealing the apparent Kd of each measurement, followed by the calculation of a Kd value for the whole dilution series. (D) Fractional dimer abundances and Kd values resulting from a dilution series of four IgG4Δhinge mutational variants, demonstrating that MP can assess affinities over a broad dynamic range.
Modeling for Dissociation Expands the Affinity Range of IgG4Δhinge Mutants Assessable by MP
MP Outperforms Native MS in the Mass Assessment of Heavily Glycosylated Antibody–Antigen Assemblies
Figure 2
Figure 2. MP and CD-MS may overcome certain limitations of native MS in the mass measurements of highly heterogeneous antibody–antigen complexes. (A) MP provides an average mass for IgG1 (upper panel) and sEGFR (middle) and is not hampered by the high micro-heterogeneity of the latter. When 2 μM IgG1 was incubated with 5 μM of sEGFR to form (IgG1)1:(sEGFR)1 and (IgG1)1:(sEGFR)2 complexes, jump dilution MP could resolve these highly heterogeneous species (lower). (B) Although native MS on samples at the same concentrations provided superior mass resolution and accuracy for free IgG1 (upper), resolving individual glycoforms (zoom), the high microheterogeneity of sEGFR, measured separately (middle) and in antibody–antigen complexes (lower), resulted in unresolved features. In these experiments, overlapping charge states prevented mass measurements of these species. (C) More accurate masses could be obtained by native CD-MS, measuring in two dimensions m/z and z (insets) for sEGFR (upper) and all co-occurring species involving IgG1 and sEGFR (lower). For these experiments, the same native MS samples were diluted 20-fold, leading to re-equilibration and thus a lower binding occupancy.
MP, SEC, and Native MS Analyses of the Monomer–Hexamer Equilibrium of Soluble IgG1-RGY Hexamers Produce Consistent Results
Figure 3
Figure 3. MP enables qualitative and quantitative characterization of the monomer–hexamer equilibrium of IgG1-RGY. (A) MP mass histogram (top) of 2 μM IgG1-RGY in PBS jump-diluted to 10 nM showing monomeric ((IgG1)1) and hexameric ((IgG1)6) species. The mass of the hexamer was consistently measured about 70 kDa too high. The relative abundance of the IgG1-RGY hexamer was measured over a dilution series spanning a concentration range of 0.1 to 8 μM (bottom), with error bars indicating the standard deviation over three technical replicate measurements. (B) SEC-MALS chromatogram of the same 2 μM IgG1-RGY sample (top) and the fractional abundance of the hexamer as measured by SEC-UV over a dilution series (bottom), revealing a similar monomer to hexamer ratio. The quantitative data in the lower panel are adapted from the work of van Kampen et al. (58) (C) Native mass spectrum (top) of 2 μM IgG1-RGY measured in 150 mM NH4OAc pH 7.5, revealing two distinct ion series for the monomer and hexamer, with ions originating from intermediate oligomeric states observed at lower abundance. While generally in good agreement with the other methods, hexamer abundances measured by MS (bottom) were less consistent and higher than expected, particularly at the lowest measured concentrations.
Characterization of Complement Component C1q by MP Exposes Shortcomings of Native MS and SEC-MALS
MP and CD-MS Tackle Mass Heterogeneity When Analyzing Immune Complexes of IgG-RGY Hexamers Bound to Highly Glycosylated Antigens and C1q
Figure 4
Figure 4. MP and CD-MS successfully determine the mass and stoichiometry of highly heterogeneous (sEGFR)12:(IgG1)6:C1q immune complexes. (A) MP measurements of IgG1-RGY incubated with C1q reveal the formation of (IgG1)6:(C1q)1 complexes, with nearly all IgG hexamers occupied. When incubating C1q with pre-formed (IgG1)6:(sEGFR)12, MP resolves a 2.35 MDa complex, likely corresponding to (sEGFR)12:(IgG1)6:(C1q)1. (B) SEC-MALS-UV-RI analysis similarly reveals the formation of ∼1.3 MDa (IgG1)6:(C1q)1 (with (IgG1)6 measured as the 0.76 MDa “protein” and C1q as a 0.49 MDa “modifier”). When sEGFR was added, SEC-MALS-UV-RI revealed the formation of larger complexes of around 1.9 MDa (1.5 MDa for (sEGFR)12:(IgG1)6 with a 0.41 MDa modifier). (C) Measurement of the same samples by native MS reveals an accurate mass for (IgG1)6:(C1q)1, but the technique struggles with complexes involving sEGFR. Larger ion species were detected in such experiments, but they could not be charge-resolved. (D) Single-particle measurements of the distribution around m/z 21,000 by CD-MS (top) revealed a mass of 2.42 MDa (bottom) corresponding to the expected mass of the full (sEGFR)12:(IgG1)6:(sEGFR)12 complex (bottom).
Comparing Advantages and Disadvantages Reveals That MP, Native MS, and SEC-MALS Are Highly Complementary
Conclusions
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.1c03656.
Supplementary methods, koff and Kd determination for IgG4Δhinge variants, SEC-MALS of IgG1 and sEGFR, native MS gas-phase dissociation of IgG1 hexamers, IgG1 hexamer dissociation upon jump dilution MP, SEC-MALS, native MS and MP characterization of C1q, SEC-MALS, native (CD) MS and MP characterization of (IgG1)6:(sEGFR)12 antibody–antigen complexes, comparison of masses measured by the assessed techniques, MP-derived kinetic rates and Kd values for IgG4Δhinge variants, comparison of advantages and disadvantages of the assessed techniques, and supplementary references (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgments
We especially thank Genmab research associates Mandy Blom and Clifford Rodriguez for their excellent work on performing the SEC-MALS experiments and fruitful discussions. We further thank members of the Heck laboratory for general support, especially Arjan Barendregt. This research received funding through the Netherlands Organization for Scientific Research (NWO) TTW-NACTAR project 16442 (A.J.R.H. and M.A.d.B.) and the Spinoza Award SPI.2017.028 to A.J.R.H. We further acknowledge funding for the large-scale proteomics facility, the Netherlands Proteomics Center, through the X-omics Road Map program (project 184.034.019) and the EU Horizon 2020 program Epic-XS (project 823839).
References
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- 9Nguyen, H.; Park, J.; Kang, S.; Kim, M. Surface plasmon resonance: a versatile technique for biosensor applications. Sensors 2015, 15, 10481– 10510, DOI: 10.3390/s150510481Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXptlKqurw%253D&md5=98cc17f36c89a34ab7db91932cbcb954Surface plasmon resonance: a versatile technique for biosensor applicationsNguyen, Hoang Hiep; Park, Jeho; Kang, Sebyung; Kim, MoonilSensors (2015), 15 (5), 10481-10510CODEN: SENSC9; ISSN:1424-8220. (MDPI AG)A review. Surface plasmon resonance (SPR) is a label-free detection method which has emerged during the last two decades as a suitable and reliable platform in clin. anal. for biomol. interactions. The technique makes it possible to measure interactions in real-time with high sensitivity and without the need of labels. This review article discusses a wide range of applications in optical-based sensors using either surface plasmon resonance (SPR) or surface plasmon resonance imaging (SPRI). Here we summarize the principles, provide examples, and illustrate the utility of SPR and SPRI through example applications from the biomedical, proteomics, genomics and bioengineering fields. In addn., SPR signal amplification strategies and surface functionalization are covered in the review.
- 10Puiu, M.; Bala, C. SPR and SPR Imaging: Recent Trends in Developing Nanodevices for Detection and Real-Time Monitoring of Biomolecular Events. Sensors 2016, 16, 870, DOI: 10.3390/s16060870Google ScholarThere is no corresponding record for this reference.
- 11Kamat, V.; Rafique, A. Designing binding kinetic assay on the bio-layer interferometry (BLI) biosensor to characterize antibody-antigen interactions. Anal. Biochem. 2017, 536, 16– 31, DOI: 10.1016/j.ab.2017.08.002Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlCntrjO&md5=863482dce4b38b09ccbe0dc33005d958Designing binding kinetic assay on the bio-layer interferometry (BLI) biosensor to characterize antibody-antigen interactionsKamat, Vishal; Rafique, AshiqueAnalytical Biochemistry (2017), 536 (), 16-31CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)The Octet biosensors provide a high-throughput alternative to the well-established surface plasmon resonance (SPR) and SPR imaging (SPRi) biosensors to characterize antibody-antigen interactions. However, the utility of the Octet biosensors for accurate and reproducible measurement of binding rate consts. of monoclonal antibodies (mAbs) is limited due to challenges such as analyte rebinding, and mass transport limitation (MTL). This study focuses on addressing these challenges and provides exptl. conditions to reliably measure kinetics of mAb-antigen interactions. The mAb capture d. of less than 0.6 nm was found to be optimal to measure a wide range of binding affinities on Octet HTX biosensor. The titrn. kinetic and single cycle kinetic assays performed on Octet HTX generated reproducible binding kinetic parameters and correlated with the values measured on Biacore 4000 and MASS-1. Kinetic assays performed on 0.1 nm d. mAb surfaces significantly reduced MTL and enabled characterization of picomolar affinity mAbs. Finally, kinetic anal. performed on 150 antibodies to 10 antigens with mol. wts. ranging from 21kD to 105kD showed concordance between Octet HTX, Biacore 4000 and MASS-1 (R2 > 0.90). The data presented in this study suggest that under optimal exptl. conditions, Octet biosensor is capable of generating kinetic values comparable to SPR/SPRi biosensors.
- 12Ogi, H. Wireless-electrodeless quartz-crystal-microbalance biosensors for studying interactions among biomolecules: a review. Proc. Jpn. Acad., Ser. B 2013, 89, 401– 417, DOI: 10.2183/pjab.89.401Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXisFKktb8%253D&md5=fba6252394f0dc5f026d3ab86b340299Wireless-electrodeless quartz-crystal-microbalance biosensors for studying interactions among biomolecules: a reviewOgi, HirotsuguProceedings of the Japan Academy, Series B: Physical and Biological Sciences (2013), 89 (9), 401-417CODEN: PJABDW; ISSN:0386-2208. (Nippon Gakushiin)A review. The mass sensitivity of quartz-crystal microbalance (QCM) was drastically improved by removing electrodes and wires attached on the quartz surfaces. Instead of wire connections, intended vibrations of quartz oscillators were excited and detected by antennas through electromagnetic waves. This noncontacting measurement is the key for ultrahigh-sensitive detection of proteins in liqs. as well as quant. measurements. This review shows the principle of wireless QCMs, their applications to studying interactions among biomols. and aggregation reactions of amyloid β peptides, and the next-generation MEMS QCM, the resonance acoustic microbalance with naked embedded quartz (RAMNE-Q).
- 13Goldschen-Ohm, M. P.; White, D. S.; Klenchin, V. A.; Chanda, B.; Goldsmith, R. H. Observing Single-Molecule Dynamics at Millimolar Concentrations. Angew. Chem., Int. Ed. 2017, 56, 2399– 2402, DOI: 10.1002/anie.201612050Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVegsb0%253D&md5=7d02aac64191833cdd8b7329a22dcd1cObserving Single-Molecule Dynamics at Millimolar ConcentrationsGoldschen-Ohm, Marcel P.; White, David S.; Klenchin, Vadim A.; Chanda, Baron; Goldsmith, Randall H.Angewandte Chemie, International Edition (2017), 56 (9), 2399-2402CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Single-mol. fluorescence microscopy is a powerful tool for revealing chem. dynamics and mol. assocn. mechanisms, but has been limited to low concns. of fluorescent species and is only suitable for studying high affinity reactions. Here, the authors combine nanophotonic zero-mode waveguides (ZMWs) with fluorescence resonance energy transfer (FRET) to resolve single-mol. assocn. dynamics at up to millimolar concns. of fluorescent species. This approach extends the resoln. of mol. dynamics to >100-fold higher concns., enabling observations at concns. relevant to biol. and chem. processes, and thus making single-mol. techniques applicable to a tremendous range of previously inaccessible mol. targets. The authors deploy this approach to show that the binding of cGMP to pacemaking ion channels is weakened by a slower internal conformational change.
- 14Aggarwal, V.; Ha, T. Single-molecule fluorescence microscopy of native macromolecular complexes. Curr. Opin. Struct. Biol. 2016, 41, 225– 232, DOI: 10.1016/j.sbi.2016.09.006Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFajsrjE&md5=fe5b02730259f80da2c3f25a18f47ad8Single-molecule fluorescence microscopy of native macromolecular complexesAggarwal, Vasudha; Ha, TaekjipCurrent Opinion in Structural Biology (2016), 41 (), 225-232CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)Macromol. complexes consisting of proteins, lipids, and/or nucleic acids are ubiquitous in biol. processes. Their compn., stoichiometry, order of assembly, and conformations can be heterogeneous or can change dynamically, making single-mol. studies best suited to measure these properties accurately. Recent single-mol. pull-down and other related approaches have combined the principles of conventional co-immunopptn. assay with single-mol. fluorescence microscopy to probe native macromol. complexes. In this review, we present the advances in single-mol. pull-down methods and biol. systems that have been investigated in such semi vivo manner.
- 15Nobbmann, U.; Connah, M.; Fish, B.; Varley, P.; Gee, C.; Mulot, S.; Chen, J.; Zhou, L.; Lu, Y.; Sheng, F.; Yi, J.; Harding, S. E. Dynamic light scattering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodies. Biotechnol. Genet. Eng. Rev. 2007, 24, 117– 128, DOI: 10.1080/02648725.2007.10648095Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlKiurjI&md5=09f5a1d27a12b0c39fa1b3679e4c5c8fDynamic light scatttering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodiesNobbmann, Ulf; Connah, Malcolm; Fish, Brendan; Varley, Paul; Gee, Chris; Mulot, Sandrine; Chen, Juntao; Zhou, Liang; Lu, Yanling; Sheng, Fei; Yi, Junming; Harding, Stephen E.Biotechnology & Genetic Engineering Reviews (2007), 24 (), 117-128CODEN: BGERES; ISSN:0264-8725. (Nottingham University Press)A review compares dynamic light scattering (DLS) to anal. ultracentrifugation. It shows that when DLS is combined online with the sepn. power of HPLC it is possible to rapidly identify sep. species in soln.: a heterogeneous material is sepd. by the material of the column and the elution peaks can be analyzed sep. downstream by the DLS.
- 16Stetefeld, J.; McKenna, S. A.; Patel, T. R. Dynamic light scattering: a practical guide and applications in biomedical sciences. Biophys. Rev. 2016, 8, 409– 427, DOI: 10.1007/s12551-016-0218-6Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1Gqs7bP&md5=468714b5413c92edb45e985fc32e8164Dynamic light scattering: a practical guide and applications in biomedical sciencesStetefeld, Jorg; McKenna, Sean A.; Patel, Trushar R.Biophysical Reviews (2016), 8 (4), 409-427CODEN: BRIECG; ISSN:1867-2450. (Springer)Dynamic light scattering (DLS), also known as photon correlation spectroscopy (PCS), is a very powerful tool for studying the diffusion behavior of macromols. in soln. The diffusion coeff., and hence the hydrodynamic radii calcd. from it, depends on the size and shape of macromols. In this review, we provide evidence of the usefulness of DLS to study the homogeneity of proteins, nucleic acids, and complexes of protein-protein or protein-nucleic acid prepns., as well as to study protein-small mol. interactions. Further, we provide examples of DLS's application both as a complementary method to anal. ultracentrifugation studies and as a screening tool to validate soln. scattering models using detd. hydrodynamic radii.
- 17Hanlon, A. D.; Larkin, M. I.; Reddick, R. M. Free-Solution, Label-Free Protein-Protein Interactions Characterized by Dynamic Light Scattering. Biophys. J. 2010, 98, 297– 304, DOI: 10.1016/j.bpj.2009.09.061Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXkslWisbo%253D&md5=2754d6e515aa45097d9a89158290e652Free-solution, label-free protein-protein interactions characterized by dynamic light scatteringHanlon, Amy D.; Larkin, Michael I.; Reddick, Ryan M.Biophysical Journal (2010), 98 (2), 297-304CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)We report a free-soln., label-free method for quant. characterization of macromol. interactions using dynamic light scattering, a temp. controlled plate reader, and a multiwell concn. gradient. This nondestructive technique enabled detn. of stoichiometry of binding, equil. dissocn. const., and thermodn. parameters, as well as the impact of temp., buffer salinity, and a small-mol. inhibitor. The low vol. capability of dynamic light scattering reduced the required sample to 426 pmol/expt., with detection limits for 150-kDa proteins anticipated to be in the low femtomole range.
- 18Berkowitz, S. A.; Philo, J. S. Characterizing Biopharmaceuticals using Analytical Ultracentrifugation. In Biophysical Characterization of Proteins in Developing Biopharmaceuticals; Houde, D. J., Berkowitz, S. A., Eds.; Elsevier: Amsterdam, 2015; pp 211– 260.Google ScholarThere is no corresponding record for this reference.
- 19Uchiyama, S.; Noda, M.; Krayukhina, E. Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodies. Biophys. Rev. 2018, 10, 259– 269, DOI: 10.1007/s12551-017-0374-3Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1Gqtg%253D%253D&md5=eff4b1bdce19b9a66e0b35ee1982bf62Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodiesUchiyama, Susumu; Noda, Masanori; Krayukhina, ElenaBiophysical Reviews (2018), 10 (2), 259-269CODEN: BRIECG; ISSN:1867-2450. (Springer)A review. Sedimentation velocity anal. ultracentrifugation (SV-AUC) coupled with direct computational fitting of the obsd. concn. profiles (sedimentating boundary) have been developed and widely used for the characterization of macromols. and nanoparticles in soln. In particular, size distribution anal. by SV-AUC has become a reliable and essential approach for the characterization of biopharmaceuticals including therapeutic antibodies. In this review, we describe the importance and advantages of SV-AUC for studying biopharmaceuticals, with an emphasis on strategies for sample prepn., data acquisition, and data anal. Recent discoveries enabled by AUC with a fluorescence detection system and potential future applications are also discussed.
- 20Schuck, P. Analytical Ultracentrifugation as a Tool for Studying Protein Interactions. Biophys. Rev. 2013, 5, 159– 171, DOI: 10.1007/s12551-013-0106-2Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXntVWqsb0%253D&md5=088ec73746d699077e6b29317ea03974Analytical ultracentrifugation as a tool for studying protein interactionsSchuck, PeterBiophysical Reviews (2013), 5 (2), 159-171CODEN: BRIECG; ISSN:1867-2450. (Springer)A review. The last two decades have led to significant progress in the field of anal. ultracentrifugation driven by instrumental, theor., and computational methods. This review will highlight key developments in sedimentation equil. (SE) and sedimentation velocity (SV) anal. For SE, this includes the anal. of tracer sedimentation equil. at high concns. with strong thermodn. non-ideality, and for ideally interacting systems, the development of strategies for the anal. of heterogeneous interactions towards global multi-signal and multi-speed SE anal. with implicit mass conservation. For SV, this includes the development and applications of numerical solns. of the Lamm equation, noise decompn. techniques enabling direct boundary fitting, diffusion deconvoluted sedimentation coeff. distributions, and multi-signal sedimentation coeff. distributions. Recently, effective particle theory has uncovered simple phys. rules for the co-migration of rapidly exchanging systems of interacting components in SV. This has opened new possibilities for the robust interpretation of the boundary patterns of heterogeneous interacting systems. Together, these SE and SV techniques have led to new approaches to study macromol. interactions across the entire spectrum of affinities, including both attractive and repulsive interactions, in both dil. and highly concd. solns., which can be applied to single-component solns. of self-assocg. proteins as well as the study of multi-protein complex formation in multi-component solns.
- 21Gandhi, A. V.; Pothecary, M. R.; Bain, D. L.; Carpenter, J. F. Some Lessons Learned From a Comparison Between Sedimentation Velocity Analytical Ultracentrifugation and Size Exclusion Chromatography to Characterize and Quantify Protein Aggregates. J. Pharm. Sci. 2017, 106, 2178– 2186, DOI: 10.1016/j.xphs.2017.04.048Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXptFClsL4%253D&md5=7b172c52f61236864ae6763d22439bbbSome Lessons Learned From a Comparison Between Sedimentation Velocity Analytical Ultracentrifugation and Size Exclusion Chromatography to Characterize and Quantify Protein AggregatesGandhi, Aditya V.; Pothecary, Mark R.; Bain, David L.; Carpenter, John F.Journal of Pharmaceutical Sciences (2017), 106 (8), 2178-2186CODEN: JPMSAE; ISSN:0022-3549. (Elsevier Inc.)There are numerous problems with size exclusion chromatog. (SEC), which often lead to inaccuracies in protein aggregate characterization. Hence, this study tested sedimentation velocity anal. ultracentrifugation (SV-AUC) as an orthogonal tool to SEC for quantifying the monomer and aggregates in i.v. Ig (IVIg) formulations. IVIg samples were subjected to agitation stress and analyzed using SEC mobile phases composed of 200 mM sodium phosphate (pH 7.0) with 0, 50, 100, 200, or 400 mM of NaCl. Surprisingly, 400 mM of NaCl was required in the mobile phase to attain complete protein recovery from the SEC column. Significant discrepancies between SEC and SV-AUC are reported when SEC anal. was performed using suboptimal concns. (e.g., 0, 50, 100, and 200 mM) of NaCl in the mobile phase. The continuous sedimentation coeff. distributions obtained with SV-AUC resolved the high mol. wt. species, whereas with SEC the high mol. wt. species eluted as a single peak. Only with the orthogonal use of SV-AUC, the authors were able to develop a robust SEC method for accurate quantitation of monomer and aggregates in unagitated and agitated IVIg samples. Addnl., this article describes a modification to an existing method of quantitating insol. aggregates from SV-AUC boundary data.
- 22Moser, A. C.; Trenhaile, S.; Frankenberg, K. Studies of antibody-antigen interactions by capillary electrophoresis: A review. Methods 2018, 146, 66– 75, DOI: 10.1016/j.ymeth.2018.03.006Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXltFWhu7k%253D&md5=a420f715c049d1659561ef685c6dc05cStudies of antibody-antigen interactions by capillary electrophoresis: A reviewMoser, Annette C.; Trenhaile, Sidney; Frankenberg, KatiMethods (Amsterdam, Netherlands) (2018), 146 (), 66-75CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)Antibody-antigen interactions are vital in immunoassay development and can det. detection limits and anal. times. Capillary electrophoresis (CE) is a powerful technique that can be used to quantify antibody-antigen interactions. These CE methods range from simple sepns. of a premixed antibody and antigen sample applied as a short plug to allow for sepn. of complex, free antibody, and free antigen to more complex systems which inject complexed samples in the presence of antibody or antigen; or even injections of antibody and antigen sequentially. The objective of this review is to identify and describe various CE techniques which have been used to study antibody-antigen interactions. A brief discussion of linear and nonlinear curve fitting is also included.
- 23Kumar, R.; Guttman, A.; Rathore, A. S. Applications of capillary electrophoresis for biopharmaceutical product characterization. Electrophoresis 2021, DOI: 10.1002/elps.202100182Google ScholarThere is no corresponding record for this reference.
- 24Leney, A. C.; Heck, A. J. R. Native Mass Spectrometry: What is in the Name?. J. Am. Soc. Mass Spectrom. 2017, 28, 5– 13, DOI: 10.1007/s13361-016-1545-3Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFylsLzF&md5=09d56e0a888ad31a6f655c4e1d840775Native Mass Spectrometry: What is in the Name?Leney, Aneika C.; Heck, Albert J. R.Journal of the American Society for Mass Spectrometry (2017), 28 (1), 5-13CODEN: JAMSEF; ISSN:1044-0305. (Springer)A review. Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomol. mass spectrometry and proteomics. Advances in sample prepn. and mass analyzers have enabled researchers to ext. much more information from biol. samples than just the mol. wt. In particular, relevant for structural biol., noncovalent protein-protein and protein-ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomol. mass spectrometry, nowadays often referred to as "native MS," has come to maturation over the last two decades, with dozens of labs. using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure-function relations. In this perspective, the authors describe the origins of and (re)define the term native MS, portraying in detail what the authors meant by "native MS," when the term was coined and also describing what it does (according to the authors) not entail. Addnl., the authors describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technol. has in solving complex biol. questions.
- 25Tamara, S.; den Boer, M. A.; Heck, A. J. R. High-Resolution Native Mass Spectrometry. Chem. Rev. 2021, DOI: 10.1021/acs.chemrev.1c00212Google ScholarThere is no corresponding record for this reference.
- 26Thompson, N. J.; Hendriks, L. J.; de Kruif, J.; Throsby, M.; Heck, A. J. Complex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometry. mAbs 2014, 6, 197– 203, DOI: 10.4161/mabs.27126Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2c3nsF2rsQ%253D%253D&md5=00569249b017ebcc1944f5af74efc52cComplex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometryThompson Natalie J; Hendriks Linda J A; de Kruif John; Throsby Mark; Heck Albert J RmAbs (2014), 6 (1), 197-203 ISSN:.Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However, parallel development of analytical technologies is required to provide fast, thorough, accurate, and robust characterization of these mixtures. Here, we evaluate the utility of native mass spectrometry on an Orbitrap platform with high mass resolving power to characterize composite mixtures of up to 15 separate antibodies. With this technique, unambiguous identification of each antibody in the mixtures was achieved. Mass measurements of the intact antibodies varied 7 ppm on average, allowing highly reproducible identification and quantitation of each compound in these complex mixtures. We show that with the high mass-resolving power and robustness of this technology, high-resolution native mass spectrometry can be used efficiently even for batch-to batch characterization.
- 27Yang, Y.; Liu, F.; Franc, V.; Halim, L. A.; Schellekens, H.; Heck, A. J. R. Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity. Nat. Commun. 2016, 7, 13397, DOI: 10.1038/ncomms13397Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvVGjsL%252FI&md5=30540791a6e2417ea0e4b9cb1dae2a59Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarityYang, Yang; Liu, Fan; Franc, Vojtech; Halim, Liem Andhyk; Schellekens, Huub; Heck, Albert J. R.Nature Communications (2016), 7 (), 13397CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Many biopharmaceutical products exhibit extensive structural micro-heterogeneity due to an array of co-occurring post-translational modifications. These modifications often effect the functionality of the product and therefore need to be characterized in detail. Here, we present an integrative approach, combining two advanced mass spectrometry-based methods, high-resoln. native mass spectrometry and middle-down proteomics, to analyze this micro-heterogeneity. Taking human erythropoietin and the human plasma properdin as model systems, we demonstrate that this strategy bridges the gap between peptide- and protein-based mass spectrometry platforms, providing the most complete profiling of glycoproteins. Integration of the two methods enabled the discovery of three undescribed C-glycosylation sites on properdin, and revealed in addn. unexpected heterogeneity in occupancies of C-mannosylation. Furthermore, using various sources of erythropoietin we define and demonstrate the usage of a biosimilarity score to quant. assess structural similarity, which would also be beneficial for profiling other therapeutic proteins and even plasma protein biomarkers.
- 28Valliere-Douglass, J. F.; McFee, W. A.; Salas-Solano, O. Native Intact Mass Determination of Antibodies Conjugated with Monomethyl Auristatin E and F at Interchain Cysteine Residues. Anal. Chem. 2012, 84, 2843– 2849, DOI: 10.1021/ac203346cGoogle Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xit1aktrk%253D&md5=8aa2b994ce97fdf12d66c83eb4bc870cNative Intact Mass Determination of Antibodies Conjugated with Monomethyl Auristatin E and F at Interchain Cysteine ResiduesValliere-Douglass, John F.; McFee, William A.; Salas-Solano, OscarAnalytical Chemistry (Washington, DC, United States) (2012), 84 (6), 2843-2849CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)We present here a method for the rapid detn. of the intact mass of noncovalently assocd. antibody heavy chains (HC) and light chains (LC) which result from the attachment of drug conjugates to interchain cysteine residues. By analyzing the antibody-drug conjugate (ADC) using native desalting conditions, we maintain the intact bivalent structure of the ADC, which ordinarily would decomp. as a consequence of denaturing chromatog. conditions typically used for liq. chromatog.-mass spectrometric (LC-MS) anal. The mass of the desalted ADC is subsequently detd. using std. desolvation and ionization conditions. Methods presented previously in the literature for analyzing interchain cysteinyl-linked ADCs are either not amenable to online mass spectrometry or result in the denaturing dissocn. of conjugated HC and LC during chromatog. sepn. and subsequent mass measurement. We have avoided this outcome with our method and have successfully and routinely obtained intact mass measurement of IgG1 mAbs conjugated with maleimidocaproyl-monomethyl Auristatin F (mcMMAF) and valine-citrulline-monomethyl Auristatin E (vcMMAE) at interchain cysteine residues. Our results thus represent the first reported direct measurement of the intact mass of an ADC conjugated at interchain cysteine residues.
- 29Hengel, S. M.; Sanderson, R.; Valliere-Douglass, J.; Nicholas, N.; Leiske, C.; Alley, S. C. Measurement of in Vivo Drug Load Distribution of Cysteine-Linked Antibody–Drug Conjugates Using Microscale Liquid Chromatography Mass Spectrometry. Anal. Chem. 2014, 86, 3420– 3425, DOI: 10.1021/ac403860cGoogle Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjt12qsrg%253D&md5=e60eaebc1ca27c3db4677eabcb55d178Measurement of in Vivo Drug Load Distribution of Cysteine-Linked Antibody-Drug Conjugates Using Microscale Liquid Chromatography Mass SpectrometryHengel, Shawna Mae; Sanderson, Russell; Valliere-Douglass, John; Nicholas, Nicole; Leiske, Chris; Alley, Stephen C.Analytical Chemistry (Washington, DC, United States) (2014), 86 (7), 3420-3425CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Anal. of samples contg. intact antibody-drug conjugates (ADC) using mass spectrometry provides a direct measurement of the drug-load distribution. Once dosed, the drug load distribution changes due to a combination of biol. and chem. factors. Liq. chromatog.-mass spectrometry (LC-MS) methods to measure the in vivo drug load distribution have been established for ADCs contg. native disulfide bonds (lysine-linked or cysteine-linked). However, because of an IgG redn. step in conjugation processes, using LC-MS to analyze intact cysteine-linked ADCs requires native conditions, thus limiting sensitivity. While this limitation has been overcome at the anal. scale, to date, these methods have not been translated to a smaller scale that is required for animal or clin. doses/sampling. In this manuscript, we describe the development of ADC specific affinity capture reagents for processing in vivo samples and optimization of native LC-MS methods at a microscale. These methods are then used to detect the changing drug load distribution over time from a set of in vivo samples, representing to our knowledge the first native mass spectra of cysteine-linked ADCs from an in vivo source.
- 30Dyachenko, A.; Wang, G.; Belov, M.; Makarov, A.; de Jong, R. N.; van den Bremer, E. T. J.; Parren, P. W. H. I.; Heck, A. J. R. Tandem Native Mass-Spectrometry on Antibody-Drug Conjugates and Submillion Da Antibody-Antigen Protein Assemblies on an Orbitrap EMR Equipped with a High-Mass Quadrupole Mass Selector. Anal. Chem. 2015, 87, 6095– 6102, DOI: 10.1021/acs.analchem.5b00788Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXosVChtrY%253D&md5=43e2f71fd709b79be7b50804993f6ea3Tandem native mass-spectrometry on antibody-drug conjugates and submillion Da antibody-antigen protein assemblies on an Orbitrap EMR equipped with a high-mass quadrupole mass selectorDyachenko, Andrey; Wang, Guanbo; Belov, Mike; Makarov, Alexander; de Jong, Rob N.; van den Bremer, Ewald T. J.; Parren, Paul W. H. I.; Heck, Albert J. R.Analytical Chemistry (Washington, DC, United States) (2015), 87 (12), 6095-6102CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Native mass spectrometry is emerging as a powerful tool for the characterization of intact antibodies and antibody-based therapeutics. Here, we demonstrate new possibilities provided by the implementation of a high mass quadrupole mass selector on the recently introduced Orbitrap Exactive EMR mass spectrometer. This configuration allows precursor ion selection, and thus tandem mass spectrometry expts., even on analytes with masses in the hundreds of kilodaltons. We apply tandem mass spectrometry to localize the drug mols. in the therapeutic antibody-drug conjugate brentuximab vedotin, which displays a heterogeneous drug load. Our tandem MS data reveal that drug conjugation takes place nonhomogeneously to cysteine residues both on the light and heavy chains. Next, we analyzed how many antigens bind to IgG hexamers, based on a recently described antibody mutant IgG1-RGY that forms hexamers and activates complement in soln. The fully satd. IgG1-RGY-antigen complexes displayed a stoichiometry of IgG:CD38 of 6:12, possessing a mol. wt. of about 1.26 MDa and demonstrating that IgG assembly does not hamper antigen binding. Through tandem MS expts., we retrieve information about the spatial arrangement and stoichiometry of the subunits within this complex. These examples underscore the potential of this further modified Orbitrap-EMR instrument esp. for the in-depth characterization by native tandem mass spectrometry of antibodies and antibody-based constructs.
- 31Atmanene, C.; Wagner-Rousset, E.; Malissard, M.; Chol, B.; Robert, A.; Corvaïa, N.; Dorsselaer, A. V.; Beck, A.; Sanglier-Cianférani, S. Extending Mass Spectrometry Contribution to Therapeutic Monoclonal Antibody Lead Optimization: Characterization of Immune Complexes Using Noncovalent ESI-MS. Anal. Chem. 2009, 81, 6364– 6373, DOI: 10.1021/ac9007557Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXot12nsLg%253D&md5=e82cff344f0429b338753105c22c46c7Extending Mass Spectrometry Contribution to Therapeutic Monoclonal Antibody Lead Optimization: Characterization of Immune Complexes Using Noncovalent ESI-MSAtmanene, Cedric; Wagner-Rousset, Elsa; Malissard, Martine; Chol, Bertrand; Robert, Alain; Corvaia, Nathalie; Van Dorsselaer, Alain; Beck, Alain; Sanglier-Cianferani, SarahAnalytical Chemistry (Washington, DC, United States) (2009), 81 (15), 6364-6373CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases including cancers, immunol. disorders, and other pathologies. These large biomols. display specific structural features, which affect their efficiency and need, therefore, to be extensively characterized using sensitive and orthogonal anal. techniques. Among them, mass spectrometry (MS) has become the method of choice to study mAb amino acid sequences as well as their post-translational modifications. In the present work, recent noncovalent MS-technologies including automated chip-based nanoelectrospray MS and traveling wave ion mobility MS were used for the first time to characterize immune complexes involving both murine and humanized mAb 6F4 directed against human JAM-A, a newly identified antigenic protein (Ag) overexpressed in tumor cells. MS-based structural insights evidenced that heterogeneous disulfide bridge pairings of recombinant JAM-A alter neither its native structure nor mAbs 6F4 recognition properties. Investigations focused on mAb:Ag complexes revealed that, similarly to murine mAb, humanized mAb 6F4 binds selectively up to four antigen mols. with a similar affinity, confirming in this way the reliability of the humanization process. Noncovalent MS appears as an addnl. supporting technique for therapeutic mAbs lead characterization and development.
- 32Debaene, F.; Wagner-Rousset, E.; Colas, O.; Ayoub, D.; Corvaïa, N.; Van Dorsselaer, A.; Beck, A.; Cianférani, S. Time Resolved Native Ion-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and “Bispecific” Monoclonal Antibody Formation. Anal. Chem. 2013, 85, 9785– 9792, DOI: 10.1021/ac402237vGoogle Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsVSit7bI&md5=8c98eab1931fb738c4a647ac61e26c64Time Resolved Native Ion-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and "Bispecific" Monoclonal Antibody FormationDebaene, Francois; Wagner-Rousset, Elsa; Colas, Olivier; Ayoub, Daniel; Corvaia, Nathalie; Van Dorsselaer, Alain; Beck, Alain; Cianferani, SarahAnalytical Chemistry (Washington, DC, United States) (2013), 85 (20), 9785-9792CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Monoclonal antibodies (mAbs) and derivs. such as antibody-drug conjugates (ADC) and bispecific antibodies (bsAb), are the fastest growing class of human therapeutics. Most of the therapeutic antibodies currently on the market and in clin. trials are chimeric, humanized, and human IgG1. An increasing no. of IgG2s and IgG4s that have distinct structural and functional properties are also investigated to develop products that lack or have diminished antibody effector functions compared to IgG1. Importantly, wild type IgG4 has been shown to form half mols. (one heavy chain and one light chain) that lack interheavy chain disulfide bonds and form intrachain disulfide bonds. Moreover, IgG4 undergoes a process of Fab-arm exchange (FAE) in which the heavy chains of antibodies of different specificities can dissoc. and recombine in bispecific antibodies both in vitro and in vivo. Here, native mass spectrometry (MS) and time-resolved traveling wave ion mobility MS (TWIM-MS) were used for the first time for online monitoring of FAE and bsAb formation using Hz6F4-2v3 and natalizumab, two humanized IgG4s which bind to human Junctional Adhesion Mol.-A (JAM-A) and alpha4 integrin, resp. In addn., native MS anal. of bsAb/JAM-A immune complexes revealed that bsAb can bind up to two antigen mols., confirming that the Hz6F4 family preferentially binds dimeric JAM-A. Our results illustrate how IM-MS can rapidly assess bsAb structural heterogeneity and be easily implemented into MS workflows for bsAb prodn. follow up and bsAb/antigen complex characterization. Altogether, these results provide new MS-based methodologies for in-depth FAE and bsAb formation monitoring. Native MS and IM-MS will play an increasing role in next generation biopharmaceutical product characterization like bsAbs, antibody mixts., and antibody-drug conjugates (ADC) as well as for biosimilar and biobetter antibodies.
- 33Wang, G.; de Jong, R. N.; van den Bremer, E. T. J.; Parren, P. W. H. I.; Heck, A. J. R. Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry. Anal. Chem. 2017, 89, 4793– 4797, DOI: 10.1021/acs.analchem.6b05129Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXls1Slurs%253D&md5=5b2d372e85bb05f5733d77e75fd21438Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass SpectrometryWang, Guanbo; de Jong, Rob N.; van den Bremer, Ewald T. J.; Parren, Paul W. H. I.; Heck, Albert J. R.Analytical Chemistry (Washington, DC, United States) (2017), 89 (9), 4793-4797CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The detn. of mol. wts. (MWs) of heavily glycosylated proteins is seriously hampered by the physicochem. characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during SDS-PAGE and size-exclusion chromatog. (SEC) anal. Std. electrospray ionization (ESI)-mass spectrometry does not provide a direct soln. as this approach is hindered by extensive interference of ion signals caused by closely spaced charge states of broadly distributed glycoforms. Here, the authors introduce a native tandem MS-based approach, enabling charge-state resoln. and charge assignment of protein ions including those that escape mass anal. under std. MS conditions. Using this method, the authors detd. the MW of two model glycoproteins, the extra-cellular domains of the highly and heterogeneously glycosylated proteins CD38 and epidermal growth factor receptor (EGFR), as well as the overall MW and binding stoichiometries of these proteins in complex with a specific antibody.
- 34Wang, G.; de Jong, R. N.; van den Bremer, E. T. J.; Beurskens, F. J.; Labrijn, A. F.; Ugurlar, D.; Gros, P.; Schuurman, J.; Parren, P. W. H. I.; Heck, A. J. R. Molecular Basis of Assembly and Activation of Complement Component C1 in Complex with Immunoglobulin G1 and Antigen. Mol. Cell 2016, 63, 135– 145, DOI: 10.1016/j.molcel.2016.05.016Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVWltrjN&md5=cf1f473fd61b1122af3e4f665f08110aMolecular Basis of Assembly and Activation of Complement Component C1 in Complex with Immunoglobulin G1 and AntigenWang, Guanbo; de Jong, Rob N.; van den Bremer, Ewald T. J.; Beurskens, Frank J.; Labrijn, Aran F.; Ugurlar, Deniz; Gros, Piet; Schuurman, Janine; Parren, Paul W. H. I.; Heck, Albert J. R.Molecular Cell (2016), 63 (1), 135-145CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)The classical complement pathway contributes to the natural immune defense against pathogens and tumors. IgG antibodies can assemble at the cell surface into hexamers via Fc:Fc interactions, which recruit complement component C1q and induce complement activation. Biophys. characterization of the C1:IgG complex has remained elusive primarily due to the low affinity of IgG-C1q binding. Using IgG variants that dynamically form hexamers efficient in C1q binding and complement activation, we could assess C1q binding in soln. by native mass spectrometry and size-exclusion chromatog. Fc-domain deglycosylation, described to abrogate complement activation, affected IgG hexamerization and C1q binding. Strikingly, antigen binding by IgG hexamers or deletion of the Fab arms substantially potentiated complement initiation, suggesting that Fab-mediated effects impact downstream Fc-mediated events. Finally, we characterized a reconstituted 2,045.3 ± 0.4-kDa complex of intact C1 bound to antigen-satd. IgG hexamer by native mass spectrometry, providing a clear visualization of a complete complement initiation complex.
- 35Wörner, T. P.; Snijder, J.; Bennett, A.; Agbandje-McKenna, M.; Makarov, A. A.; Heck, A. J. R. Resolving heterogeneous macromolecular assemblies by Orbitrap-based single-particle charge detection mass spectrometry. Nat. Methods 2020, 17, 395– 398, DOI: 10.1038/s41592-020-0770-7Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXks1Kgs7w%253D&md5=01b8a2d8e1d482367755ce3f107c6667Resolving heterogeneous macromolecular assemblies by Orbitrap-based single-particle charge detection mass spectrometryWorner, Tobias P.; Snijder, Joost; Bennett, Antonette; Agbandje-McKenna, Mavis; Makarov, Alexander A.; Heck, Albert J. R.Nature Methods (2020), 17 (4), 395-398CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)We demonstrate single-particle charge detection mass spectrometry on an Orbitrap for the anal. of megadalton biomol. assemblies. We establish that the signal amplitudes of individual ions scale linearly with their charge, which can be used to resolve mixed ion populations, det. charge states and thus also det. the masses of individual ions. This enables the ultrasensitive anal. of heterogeneous protein assemblies including Ig oligomers, ribosomes, proteinaceous nanocontainers and genome-packed adeno-assocd. viruses.
- 36Eschweiler, J. D.; Kerr, R.; Rabuck-Gibbons, J.; Ruotolo, B. T. Sizing Up Protein–Ligand Complexes: The Rise of Structural Mass Spectrometry Approaches in the Pharmaceutical Sciences. Annu. Rev. Anal. Chem. 2017, 10, 25– 44, DOI: 10.1146/annurev-anchem-061516-045414Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXksVOksrY%253D&md5=15c08f3dc25938a1436d70d24401347fSizing Up Protein-Ligand Complexes: The Rise of Structural Mass Spectrometry Approaches in the Pharmaceutical SciencesEschweiler, Joseph D.; Kerr, Richard; Rabuck-Gibbons, Jessica; Ruotolo, Brandon T.Annual Review of Analytical Chemistry (2017), 10 (), 25-44CODEN: ARACFU; ISSN:1936-1327. (Annual Reviews)Capturing the dynamic interplay between proteins and their myriad interaction partners is critically important for advancing our understanding of almost every biochem. process and human disease. The importance of this general area has spawned many measurement methods capable of assaying such protein complexes, and the mass spectrometry-based structural biol. methods described in this review form an important part of that anal. arsenal. Here, we survey the basic principles of such measurements, cover recent applications of the technol. that have focused on protein-small-mol. complexes, and discuss the bright future awaiting this group of technologies.
- 37Mathur, S.; Badertscher, M.; Scott, M.; Zenobi, R. Critical evaluation of mass spectrometric measurement of dissociation constants: accuracy and cross-validation against surface plasmon resonance and circular dichroism for the calmodulin–melittin system. Phys. Chem. Chem. Phys. 2007, 9, 6187– 6198, DOI: 10.1039/b707946jGoogle Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlKht7rJ&md5=e9069d5a5561deca0039928297af8d55Critical evaluation of mass spectrometric measurement of dissociation constants: accuracy and cross-validation against surface plasmon resonance and circular dichroism for the calmodulin-melittin systemMathur, Sonal; Badertscher, Martin; Scott, Michael; Zenobi, RenatoPhysical Chemistry Chemical Physics (2007), 9 (47), 6187-6198CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)The authors present a comprehensive study for detg. the binding affinity of a protein-ligand complex, using mass spectrometric methods. Mass spectrometry has been used to study noncovalent interactions for a no. of years. However, the use of soft ionization mass spectrometry for quant. anal. of noncovalently bound complexes is not widely accepted. This paper reports a comparison of MS methods against established methods such as surface plasmon resonance (SPR) and CD whose suitability for the quant. assessment of noncovalent interactions is well known. ESI titrn. and MALDI-SUPREX were used as representative mass spectrometric methods for this work. The authors chose to study the calmodulin-melittin complex that presents three challenges: (i) it exhibits a high affinity (low nanomolar KD); (ii) complexes are formed only in the presence of a coactivator, calcium ions in this case; and (iii) the protein and the complex show a different ionization efficiency. Dissocn. consts. were obtained from each method for the selected system and compared thoroughly to elucidate pros and cons of the selected methodologies in terms of their ability for the detn. of binding consts. of protein-ligand complexes. ESI titrn., SPR, CD and MALDI-SUPREX yielded KD values in the low nanomolar range that are in general agreement with an older value reported in the literature. The authors also critically evaluated the limitations in particular of the MS methods and the assocd. data evaluation procedures. The authors present an improved evaluation of SUPREX data, as well as a detailed error anal. for all methods used.
- 38Kitova, E. N.; El-Hawiet, A.; Schnier, P. D.; Klassen, J. S. Reliable Determinations of Protein–Ligand Interactions by Direct ESI-MS Measurements. Are We There Yet?. J. Am. Soc. Mass Spectrom. 2012, 23, 431– 441, DOI: 10.1007/s13361-011-0311-9Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XltVahs7k%253D&md5=2059dccb131ddbff901a9e193ecde795Reliable determinations of protein-ligand interactions by direct ESI-MS measurements. Are we there yet?Kitova, Elena N.; El-Hawiet, Amr; Schnier, Paul D.; Klassen, John S.Journal of the American Society for Mass Spectrometry (2012), 23 (3), 431-441CODEN: JAMSEF; ISSN:1044-0305. (Springer)A review. The assocn.-dissocn. of noncovalent interactions between protein and ligands, such as other proteins, carbohydrates, lipids, DNA, or small mols., are crit. events in many biol. processes. The discovery and characterization of these interactions is essential to a complete understanding of biochem. reactions and pathways and to the design of novel therapeutic agents that may be used to treat a variety of diseases and infections. Over the last 20 y, electrospray ionization mass spectrometry (ESI-MS) has emerged as a versatile tool for the identification and quantification of protein-ligand interactions in vitro. Here, the authors describe the implementation of the direct ESI-MS assay for the detn. of protein-ligand binding stoichiometry and affinity. Addnl., the authors outline common sources of error encountered with these measurements and various strategies to overcome them. Finally, the authors comment on some of the outstanding challenges assocd. with the implementation of the assay and highlight new areas where direct ESI-MS measurements are expected to make significant contributions in the future.
- 39Daniel, J. M.; Friess, S. D.; Rajagopalan, S.; Wendt, S.; Zenobi, R. Quantitative determination of noncovalent binding interactions using soft ionization mass spectrometry. Int. J. Mass Spectrom. 2002, 216, 1– 27, DOI: 10.1016/s1387-3806(02)00585-7Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xis1enur4%253D&md5=2b46b1d642f0e21f6948ce0064a1ef25Quantitative determination of noncovalent binding interactions using soft ionization mass spectrometryDaniel, Jurg M.; Friess, Sebastian D.; Rajagopalan, Sudha; Wendt, Silke; Zenobi, RenatoInternational Journal of Mass Spectrometry (2002), 216 (1), 1-27CODEN: IMSPF8; ISSN:1387-3806. (Elsevier Science B.V.)A review. For a no. of years, soft ionization mass spectrometry was used for studying noncovalently bound complexes. An intriguing question in this context is whether MS expts. can be used to measure the interaction strength. A no. of recent studies have addressed this question. The results of these studies, as well as the methods employed are reviewed here. We distinguish between liq.-phase methods such as mass spectrometrically detected melting curves, titrn. expts., or competition expts., and gas-phase methods such as cone voltage-driven dissocn., collision-induced dissocn., blackbody IR radiative dissocn., or thermal dissocn. of gas-phase complex ions. With a few exceptions, no agreement exists between soln.-phase and gas-phase binding energies. The main reason is that electrostatic and dipolar noncovalent interactions are strengthened in the absence of solvent shielding, while other noncovalent interactions, in particular hydrophobic interactions, become less important in the absence of solvent. The possibility to quant. measure soln.-phase as well as gas-phase noncovalent interaction strengths by mass spectrometry opens fascinating perspectives for very high sensitivity screening assays as well as for improved fundamental understanding of the nature of noncovalent interactions.
- 40Rose, R. J.; Labrijn, A. F.; van den Bremer, E. T. J.; Loverix, S.; Lasters, I.; van Berkel, P. H. C.; van de Winkel, J. G. J.; Schuurman, J.; Parren, P. W. H. I.; Heck, A. J. R.; Schuurman, J.; Parren, P. W. H. I.; Heck, A. J. R. Quantitative Analysis of the Interaction Strength and Dynamics of Human IgG4 Half Molecules by Native Mass Spectrometry. Structure 2011, 19, 1274– 1282, DOI: 10.1016/j.str.2011.06.016Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFaru7fL&md5=ecca0a3dd3826087d4881565fa423c4bQuantitative Analysis of the Interaction Strength and Dynamics of Human IgG4 Half Molecules by Native Mass SpectrometryRose, Rebecca J.; Labrijn, Aran F.; van den Bremer, Ewald T. J.; Loverix, Stefan; Lasters, Ignace; van Berkel, Patrick H. C.; van de Winkel, Jan G. J.; Schuurman, Janine; Parren, Paul W. H. I.; Heck, Albert J. R.Structure (Cambridge, MA, United States) (2011), 19 (9), 1274-1282CODEN: STRUE6; ISSN:0969-2126. (Cell Press)Native mass spectrometry (MS) is a powerful technique for studying noncovalent protein-protein interactions. Here, native MS was employed to examine the noncovalent interactions involved in homodimerization of antibody half mols. (HL) in hinge-deleted human IgG4 (IgG4Δhinge). By analyzing the concn. dependence of the relative distribution of monomer HL and dimer (HL)2 species, the apparent dissocn. const. (KD) for this interaction was detd. In combination with site-directed mutagenesis, the relative contributions of residues at the CH3-CH3 interface to this interaction could be characterized and corresponding KD values quantified over a range of 10-10-10-4 M. The crit. importance of this noncovalent interaction in maintaining the intact dimeric structure was also proven for the full-length IgG4 backbone. Using time-resolved MS, the kinetics of the interaction could be measured, reflecting the dynamics of IgG4 HL exchange. Hence, native MS has provided a quant. view of local structural features that define biol. properties of IgG4.
- 41Belov, A. M.; Viner, R.; Santos, M. R.; Horn, D. M.; Bern, M.; Karger, B. L.; Ivanov, A. R. Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2017, 28, 2614– 2634, DOI: 10.1007/s13361-017-1781-1Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVOjsLnP&md5=6c6178d79eaa15c6ae8eb8830d78de42Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass SpectrometryBelov, Arseniy M.; Viner, Rosa; Santos, Marcia R.; Horn, David M.; Bern, Marshall; Karger, Barry L.; Ivanov, Alexander R.Journal of the American Society for Mass Spectrometry (2017), 28 (12), 2614-2634CODEN: JAMSEF; ISSN:1044-0305. (Springer)Native mass spectrometry (MS) is a rapidly advancing field in the anal. of proteins, protein complexes, and macromol. species of various types. The majority of native MS expts. reported to-date has been conducted using direct infusion of purified analytes into a mass spectrometer. Capillary zone electrophoresis (CZE) was coupled online to Orbitrap mass spectrometers using a com. sheathless interface to enable high-performance sepn., identification, and structural characterization of limited amts. of purified proteins and protein complexes, the latter with preserved noncovalent assocns. under native conditions. The performance of both bare-fused silica and polyacrylamide-coated capillaries was assessed using mixts. of protein stds. known to form noncovalent protein-protein and protein-ligand complexes. High-efficiency sepn. of native complexes is demonstrated using both capillary types, while the polyacrylamide neutral-coated capillary showed better reproducibility and higher efficiency for more complex samples. The platform was then evaluated for the detn. of monoclonal antibody aggregation and for anal. of proteomes of limited complexity using a ribosomal isolate from E. coli. Native CZE-MS, using accurate single stage and tandem-MS measurements, enabled identification of proteoforms and noncovalent complexes at femtomole levels. Native CZE-MS can serve as an orthogonal and complementary technique to conventional native MS methodologies with the advantages of low sample consumption, minimal sample processing and losses, and high throughput and sensitivity. This study presents a novel platform for anal. of ribosomes and other macromol. complexes and organelles, with the potential for discovery of novel structural features defining cellular phenotypes (e.g., specialized ribosomes).
- 42VanAernum, Z. L.; Busch, F.; Jones, B. J.; Jia, M.; Chen, Z.; Boyken, S. E.; Sahasrabuddhe, A.; Baker, D.; Wysocki, V. H. Rapid online buffer exchange for screening of proteins, protein complexes and cell lysates by native mass spectrometry. Nat. Protoc. 2020, 15, 1132– 1157, DOI: 10.1038/s41596-019-0281-0Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB38%252FmvFWgug%253D%253D&md5=c646764d754642a085d18013fb986ae2Rapid online buffer exchange for screening of proteins, protein complexes and cell lysates by native mass spectrometryVanAernum Zachary L; Busch Florian; Jones Benjamin J; Jia Mengxuan; Sahasrabuddhe Aniruddha; Wysocki Vicki H; VanAernum Zachary L; Busch Florian; Jones Benjamin J; Jia Mengxuan; Wysocki Vicki H; Chen Zibo; Boyken Scott E; Baker David; Chen Zibo; Boyken Scott E; Baker David; Chen Zibo; Boyken Scott E; Sahasrabuddhe Aniruddha; Baker DavidNature protocols (2020), 15 (3), 1132-1157 ISSN:.It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.
- 43Keifer, D. Z.; Jarrold, M. F. Single-molecule mass spectrometry. Mass Spectrom. Rev. 2017, 36, 715– 733, DOI: 10.1002/mas.21495Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1Wisb3I&md5=5f6d139ce05f4572c3717daee02d1f6dSingle-molecule mass spectrometryKeifer, David Z.; Jarrold, Martin F.Mass Spectrometry Reviews (2017), 36 (6), 715-733CODEN: MSRVD3; ISSN:0277-7037. (John Wiley & Sons, Inc.)A review. In single-mol. mass spectrometry, the mass of each ion is measured individually; making it suitable for the anal. of very large, heterogeneous objects that cannot be analyzed by conventional means. A range of single-mol. mass spectrometry techniques has been developed, including time-of-flight with cryogenic detectors, a quadrupole ion trap with optical detection, single-mol. Fourier transform ion cyclotron resonance, charge detection mass spectrometry, quadrupole ion traps coupled to charge detector plates, and nanomech. oscillators. In addn. to providing information on mass and heterogeneity, these techniques have been used to study impact craters from cosmic dust, monitor the assembly of viruses, elucidate the fluorescence dynamics of quantum dots, and much more. This review focuses on the merits of each of these technologies, their limitations, and their applications. Mass Spec Rev 36:715-733, 2017.
- 44Kafader, J. O.; Melani, R. D.; Durbin, K. R.; Ikwuagwu, B.; Early, B. P.; Fellers, R. T.; Beu, S. C.; Zabrouskov, V.; Makarov, A. A.; Maze, J. T.; Shinholt, D. L.; Yip, P. F.; Tullman-Ercek, D.; Senko, M. W.; Compton, P. D.; Kelleher, N. L. Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes. Nat. Methods 2020, 17, 391– 394, DOI: 10.1038/s41592-020-0764-5Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXktFelsro%253D&md5=8c506e953825257f71b5721adfa4d24cMultiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexesKafader, Jared O.; Melani, Rafael D.; Durbin, Kenneth R.; Ikwuagwu, Bon; Early, Bryan P.; Fellers, Ryan T.; Beu, Steven C.; Zabrouskov, Vlad; Makarov, Alexander A.; Maze, Joshua T.; Shinholt, Deven L.; Yip, Ping F.; Tullman-Ercek, Danielle; Senko, Michael W.; Compton, Philip D.; Kelleher, Neil L.Nature Methods (2020), 17 (4), 391-394CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)An Orbitrap-based ion anal. procedure dets. the direct charge for numerous individual protein ions to generate true mass spectra. This individual ion mass spectrometry (I2MS) method for charge detection enables the characterization of highly complicated mixts. of proteoforms and their complexes in both denatured and native modes of operation, revealing information not obtainable by typical measurements of ensembles of ions.
- 45Young, G.; Hundt, N.; Cole, D.; Fineberg, A.; Andrecka, J.; Tyler, A.; Olerinyova, A.; Ansari, A.; Marklund, E. G.; Collier, M. P.; Chandler, S. A.; Tkachenko, O.; Allen, J.; Crispin, M.; Billington, N.; Takagi, Y.; Sellers, J. R.; Eichmann, C.; Selenko, P.; Frey, L.; Riek, R.; Galpin, M. R.; Struwe, W. B.; Benesch, J. L. P.; Kukura, P. Quantitative mass imaging of single biological macromolecules. Science 2018, 360, 423– 427, DOI: 10.1126/science.aar5839Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXotFyku7k%253D&md5=38df454f60283833ea53eb4e1577e31dQuantitative mass imaging of single biological macromoleculesYoung, Gavin; Hundt, Nikolas; Cole, Daniel; Fineberg, Adam; Andrecka, Joanna; Tyler, Andrew; Olerinyova, Anna; Ansari, Ayla; Marklund, Erik G.; Collier, Miranda P.; Chandler, Shane A.; Tkachenko, Olga; Allen, Joel; Crispin, Max; Billington, Neil; Takagi, Yasuharu; Sellers, James R.; Eichmann, Cedric; Selenko, Philipp; Frey, Lukas; Riek, Roland; Galpin, Martin R.; Struwe, Weston B.; Benesch, Justin L. P.; Kukura, PhilippScience (Washington, DC, United States) (2018), 360 (6387), 423-427CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The cellular processes underpinning life are orchestrated by proteins and their interactions. The assocd. structural and dynamic heterogeneity, despite being key to function, poses a fundamental challenge to existing anal. and structural methodologies. The authors used interferometric scattering microscopy to quantify the mass of single biomols. in soln. with 2% sequence mass accuracy, up to 19-kDa resoln., and 1-kDa precision. The authors resolved oligomeric distributions at high dynamic range, detected small-mol. binding, and mass-imaged proteins with assocd. lipids and sugars. These capabilities enabled us to characterize the mol. dynamics of processes as diverse as glycoprotein crosslinking, amyloidogenic protein aggregation, and actin polymn. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomol. interactions, one mol. at a time.
- 46Ortega Arroyo, J.; Andrecka, J.; Spillane, K. M.; Billington, N.; Takagi, Y.; Sellers, J. R.; Kukura, P. Label-Free, All-Optical Detection, Imaging, and Tracking of a Single Protein. Nano Lett. 2014, 14, 2065– 2070, DOI: 10.1021/nl500234tGoogle Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjsFOksbs%253D&md5=89e8424b8ccc5aa9a2aac0817cbcb8f1Label-Free, All-Optical Detection, Imaging, and Tracking of a Single ProteinOrtega Arroyo, J.; Andrecka, J.; Spillane, K. M.; Billington, N.; Takagi, Y.; Sellers, J. R.; Kukura, P.Nano Letters (2014), 14 (4), 2065-2070CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Optical detection of individual proteins requires fluorescent labeling. Cavity and plasmonic methodologies enhance single mol. signatures in the absence of any labels but have struggled to demonstrate routine and quant. single protein detection. Here, we used interferometric scattering microscopy not only to detect but also to image and nanometrically track the motion of single myosin 5a heavy meromyosin mols. without the use of labels or any nanoscopic amplification. Together with the simple exptl. arrangement, an intrinsic independence from strong electronic transition dipoles and a detection limit of <60 kDa, our approach paves the way toward nonresonant, label-free sensing and imaging of nanoscopic objects down to the single protein level.
- 47Piliarik, M.; Sandoghdar, V. Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites. Nat. Commun. 2014, 5, 4495, DOI: 10.1038/ncomms5495Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXksVChsbc%253D&md5=376e935af25d8584b338908df79e8b97Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sitesPiliarik, Marek; Sandoghdar, VahidNature Communications (2014), 5 (), 4495CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Detection of single analyte mols. without the use of any label would improve the sensitivity of current biosensors by orders of magnitude to the ultimate graininess of biol. matter. Over two decades, scientists have succeeded in pushing the limits of optical detection to single mols. using fluorescence. However, restrictions in photophysics and labeling protocols make this technique less attractive for biosensing. Recently, mechanisms based on vibrational spectroscopy, photothermal detection, plasmonics and microcavities have been explored for fluorescence-free detection of single biomols. Here, we show that interferometric detection of scattering (iSCAT) can achieve this goal in a direct and label-free fashion. In particular, we demonstrate detection of cancer marker proteins in buffer soln. and in the presence of other abundant proteins. Furthermore, we present super-resoln. imaging of protein binding with nanometer localization precision. The ease of iSCAT instrumentation promises a breakthrough for label-free studies of interactions involving proteins and other small biomols.
- 48Cole, D.; Young, G.; Weigel, A.; Sebesta, A.; Kukura, P. Label-Free Single-Molecule Imaging with Numerical-Aperture-Shaped Interferometric Scattering Microscopy. ACS Photonics 2017, 4, 211– 216, DOI: 10.1021/acsphotonics.6b00912Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFWntr4%253D&md5=d3d61d58c3f8de0f3f523c11cdc6265aLabel-Free Single-Molecule Imaging with Numerical-Aperture-Shaped Interferometric Scattering MicroscopyCole, Daniel; Young, Gavin; Weigel, Alexander; Sebesta, Aleksandar; Kukura, PhilippACS Photonics (2017), 4 (2), 211-216CODEN: APCHD5; ISSN:2330-4022. (American Chemical Society)Our ability to optically interrogate nanoscopic objects is controlled by the difference between their extinction cross sections and the diffraction-limited area to which light can be confined in the far field. We show that a partially transmissive spatial mask placed near the back focal plane of a high numerical aperture microscope objective enhances the extinction contrast of a scatterer near an interface by approx. T-1/2, where T is the transmissivity of the mask. Numerical-aperture-based differentiation of background from scattered light represents a general approach to increasing extinction contrast and enables routine label-free imaging down to the single-mol. level.
- 49Young, G.; Kukura, P. Interferometric Scattering Microscopy. Annu. Rev. Phys. Chem. 2019, 70, 301– 322, DOI: 10.1146/annurev-physchem-050317-021247Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXnsVSlsLs%253D&md5=4aeb584a1b7322c88b48047395fb29daInterferometric Scattering MicroscopyYoung, Gavin; Kukura, PhilippAnnual Review of Physical Chemistry (2019), 70 (), 301-322CODEN: ARPLAP; ISSN:0066-426X. (Annual Reviews)Interferometric scattering microscopy (iSCAT) is an extremely sensitive imaging method based on the efficient detection of light scattered by nanoscopic objects. The ability to, at least in principle, maintain high imaging contrast independent of the exposure time or the scattering cross section of the object allows for unique applications in single-particle tracking, label-free imaging of nanoscopic (dis)assembly, and quant. single-mol. characterization. We illustrate these capabilities in areas as diverse as mechanistic studies of motor protein function, viral capsid assembly, and single-mol. mass measurement in soln. We anticipate that iSCAT will become a widely used approach to unravel previously hidden details of biomol. dynamics and interactions.
- 50Lai, S.-H.; Tamara, S.; Heck, A. J. R. Single-particle mass analysis of intact ribosomes by mass photometry and Orbitrap-based charge detection mass spectrometry. iScience 2021, 24, 103211, DOI: 10.1016/j.isci.2021.103211Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xlt1Ortbc%253D&md5=51d6e5bbaac3478205ec360ba0c9369bSingle-particle mass analysis of intact ribosomes by mass photometry and Orbitrap-based charge detection mass spectrometryLai, Szu-Hsueh; Tamara, Sem; Heck, Albert J. R.iScience (2021), 24 (11), 103211CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)Std. methods for mass anal. measure ensembles of thousand to millions of mols. This approach enables anal. of monodisperse recombinant proteins, whereas some heterogeneous protein assemblies pose a significant challenge, whereby co-occurring stoichiometries, sub-complexes, and modifications hamper anal. using native mass spectrometry. To tackle the challenges posed by mass heterogeneity, single-particle methods may come to the rescue. Recently, two such approaches have been introduced, namely, mass photometry (MP) and Orbitrap-based charge detection mass spectrometry (CDMS). Both methods assess masses of individual mols., albeit adhering to distinct phys. principles. To evaluate these methods side by side, we analyzed a set of ribosomal particles, representing polydisperse ribonucleoprotein assemblies in the MDa range. MP and CDMS provide accurate masses for intact ribosomes and enable quant. anal. of concomitant distinct particles within each ribosome sample. Here, we discuss pros and cons of these single-mol. techniques, also in the context of other techniques used for mass anal.
- 51Liebel, M.; Hugall, J. T.; van Hulst, N. F. Ultrasensitive Label-Free Nanosensing and High-Speed Tracking of Single Proteins. Nano Lett. 2017, 17, 1277– 1281, DOI: 10.1021/acs.nanolett.6b05040Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXps1eqtQ%253D%253D&md5=e1a54380192acc8d088626422814adccUltrasensitive Label-Free Nanosensing and High-Speed Tracking of Single ProteinsLiebel, Matz; Hugall, James T.; van Hulst, Niek F.Nano Letters (2017), 17 (2), 1277-1281CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Label-free detection, anal., and rapid tracking of nanoparticles is crucial for future ultrasensitive sensing applications, ranging from understanding of biol. interactions to the study of size-dependent classical-quantum transitions. Yet optical techniques to distinguish nanoparticles directly among their background remain challenging. Here the authors present amplified interferometric scattering microscopy (a-iSCAT) as a new all-optical method capable of detecting individual nanoparticles as small as 15 kDa proteins that is equiv. to half a GFP. By balancing scattering and reflection amplitudes the interference contrast of the nanoparticle signal is amplified 1 to 2 orders of magnitude. Beyond high sensitivity, a-iSCAT allows high-speed image acquisition exceeding several hundreds of frames-per-second. The authors showcase the performance of their approach by detecting single Streptavidin binding events and by tracking single Ferritin proteins at 400 frames-per-second with 12 nm localization precision over seconds. Moreover, due to its extremely simple exptl. realization, this advancement finally enables a cheap and routine implementation of label-free all-optical single nanoparticle detection platforms with sensitivity operating at the single protein level.
- 52Soltermann, F.; Foley, E. D. B.; Pagnoni, V.; Galpin, M.; Benesch, J. L. P.; Kukura, P.; Struwe, W. B. Quantifying Protein–Protein Interactions by Molecular Counting with Mass Photometry. Angew. Chem., Int. Ed. 2020, 59, 10774– 10779, DOI: 10.1002/anie.202001578Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmt1Gnsbw%253D&md5=d8a6c0cf086ae98d816a799c723b9f61Quantifying Protein-Protein Interactions by Molecular Counting with Mass PhotometrySoltermann, Fabian; Foley, Eric D. B.; Pagnoni, Veronica; Galpin, Martin; Benesch, Justin L. P.; Kukura, Philipp; Struwe, Weston B.Angewandte Chemie, International Edition (2020), 59 (27), 10774-10779CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Interactions between biomols. control the processes of life in health and their malfunction in disease, making their characterization and quantification essential. Immobilization- and label-free anal. techniques are desirable because of their simplicity and minimal invasiveness, but they struggle with quantifying tight interactions. Here, the authors show that mass photometry can accurately count, distinguish by mol. mass, and thereby reveal the relative abundance of different unlabeled biomols. and their complexes in mixts. at the single-mol. level. These measurements det. binding affinities over four orders of magnitude at equil. for both simple and complex stoichiometries within minutes, as well as the assocd. kinetics. These results introduce mass photometry as a rapid, simple and label-free method for studying sub-micromolar binding affinities, with potential for extension towards a universal approach for characterizing complex biomol. interactions.
- 53Wu, D.; Piszczek, G. Measuring the affinity of protein-protein interactions on a single-molecule level by mass photometry. Anal. Biochem. 2020, 592, 113575, DOI: 10.1016/j.ab.2020.113575Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtFCgs7Y%253D&md5=86f67da91b8c3d84c6bef22c68f76dcdMeasuring the affinity of protein-protein interactions on a single-molecule level by mass photometryWu, Di; Piszczek, GrzegorzAnalytical Biochemistry (2020), 592 (), 113575CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)Measurements of biomol. interactions are crucial to understand the mechanisms of the biol. processes they facilitate. Bulk-based methods such as ITC and SPR provide important information on binding affinities, stoichiometry, and kinetics of interactions. However, the ensemble averaging approaches are not able to probe the intrinsic heterogeneity often displayed by biol. systems. Interactions that involve cooperativity or gave multicomponent complexes pose addnl. exptl. challenges. Single-mol. techniques have previously been applied to solve these problems. However, single-mol. expts. are often tech. demanding and require labeling or immobilization of the mols. under study. A recently developed single-mol. method, mass photometry (MP), overcomes these limitations. Here the authors applied MP to measure the affinities of biomol. interactions. MP allows the user to study multivalent complexes and quantify the affinities of different binding sites in a single measurement. Results obtained from this single-mol. technique have been validated by ITC and BLI. The quality and information content of the MP data, combined with simple and fast measurements and low sample consumption makes MP a new preferred method for measuring strong protein-protein interactions.
- 54Bleeker, W. K.; Lammerts van Bueren, J. J.; van Ojik, H. H.; Gerritsen, A. F.; Pluyter, M.; Houtkamp, M.; Halk, E.; Goldstein, J.; Schuurman, J.; van Dijk, M. A.; van de Winkel, J. G. J.; Parren, P. W. H. I. Dual Mode of Action of a Human Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Cancer Therapy. J. Immunol. 2004, 173, 4699– 4707, DOI: 10.4049/jimmunol.173.7.4699Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXnslWmtbs%253D&md5=8fd2e530117b697dea49b8b97b0c3521Dual Mode of Action of a Human Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Cancer TherapyBleeker, Wim K.; Lammerts van Bueren, Jeroen J.; van Ojik, Heidi H.; Gerritsen, Arnout F.; Pluyter, Marielle; Houtkamp, Mischa; Halk, Ed; Goldstein, Joel; Schuurman, Janine; van Dijk, Marc A.; van de Winkel, Jan G. J.; Parren, Paul W. H. I.Journal of Immunology (2004), 173 (7), 4699-4707CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)Epidermal growth factor receptor (EGF-R) overexpression is common in a large no. of solid tumors and represents a neg. prognostic indicator. Overexpression of EGF-R is strongly tumor assocd., and this tyrosine kinase type receptor is considered an attractive target for Ab therapy. In this study, we describe the evaluation of mAb 2F8, a high avidity human mAb (IgG1κ) directed against EGF-R, developed using human Ig transgenic mice. MAb 2F8 effectively blocked binding of EGF and TGF-α to the EGF-R. At satg. concns., 2F8 completely blocked EGF-R signaling and inhibited the in vitro proliferation of EGF-R-overexpressing A431 cells. At much lower concns., assocd. with low receptor occupancy, 2F8 induced efficient Ab-dependent cell-mediated cytotoxicity (ADCC) in vitro. In vivo studies showed potent antitumor effects in models with A431 tumor xenografts in athymic mice. Ex vivo anal. of the EGF-R status in tumor xenografts in 2F8-treated mice revealed that there are two therapeutic mechanisms. First, blocking of EGF-R signaling, which is most effective at complete receptor satn. and therefore requires a relatively high Ab dose. Second, at very low 2F8 receptor occupancy, we obsd. potent antitumor effects in mice, which are likely based on the engagement of immune effector mechanisms, in particular ADCC. Taken together, our findings indicate that ADCC represents an important effector mechanism of this Ab, which is effective at relatively low dose.
- 55de Jong, R. N.; Beurskens, F. J.; Verploegen, S.; Strumane, K.; van Kampen, M. D.; Horstman, W.; Engelberts, P. J.; Oostindie, S. C.; Wang, G.; Heck, A. J. R.; Schuurman, J.; Parren, P. W. H. I. A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface. PLoS Biol. 2016, 14, e1002344 DOI: 10.1371/journal.pbio.1002344Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVKlurbI&md5=c0c494715c1bd4f6102429baf06bfaf9A novel platform for the potentiation of therapeutic antibodies based on antigen-dependent formation of IgG hexamers at the cell surfacede Jong, Rob N.; Beurskens, Frank J.; Verploegen, Sandra; Strumane, Kristin; van Kampen, Muriel D.; Voorhorst, Marleen; Horstman, Wendy; Engelberts, Patrick J.; Oostindie, Simone C.; Wang, Guanbo; Heck, Albert J. R.; Schuurman, Janine; Parren, Paul W. H. I.PLoS Biology (2016), 14 (1), e1002344/1-e1002344/24CODEN: PBLIBG; ISSN:1545-7885. (Public Library of Science)IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technol. platform (HexaBody technol.) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematol. and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell-expressed antigen.
- 56Wu, S.-L.; Taylor, A. D.; Lu, Q.; Hanash, S. M.; Im, H.; Snyder, M.; Hancock, W. S. Identification of Potential Glycan Cancer Markers with Sialic Acid Attached to Sialic Acid and Up-regulated Fucosylated Galactose Structures in Epidermal Growth Factor Receptor Secreted from A431 Cell Line. Mol. Cell. Proteomics 2013, 12, 1239– 1249, DOI: 10.1074/mcp.m112.024554Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXntVGnur0%253D&md5=38f1d9b9ba4445dbcd6fd3853605ee0bIdentification of Potential Glycan Cancer Markers with Sialic Acid Attached to Sialic Acid and Up-regulated Fucosylated Galactose Structures in Epidermal Growth Factor Receptor Secreted from A431 Cell LineWu, Shiaw-Lin; Taylor, Allen D.; Lu, Qiaozhen; Hanash, Samir M.; Im, Hogune; Snyder, Michael; Hancock, William S.Molecular & Cellular Proteomics (2013), 12 (5), 1239-1249CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)We have used powerful HPLC-mass spectrometric approaches to characterize the secreted form of epidermal growth factor receptor (sEGFR). We demonstrated that the amino acid sequence lacked the cytoplasmic domain and was consistent with the primary sequence reported for EGFR purified from a human plasma pool. One of the sEGFR forms, attributed to the alternative RNA splicing, was also confirmed by transcriptional anal. (RNA sequencing). Two unusual types of glycan structures were obsd. in sEGFR as compared with membrane-bound EGFR from the A431 cell line. The unusual glycan structures were di-sialylated glycans (sialic acid attached to sialic acid) at Asn-151 and N-acetylhexosamine attached to a branched fucosylated galactose with N-acetylglucosamine moieties (HexNAc-(Fuc)Gal-GlcNAc) at Asn-420. These unusual glycans at specific sites were either present at a much lower level or were not observable in membrane-bound EGFR present in the A431 cell lysate. The observation of these di-sialylated glycan structures was consistent with the obsd. expression of the corresponding α-N-acetylneuraminide α-2,8-sialyltransferase 2 (ST8SiA2) and α-N-acetylneuraminide α-2,8-sialyltransferase 4 (ST8SiA4), by quant. real time RT-PCR. The connectivity present at the branched fucosylated galactose was also confirmed by methylation of the glycans followed by anal. with sequential fragmentation in mass spectrometry. We hypothesize that the presence of such glycan structures could promote secretion via anionic or steric repulsion mechanisms and thus facilitate the observation of these glycan forms in the secreted fractions. We plan to use this model system to facilitate the search for novel glycan structures present at specific sites in sEGFR as well as other secreted oncoproteins such as Erbb2 as markers of disease progression in blood samples from cancer patients.
- 57Cruz, A. R.; Boer, M. A. d.; Strasser, J.; Zwarthoff, S. A.; Beurskens, F. J.; de Haas, C. J. C.; Aerts, P. C.; Wang, G.; de Jong, R. N.; Bagnoli, F.; van Strijp, J. A. G.; van Kessel, K. P. M.; Schuurman, J.; Preiner, J.; Heck, A. J. R.; Rooijakkers, S. H. M. Staphylococcal protein A inhibits complement activation by interfering with IgG hexamer formation. Proc. Natl. Acad. Sci. U.S.A. 2021, 118, e2016772118 DOI: 10.1073/pnas.2016772118Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXktlGju7g%253D&md5=f679d81dba72a6829863ce0ef89d4cabStaphylococcal protein A inhibits complement activation by interfering with IgG hexamer formationCruz, Ana Rita; den Boer, Maurits A.; Strasser, Juergen; Zwarthoff, Seline A.; Beurskens, Frank J.; de Haas, Carla J. C.; Aerts, Piet C.; Wang, Guanbo; de Jong, Rob N.; Bagnoli, Fabio; van Strijp, Jos A. G.; van Kessel, Kok P. M.; Schuurman, Janine; Preiner, Johannes; Heck, Albert J. R.; Rooijakkers, Suzan H. M.Proceedings of the National Academy of Sciences of the United States of America (2021), 118 (7), e2016772118CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Ig (Ig) G mols. are essential players in the human immune response against bacterial infections. An important effector of IgG-dependent immunity is the induction of complement activation, a reaction that triggers a variety of responses that help kill bacteria. Antibody-dependent complement activation is promoted by the organization of target-bound IgGs into hexamers that are held together via noncovalent Fc-Fc interactions. Here we show that staphylococcal protein A (SpA), an important virulence factor and vaccine candidate of Staphylococcus aureus, effectively blocks IgG hexamerization and subsequent complement activation. Using native mass spectrometry and high-speed at. force microscopy, we demonstrate that SpA blocks IgG hexamerization through competitive binding to the Fc-Fc interaction interface on IgG monomers. In concordance, we show that SpA interferes with the formation of (IgG)6:C1q complexes and prevents downstream complement activation on the surface of S. aureus. Finally, we demonstrate that IgG3 antibodies against S. aureus can potently induce complement activation and opsonophagocytic killing even in the presence of SpA. Together, our findings identify SpA as an immune evasion protein that specifically blocks IgG hexamerization.
- 58van Kampen, M. D.; Kuipers-De Wilt, L. H. A. M.; van Egmond, M. L.; Reinders-Blankert, P.; van den Bremer, E. T. J.; Wang, G.; Heck, A. J. R.; Parren, P. W. H. I.; Beurskens, F. J.; Schuurman, J.; de Jong, R. N. Biophysical Characterization and Stability of IgG1 Variants with Different Hexamerization Propensities; Genmab: Utrecht, the Netherlands, 2021, Unpublished work.Google ScholarThere is no corresponding record for this reference.
- 59Gaboriaud, C.; Thielens, N. M.; Gregory, L. A.; Rossi, V.; Fontecilla-Camps, J. C.; Arlaud, G. J. Structure and activation of the C1 complex of complement: unraveling the puzzle. Trends Immunol. 2004, 25, 368– 373, DOI: 10.1016/j.it.2004.04.008Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXltVKjtb4%253D&md5=7034d86f62bf41ce7444e21125208a19Structure and activation of the C1 complex of complement: unraveling the puzzleGaboriaud, Christine; Thielens, Nicole M.; Gregory, Lynn A.; Rossi, Veronique; Fontecilla-Camps, Juan C.; Arlaud, Gerard J.Trends in Immunology (2004), 25 (7), 368-373CODEN: TIRMAE; ISSN:1471-4906. (Elsevier Science Ltd.)A review. C1, the multimol. protease that triggers the classical pathway of complement, has a major role in the host defense against pathogens. It also participates in other biol. functions, such as immune tolerance, owing to the ability of its binding subunit, C1q, to recognize abnormal structures from self, including apoptotic cells. Structural biol. has been used over the past few years to elucidate the structure of its three subunits: C1q, C1r and C1s. These new advances have led to a comprehensive, three-dimensional model of C1 and provide insights into the mechanisms underlying its activation and the extraordinarily versatile recognition properties of its C1q subunit.
- 60Mortensen, S. A.; Sander, B.; Jensen, R. K.; Pedersen, J. S.; Golas, M. M.; Jensenius, J. C.; Hansen, A. G.; Thiel, S.; Andersen, G. R. Structure and activation of C1, the complex initiating the classical pathway of the complement cascade. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 986– 991, DOI: 10.1073/pnas.1616998114Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1Crsbk%253D&md5=e060af7ce5c8b8417192cf22d0067e52Structure and activation of C1, the complex initiating the classical pathway of the complement cascadeMortensen, Simon A.; Sander, Bjoern; Jensen, Rasmus K.; Pedersen, Jan Skov; Golas, Monika M.; Jensenius, Jens C.; Hansen, Annette G.; Thiel, Steffen; Andersen, Gregers R.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (5), 986-991CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The complement system is an important antimicrobial and inflammation-generating component of the innate immune system. The classical pathway of complement is activated upon binding of the 774-kDa C1 complex, consisting of the recognition mol. C1q and the tetrameric protease complex C1r2s2, to a variety of activators presenting specific mol. patterns such as IgG- and IgM-contg. immune complexes. A canonical model entails a C1r2s2 with its serine protease domains tightly packed together in the center of C1 and an intricate intramol. reaction mechanism for activation of C1r and C1s, induced upon C1 binding to the activator. Here, the authors show that the serine protease domains of C1r and C1s are located at the periphery of the C1r2s2 tetramer both when alone or within the nonactivated C1 complex. The authors' structural studies indicate that the C1 complex adopts a conformation incompatible with intramol. activation of C1, suggesting instead that intermol. proteolytic activation between neighboring C1 complexes bound to a complement activating surface occurs. The authors' results rationalize how a multitude of structurally unrelated mol. patterns can activate C1 and suggests a conserved mechanism for complement activation through the classical and the related lectin pathway.
- 61Hughes-Jones, N. C.; Gardner, B. Reaction between the isolated globular sub-units of the complement component Clq and IgG-complexes. Mol. Immunol. 1979, 16, 697– 701, DOI: 10.1016/0161-5890(79)90010-5Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3cXpt1GhtQ%253D%253D&md5=aa3f9f8733467c26e8a89d46e38b8a48Reaction between the isolated globular subunits of the complement component C1q and IgG-complexesHughes-Jones, N. C.; Gardner, BrigitteMolecular Immunology (1979), 16 (9), 697-701CODEN: MOIMD5; ISSN:0161-5890.The globular subunits of the C1q complement component isolated by collagenase digestion of purified C1q followed by gel filtration had mol. wt. 37,000. The globular subunits reacted with IgG immune complexes as shown by their ability to inhibit the reaction of the latter with C1q-125I; the functional affinity const. for the subunit IgG complex reaction was estd. to be 1.8-5.8 × 104M-1, values similar to those previously found for the reaction between monomeric IgG and intact C1q. C1q thus binds to immune complexes through the peripheral globular subunits.
- 62Feinstein, A.; Richardson, N.; Taussig, M. I. Immunoglobulin flexibility in complement activation. Immunol. Today 1986, 7, 169– 174, DOI: 10.1016/0167-5699(86)90168-4Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XksF2jt7s%253D&md5=3b8e73d0c6242c42f8a122e1a17b94c6Immunoglobulin flexibility in complement activationFeinstein, Arnold; Richardson, Neil; Taussig, Michael J.Immunology Today (1986), 7 (6), 169-74CODEN: IMTOD8; ISSN:0167-4919.A review with 40 refs. of the activation of complement C1 after it has bound to a site on the Fc portion of an antibody mol. and how this site is made available in different antibody classes.
- 63Burton, D. R. Immunoglobulin G: Functional sites. Mol. Immunol. 1985, 22, 161– 206, DOI: 10.1016/0161-5890(85)90151-8Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXhs1Cqs7k%253D&md5=77689b7b3b61fcb16eee95f1dde8d923Immunoglobulin G: functional sitesBurton, Dennis R.Molecular Immunology (1985), 22 (3), 161-206CODEN: MOIMD5; ISSN:0161-5890.A review with ∼240 refs. on the functional binding sites of the IgG mol. Data from crystallog. studies and of IgG interactions are considered with respect to the localization of the binding sites.
- 64Sharp, T. H.; Boyle, A. L.; Diebolder, C. A.; Kros, A.; Koster, A. J.; Gros, P. Insights into IgM-mediated complement activation based on in situ structures of IgM-C1-C4b. Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 11900– 11905, DOI: 10.1073/pnas.1901841116Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtFeqsLjP&md5=a295722047ff0a169ab2ec96e15d375bInsights into IgM-mediated complement activation based on in situ structures of IgM-C1-C4bSharp, Thomas H.; Boyle, Aimee L.; Diebolder, Christoph A.; Kros, Alexander; Koster, Abraham J.; Gros, PietProceedings of the National Academy of Sciences of the United States of America (2019), 116 (24), 11900-11905CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Antigen binding by serum Ig-M (IgM) protects against microbial infections and helps to prevent autoimmunity, but causes life-threatening diseases when mistargeted. How antigen-bound IgM activates complement-immune responses remains unclear. We present cryoelectron tomog. structures of IgM, C1, and C4b complexes formed on antigen-bearing lipid membranes by normal human serum at 4°C. The IgM-C1-C4b complexes revealed C4b product release as the temp.-limiting step in complement activation. Both IgM hexamers and pentamers adopted hexagonal, dome-shaped structures with Fab pairs, dimerized by hinge domains, bound to surface antigens that support a platform of Fc regions. C1 binds IgM through widely spread C1q-collagen helixes, with C1r proteases pointing outward and C1s bending downward and interacting with surface-attached C4b, which further interacts with the adjacent IgM-Fab2 and globular C1q-recognition unit. Based on these data, we present mechanistic models for antibody-mediated, C1q-transmitted activation of C1 and for C4b deposition, while further conformational rearrangements are required to form C3 convertases.
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Abstract
Figure 1
Figure 1. Qualitative and quantitative characterization of IgG4Δhinge mutants by MP. (A) Mass histogram showing particle counts of “wt” IgG4Δhinge in PBS, jump-diluted from 16 μM and measured at 4 nM, with normal distributions fitted for HL (bright red, 26%) and (HL)2 (dark red, 74%). This histogram corresponds to the first 80 s after jump dilution. Masses are indicated as the mean of a fitted normal distribution. (B) Monomer–dimer distribution during an extended experiment in triplicate (shades of red) revealed that the abundance of “wt” (HL)2 decreased during the analysis time window. Data were split into bins of 100 events, and an exponential decay function was fitted to the dimer abundance within the bin to determine the koff. (C) Determined koff was used to estimate the ratio between HL and (HL)2 at the instant of jump dilution for a dilution series of the “wt” measured in triplicate, revealing the apparent Kd of each measurement, followed by the calculation of a Kd value for the whole dilution series. (D) Fractional dimer abundances and Kd values resulting from a dilution series of four IgG4Δhinge mutational variants, demonstrating that MP can assess affinities over a broad dynamic range.
Figure 2
Figure 2. MP and CD-MS may overcome certain limitations of native MS in the mass measurements of highly heterogeneous antibody–antigen complexes. (A) MP provides an average mass for IgG1 (upper panel) and sEGFR (middle) and is not hampered by the high micro-heterogeneity of the latter. When 2 μM IgG1 was incubated with 5 μM of sEGFR to form (IgG1)1:(sEGFR)1 and (IgG1)1:(sEGFR)2 complexes, jump dilution MP could resolve these highly heterogeneous species (lower). (B) Although native MS on samples at the same concentrations provided superior mass resolution and accuracy for free IgG1 (upper), resolving individual glycoforms (zoom), the high microheterogeneity of sEGFR, measured separately (middle) and in antibody–antigen complexes (lower), resulted in unresolved features. In these experiments, overlapping charge states prevented mass measurements of these species. (C) More accurate masses could be obtained by native CD-MS, measuring in two dimensions m/z and z (insets) for sEGFR (upper) and all co-occurring species involving IgG1 and sEGFR (lower). For these experiments, the same native MS samples were diluted 20-fold, leading to re-equilibration and thus a lower binding occupancy.
Figure 3
Figure 3. MP enables qualitative and quantitative characterization of the monomer–hexamer equilibrium of IgG1-RGY. (A) MP mass histogram (top) of 2 μM IgG1-RGY in PBS jump-diluted to 10 nM showing monomeric ((IgG1)1) and hexameric ((IgG1)6) species. The mass of the hexamer was consistently measured about 70 kDa too high. The relative abundance of the IgG1-RGY hexamer was measured over a dilution series spanning a concentration range of 0.1 to 8 μM (bottom), with error bars indicating the standard deviation over three technical replicate measurements. (B) SEC-MALS chromatogram of the same 2 μM IgG1-RGY sample (top) and the fractional abundance of the hexamer as measured by SEC-UV over a dilution series (bottom), revealing a similar monomer to hexamer ratio. The quantitative data in the lower panel are adapted from the work of van Kampen et al. (58) (C) Native mass spectrum (top) of 2 μM IgG1-RGY measured in 150 mM NH4OAc pH 7.5, revealing two distinct ion series for the monomer and hexamer, with ions originating from intermediate oligomeric states observed at lower abundance. While generally in good agreement with the other methods, hexamer abundances measured by MS (bottom) were less consistent and higher than expected, particularly at the lowest measured concentrations.
Figure 4
Figure 4. MP and CD-MS successfully determine the mass and stoichiometry of highly heterogeneous (sEGFR)12:(IgG1)6:C1q immune complexes. (A) MP measurements of IgG1-RGY incubated with C1q reveal the formation of (IgG1)6:(C1q)1 complexes, with nearly all IgG hexamers occupied. When incubating C1q with pre-formed (IgG1)6:(sEGFR)12, MP resolves a 2.35 MDa complex, likely corresponding to (sEGFR)12:(IgG1)6:(C1q)1. (B) SEC-MALS-UV-RI analysis similarly reveals the formation of ∼1.3 MDa (IgG1)6:(C1q)1 (with (IgG1)6 measured as the 0.76 MDa “protein” and C1q as a 0.49 MDa “modifier”). When sEGFR was added, SEC-MALS-UV-RI revealed the formation of larger complexes of around 1.9 MDa (1.5 MDa for (sEGFR)12:(IgG1)6 with a 0.41 MDa modifier). (C) Measurement of the same samples by native MS reveals an accurate mass for (IgG1)6:(C1q)1, but the technique struggles with complexes involving sEGFR. Larger ion species were detected in such experiments, but they could not be charge-resolved. (D) Single-particle measurements of the distribution around m/z 21,000 by CD-MS (top) revealed a mass of 2.42 MDa (bottom) corresponding to the expected mass of the full (sEGFR)12:(IgG1)6:(sEGFR)12 complex (bottom).
References
This article references 64 other publications.
- 1Schroeder, H. W., Jr.; Cavacini, L. Structure and function of immunoglobulins. J. Allergy Clin. Immunol. 2010, 125, S41– S52, DOI: 10.1016/j.jaci.2009.09.0461https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC3c7lsFCrtw%253D%253D&md5=b10939b641dc1a447cef6fd319db0bf1Structure and function of immunoglobulinsSchroeder Harry W Jr; Cavacini LisaThe Journal of allergy and clinical immunology (2010), 125 (2 Suppl 2), S41-52 ISSN:.Immunoglobulins are heterodimeric proteins composed of 2 heavy and 2 light chains. They can be separated functionally into variable domains that bind antigens and constant domains that specify effector functions, such as activation of complement or binding to Fc receptors. The variable domains are created by means of a complex series of gene rearrangement events and can then be subjected to somatic hypermutation after exposure to antigen to allow affinity maturation. Each variable domain can be split into 3 regions of sequence variability termed the complementarity-determining regions (CDRs) and 4 regions of relatively constant sequence termed the framework regions. The 3 CDRs of the heavy chain are paired with the 3 CDRs of the light chain to form the antigen-binding site, as classically defined. The constant domains of the heavy chain can be switched to allow altered effector function while maintaining antigen specificity. There are 5 main classes of heavy chain constant domains. Each class defines the IgM, IgG, IgA, IgD, and IgE isotypes. IgG can be split into 4 subclasses, IgG1, IgG2, IgG3, and IgG4, each with its own biologic properties, and IgA can similarly be split into IgA1 and IgA2.
- 2Peng, H.-P.; Lee, K. H.; Jian, J.-W.; Yang, A.-S. Origins of specificity and affinity in antibody-protein interactions. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, E2656– E2665, DOI: 10.1073/pnas.14011311112https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXpvVSlsLY%253D&md5=7dd3007af6e46f33699b20d18c1f35f6Origins of specificity and affinity in antibody-protein interactionsPeng, Hung-Pin; Lee, Kuo Hao; Jian, Jhih-Wei; Yang, An-SueiProceedings of the National Academy of Sciences of the United States of America (2014), 111 (26), E2656-E2665CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Natural antibodies are frequently elicited to recognize diverse protein surfaces, where the sequence features of the epitopes are frequently indistinguishable from those of nonepitope protein surfaces. It is not clearly understood how the paratopes are able to recognize sequence-wise featureless epitopes and how a natural antibody repertoire with limited variants can recognize seemingly unlimited protein antigens foreign to the host immune system. In this work, computational methods were used to predict the functional paratopes with the 3D antibody variable domain structure as input. The predicted functional paratopes were reasonably validated by the hot spot residues known from exptl. alanine scanning measurements. The functional paratope (hot spot) predictions on a set of 111 antibody-antigen complex structures indicate that arom., mostly tyrosyl, side chains constitute the major part of the predicted functional paratopes, with short-chain hydrophilic residues forming the minor portion of the predicted functional paratopes. These arom. side chains interact mostly with the epitope main chain atoms and side-chain carbons. The functional paratopes are surrounded by favorable polar atomistic contacts in the structural paratope-epitope interfaces; more that 80% these polar contacts are electrostatically favorable and about 40% of these polar contacts form direct hydrogen bonds across the interfaces. These results indicate that a limited repertoire of antibodies bearing paratopes with diverse structural contours enriched with arom. side chains among short-chain hydrophilic residues can recognize all sorts of protein surfaces, because the determinants for antibody recognition are common physicochem. features ubiquitously distributed over all protein surfaces.
- 3Sela-Culang, I.; Kunik, V.; Ofran, Y. The Structural Basis of Antibody-Antigen Recognition. Front. Immunol. 2013, 4, 302, DOI: 10.3389/fimmu.2013.003023https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2cfltlahtg%253D%253D&md5=130f71681db9d6aa55eabc2434160ed5The structural basis of antibody-antigen recognitionSela-Culang Inbal; Kunik Vered; Ofran YanayFrontiers in immunology (2013), 4 (), 302 ISSN:1664-3224.The function of antibodies (Abs) involves specific binding to antigens (Ags) and activation of other components of the immune system to fight pathogens. The six hypervariable loops within the variable domains of Abs, commonly termed complementarity determining regions (CDRs), are widely assumed to be responsible for Ag recognition, while the constant domains are believed to mediate effector activation. Recent studies and analyses of the growing number of available Ab structures, indicate that this clear functional separation between the two regions may be an oversimplification. Some positions within the CDRs have been shown to never participate in Ag binding and some off-CDRs residues often contribute critically to the interaction with the Ag. Moreover, there is now growing evidence for non-local and even allosteric effects in Ab-Ag interaction in which Ag binding affects the constant region and vice versa. This review summarizes and discusses the structural basis of Ag recognition, elaborating on the contribution of different structural determinants of the Ab to Ag binding and recognition. We discuss the CDRs, the different approaches for their identification and their relationship to the Ag interface. We also review what is currently known about the contribution of non-CDRs regions to Ag recognition, namely the framework regions (FRs) and the constant domains. The suggested mechanisms by which these regions contribute to Ag binding are discussed. On the Ag side of the interaction, we discuss attempts to predict B-cell epitopes and the suggested idea to incorporate Ab information into B-cell epitope prediction schemes. Beyond improving the understanding of immunity, characterization of the functional role of different parts of the Ab molecule may help in Ab engineering, design of CDR-derived peptides, and epitope prediction.
- 4Hayes, J.; Wormald, M.; Rudd, P.; Davey, G. Fc gamma receptors: glycobiology and therapeutic prospects. J. Inflammation Res. 2016, 9, 209– 219, DOI: 10.2147/jir.s1212334https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpvFOjsrs%253D&md5=edef75d96d3a6eebcb8cd99542282d0eFc gamma receptors: glycobiology and therapeutic prospectsHayes, Jerrard M.; Wormald, Mark R.; Rudd, Pauline M.; Davey, Gavin P.Journal of Inflammation Research (2016), 9 (), 209-219CODEN: JIROAC; ISSN:1178-7031. (Dove Medical Press Ltd.)Therapeutic antibodies hold great promise for the treatment of cancer and autoimmune diseases, and developments in antibody-drug conjugates and bispecific antibodies continue to enhance treatment options for patients. Ig (Ig) G antibodies are proteins with complex modifications, which have a significant impact on their function. The most important of these modifications is glycosylation, the addn. of conserved glycans to the antibody Fc region, which is crit. for its interaction with the immune system and induction of effector activities such as antibody-dependent cell cytotoxicity, complement activation and phagocytosis. Communication of IgG antibodies with the immune system is controlled and mediated by Fc gamma receptors (FcγRs), membrane-bound proteins, which relay the information sensed and gathered by antibodies to the immune system. These receptors are also glycoproteins and provide a link between the innate and adaptive immune systems. Recent information suggests that this receptor glycan modification is also important for the interaction with antibodies and downstream immune response. In this study, the current knowledge on FcγR glycosylation is discussed, and some insight into its role and influence on the interaction properties with IgG, particularly in the context of biotherapeutics, is provided. For the purpose of this study, other Fc receptors such as FcαR, FcεR or FcRn are not discussed extensively, as IgG-based antibodies are currently the only therapeutic antibody-based products on the market. In addn., FcγRs as therapeutics and therapeutic targets are discussed, and insight into and comment on the therapeutic aspects of receptor glycosylation are provided.
- 5Mellor, J. D.; Brown, M. P.; Irving, H. R.; Zalcberg, J. R.; Dobrovic, A. A critical review of the role of Fc gamma receptor polymorphisms in the response to monoclonal antibodies in cancer. J. Hematol. Oncol. 2013, 6, 1, DOI: 10.1186/1756-8722-6-15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXitlSmsr0%253D&md5=75ffe82f33df12688942b58224ec9230A critical review of the role of Fc gamma receptor polymorphisms in the response to monoclonal antibodies in cancerMellor, James D.; Brown, Michael P.; Irving, Helen R.; Zalcberg, John R.; Dobrovic, AlexanderJournal of Hematology & Oncology (2013), 6 (), 1CODEN: JHOOAO; ISSN:1756-8722. (BioMed Central Ltd.)A review. Antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism of action of therapeutic monoclonal antibodies (mAbs) such as cetuximab, rituximab and trastuzumab. Fc gamma receptors (FcgR) on human white blood cells are an integral part of the ADCC pathway. Differential response to therapeutic mAbs has been reported to correlate with specific polymorphisms in two of these genes: FCGR2A (H131R) and FCGR3A (V158F). These polymorphisms are assocd. with differential affinity of the receptors for mAbs. This review critically examines the current evidence for genotyping the corresponding single nucleotide polymorphisms (SNPs) to predict response to mAbs in patients with cancer.
- 6Ugurlar, D.; Howes, S. C.; de Kreuk, B.-J.; de Jong, R. N.; Beurskens, F. J.; Schuurman, J.; Koster, A. J.; Sharp, T. H.; Parren, P. W. H. I.; Gros, P. Structures of C1-IgG1 provide insights into how danger pattern recognition activates complement. Science 2018, 359, 794– 797, DOI: 10.1126/science.aao49886https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXivVaisb0%253D&md5=7dbf8ff47e23f7dcc8dc77e40f74e35fStructures of C1-IgG1 provide insights into how danger pattern recognition activates complementUgurlar, Deniz; Howes, Stuart C.; de Kreuk, Bart-Jan; Koning, Roman I.; de Jong, Rob N.; Beurskens, Frank J.; Schuurman, Janine; Koster, Abraham J.; Sharp, Thomas H.; Parren, Paul W. H. I.; Gros, PietScience (Washington, DC, United States) (2018), 359 (6377), 794-797CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Danger patterns on microbes or damaged host cells bind and activate C1, inducing innate immune responses and clearance through the complement cascade. How these patterns trigger complement initiation remains elusive. Here, we present cryo-electron microscopy analyses of C1 bound to monoclonal antibodies in which we obsd. heterogeneous structures of single and clustered C1-IgG1 hexamer complexes. Distinct C1q binding sites are obsd. on the two Fc-CH2 domains of each IgG mol. These are consistent with known interactions and also reveal addnl. interactions, which are supported by functional IgG1-mutant anal. Upon antibody binding, the C1q arms condense, inducing rearrangements of the C1r2s2 proteases and tilting C1q's cone-shaped stalk. The data suggest that C1r may activate C1s within single, strained C1 complexes or between neighboring C1 complexes on surfaces.
- 7Lu, L. L.; Suscovich, T. J.; Fortune, S. M.; Alter, G. Beyond binding: antibody effector functions in infectious diseases. Nat. Rev. Immunol. 2018, 18, 46– 61, DOI: 10.1038/nri.2017.1067https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslehtr%252FN&md5=29f715b374b845db613d5eb8a40f6035Beyond binding: antibody effector functions in infectious diseasesLu, Lenette L.; Suscovich, Todd J.; Fortune, Sarah M.; Alter, GalitNature Reviews Immunology (2018), 18 (1), 46-61CODEN: NRIABX; ISSN:1474-1733. (Nature Research)Antibodies play an essential role in host defense against pathogens by recognizing microorganisms or infected cells. Although preventing pathogen entry is one potential mechanism of protection, antibodies can control and eradicate infections through a variety of other mechanisms. In addn. to binding and directly neutralizing pathogens, antibodies drive the clearance of bacteria, viruses, fungi and parasites via their interaction with the innate and adaptive immune systems, leveraging a remarkable diversity of antimicrobial processes locked within our immune system. Specifically, antibodies collaboratively form immune complexes that drive sequestration and uptake of pathogens, clear toxins, eliminate infected cells, increase antigen presentation and regulate inflammation. The diverse effector functions that are deployed by antibodies are dynamically regulated via differential modification of the antibody const. domain, which provides specific instructions to the immune system. Here, we review mechanisms by which antibody effector functions contribute to the balance between microbial clearance and pathol. and discuss tractable lessons that may guide rational vaccine and therapeutic design to target gaps in our infectious disease armamentarium.
- 8Diebolder, C. A.; Beurskens, F. J.; de Jong, R. N.; Koning, R. I.; Strumane, K.; Lindorfer, M. A.; Voorhorst, M.; Ugurlar, D.; Rosati, S.; Heck, A. J. R.; van de Winkel, J. G. J.; Wilson, I. A.; Koster, A. J.; Taylor, R. P.; Ollmann Saphire, E.; Burton, D. R.; Schuurman, J.; Gros, P.; Parren, P. W. H. I. Complement is activated by IgG hexamers assembled at the cell surface. Science 2014, 343, 1260– 1263, DOI: 10.1126/science.12489438https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjvVygsrs%253D&md5=db48d5a038a5d64810b40d634840e73bComplement Is Activated by IgG Hexamers Assembled at the Cell SurfaceDiebolder, Christoph A.; Beurskens, Frank J.; de Jong, Rob N.; Koning, Roman I.; Strumane, Kristin; Lindorfer, Margaret A.; Voorhorst, Marleen; Ugurlar, Deniz; Rosati, Sara; Heck, Albert J. R.; van de Winkel, Jan G. J.; Wilson, Ian A.; Koster, Abraham J.; Taylor, Ronald P.; Ollmann Saphire, Erica; Burton, Dennis R.; Schuurman, Janine; Gros, Piet; Parren, Paul W. H. I.Science (Washington, DC, United States) (2014), 343 (6176), 1260-1263CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Complement activation by antibodies bound to pathogens, tumors, and self antigens is a crit. feature of natural immune defense, a no. of disease processes, and immunotherapies. How antibodies activate the complement cascade, however, is poorly understood. The authors found that specific noncovalent interactions between Fc segments of IgG antibodies resulted in the formation of ordered antibody hexamers after antigen binding on cells. These hexamers recruited and activated C1, the first component of complement, thereby triggering the complement cascade. The interactions between neighboring Fc segments could be manipulated to block, reconstitute, and enhance complement activation and killing of target cells, using all four human IgG subclasses. The authors offer a general model for understanding antibody-mediated complement activation and the design of antibody therapeutics with enhanced efficacy.
- 9Nguyen, H.; Park, J.; Kang, S.; Kim, M. Surface plasmon resonance: a versatile technique for biosensor applications. Sensors 2015, 15, 10481– 10510, DOI: 10.3390/s1505104819https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXptlKqurw%253D&md5=98cc17f36c89a34ab7db91932cbcb954Surface plasmon resonance: a versatile technique for biosensor applicationsNguyen, Hoang Hiep; Park, Jeho; Kang, Sebyung; Kim, MoonilSensors (2015), 15 (5), 10481-10510CODEN: SENSC9; ISSN:1424-8220. (MDPI AG)A review. Surface plasmon resonance (SPR) is a label-free detection method which has emerged during the last two decades as a suitable and reliable platform in clin. anal. for biomol. interactions. The technique makes it possible to measure interactions in real-time with high sensitivity and without the need of labels. This review article discusses a wide range of applications in optical-based sensors using either surface plasmon resonance (SPR) or surface plasmon resonance imaging (SPRI). Here we summarize the principles, provide examples, and illustrate the utility of SPR and SPRI through example applications from the biomedical, proteomics, genomics and bioengineering fields. In addn., SPR signal amplification strategies and surface functionalization are covered in the review.
- 10Puiu, M.; Bala, C. SPR and SPR Imaging: Recent Trends in Developing Nanodevices for Detection and Real-Time Monitoring of Biomolecular Events. Sensors 2016, 16, 870, DOI: 10.3390/s16060870There is no corresponding record for this reference.
- 11Kamat, V.; Rafique, A. Designing binding kinetic assay on the bio-layer interferometry (BLI) biosensor to characterize antibody-antigen interactions. Anal. Biochem. 2017, 536, 16– 31, DOI: 10.1016/j.ab.2017.08.00211https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlCntrjO&md5=863482dce4b38b09ccbe0dc33005d958Designing binding kinetic assay on the bio-layer interferometry (BLI) biosensor to characterize antibody-antigen interactionsKamat, Vishal; Rafique, AshiqueAnalytical Biochemistry (2017), 536 (), 16-31CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)The Octet biosensors provide a high-throughput alternative to the well-established surface plasmon resonance (SPR) and SPR imaging (SPRi) biosensors to characterize antibody-antigen interactions. However, the utility of the Octet biosensors for accurate and reproducible measurement of binding rate consts. of monoclonal antibodies (mAbs) is limited due to challenges such as analyte rebinding, and mass transport limitation (MTL). This study focuses on addressing these challenges and provides exptl. conditions to reliably measure kinetics of mAb-antigen interactions. The mAb capture d. of less than 0.6 nm was found to be optimal to measure a wide range of binding affinities on Octet HTX biosensor. The titrn. kinetic and single cycle kinetic assays performed on Octet HTX generated reproducible binding kinetic parameters and correlated with the values measured on Biacore 4000 and MASS-1. Kinetic assays performed on 0.1 nm d. mAb surfaces significantly reduced MTL and enabled characterization of picomolar affinity mAbs. Finally, kinetic anal. performed on 150 antibodies to 10 antigens with mol. wts. ranging from 21kD to 105kD showed concordance between Octet HTX, Biacore 4000 and MASS-1 (R2 > 0.90). The data presented in this study suggest that under optimal exptl. conditions, Octet biosensor is capable of generating kinetic values comparable to SPR/SPRi biosensors.
- 12Ogi, H. Wireless-electrodeless quartz-crystal-microbalance biosensors for studying interactions among biomolecules: a review. Proc. Jpn. Acad., Ser. B 2013, 89, 401– 417, DOI: 10.2183/pjab.89.40112https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXisFKktb8%253D&md5=fba6252394f0dc5f026d3ab86b340299Wireless-electrodeless quartz-crystal-microbalance biosensors for studying interactions among biomolecules: a reviewOgi, HirotsuguProceedings of the Japan Academy, Series B: Physical and Biological Sciences (2013), 89 (9), 401-417CODEN: PJABDW; ISSN:0386-2208. (Nippon Gakushiin)A review. The mass sensitivity of quartz-crystal microbalance (QCM) was drastically improved by removing electrodes and wires attached on the quartz surfaces. Instead of wire connections, intended vibrations of quartz oscillators were excited and detected by antennas through electromagnetic waves. This noncontacting measurement is the key for ultrahigh-sensitive detection of proteins in liqs. as well as quant. measurements. This review shows the principle of wireless QCMs, their applications to studying interactions among biomols. and aggregation reactions of amyloid β peptides, and the next-generation MEMS QCM, the resonance acoustic microbalance with naked embedded quartz (RAMNE-Q).
- 13Goldschen-Ohm, M. P.; White, D. S.; Klenchin, V. A.; Chanda, B.; Goldsmith, R. H. Observing Single-Molecule Dynamics at Millimolar Concentrations. Angew. Chem., Int. Ed. 2017, 56, 2399– 2402, DOI: 10.1002/anie.20161205013https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVegsb0%253D&md5=7d02aac64191833cdd8b7329a22dcd1cObserving Single-Molecule Dynamics at Millimolar ConcentrationsGoldschen-Ohm, Marcel P.; White, David S.; Klenchin, Vadim A.; Chanda, Baron; Goldsmith, Randall H.Angewandte Chemie, International Edition (2017), 56 (9), 2399-2402CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Single-mol. fluorescence microscopy is a powerful tool for revealing chem. dynamics and mol. assocn. mechanisms, but has been limited to low concns. of fluorescent species and is only suitable for studying high affinity reactions. Here, the authors combine nanophotonic zero-mode waveguides (ZMWs) with fluorescence resonance energy transfer (FRET) to resolve single-mol. assocn. dynamics at up to millimolar concns. of fluorescent species. This approach extends the resoln. of mol. dynamics to >100-fold higher concns., enabling observations at concns. relevant to biol. and chem. processes, and thus making single-mol. techniques applicable to a tremendous range of previously inaccessible mol. targets. The authors deploy this approach to show that the binding of cGMP to pacemaking ion channels is weakened by a slower internal conformational change.
- 14Aggarwal, V.; Ha, T. Single-molecule fluorescence microscopy of native macromolecular complexes. Curr. Opin. Struct. Biol. 2016, 41, 225– 232, DOI: 10.1016/j.sbi.2016.09.00614https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFajsrjE&md5=fe5b02730259f80da2c3f25a18f47ad8Single-molecule fluorescence microscopy of native macromolecular complexesAggarwal, Vasudha; Ha, TaekjipCurrent Opinion in Structural Biology (2016), 41 (), 225-232CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)Macromol. complexes consisting of proteins, lipids, and/or nucleic acids are ubiquitous in biol. processes. Their compn., stoichiometry, order of assembly, and conformations can be heterogeneous or can change dynamically, making single-mol. studies best suited to measure these properties accurately. Recent single-mol. pull-down and other related approaches have combined the principles of conventional co-immunopptn. assay with single-mol. fluorescence microscopy to probe native macromol. complexes. In this review, we present the advances in single-mol. pull-down methods and biol. systems that have been investigated in such semi vivo manner.
- 15Nobbmann, U.; Connah, M.; Fish, B.; Varley, P.; Gee, C.; Mulot, S.; Chen, J.; Zhou, L.; Lu, Y.; Sheng, F.; Yi, J.; Harding, S. E. Dynamic light scattering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodies. Biotechnol. Genet. Eng. Rev. 2007, 24, 117– 128, DOI: 10.1080/02648725.2007.1064809515https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlKiurjI&md5=09f5a1d27a12b0c39fa1b3679e4c5c8fDynamic light scatttering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodiesNobbmann, Ulf; Connah, Malcolm; Fish, Brendan; Varley, Paul; Gee, Chris; Mulot, Sandrine; Chen, Juntao; Zhou, Liang; Lu, Yanling; Sheng, Fei; Yi, Junming; Harding, Stephen E.Biotechnology & Genetic Engineering Reviews (2007), 24 (), 117-128CODEN: BGERES; ISSN:0264-8725. (Nottingham University Press)A review compares dynamic light scattering (DLS) to anal. ultracentrifugation. It shows that when DLS is combined online with the sepn. power of HPLC it is possible to rapidly identify sep. species in soln.: a heterogeneous material is sepd. by the material of the column and the elution peaks can be analyzed sep. downstream by the DLS.
- 16Stetefeld, J.; McKenna, S. A.; Patel, T. R. Dynamic light scattering: a practical guide and applications in biomedical sciences. Biophys. Rev. 2016, 8, 409– 427, DOI: 10.1007/s12551-016-0218-616https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1Gqs7bP&md5=468714b5413c92edb45e985fc32e8164Dynamic light scattering: a practical guide and applications in biomedical sciencesStetefeld, Jorg; McKenna, Sean A.; Patel, Trushar R.Biophysical Reviews (2016), 8 (4), 409-427CODEN: BRIECG; ISSN:1867-2450. (Springer)Dynamic light scattering (DLS), also known as photon correlation spectroscopy (PCS), is a very powerful tool for studying the diffusion behavior of macromols. in soln. The diffusion coeff., and hence the hydrodynamic radii calcd. from it, depends on the size and shape of macromols. In this review, we provide evidence of the usefulness of DLS to study the homogeneity of proteins, nucleic acids, and complexes of protein-protein or protein-nucleic acid prepns., as well as to study protein-small mol. interactions. Further, we provide examples of DLS's application both as a complementary method to anal. ultracentrifugation studies and as a screening tool to validate soln. scattering models using detd. hydrodynamic radii.
- 17Hanlon, A. D.; Larkin, M. I.; Reddick, R. M. Free-Solution, Label-Free Protein-Protein Interactions Characterized by Dynamic Light Scattering. Biophys. J. 2010, 98, 297– 304, DOI: 10.1016/j.bpj.2009.09.06117https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXkslWisbo%253D&md5=2754d6e515aa45097d9a89158290e652Free-solution, label-free protein-protein interactions characterized by dynamic light scatteringHanlon, Amy D.; Larkin, Michael I.; Reddick, Ryan M.Biophysical Journal (2010), 98 (2), 297-304CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)We report a free-soln., label-free method for quant. characterization of macromol. interactions using dynamic light scattering, a temp. controlled plate reader, and a multiwell concn. gradient. This nondestructive technique enabled detn. of stoichiometry of binding, equil. dissocn. const., and thermodn. parameters, as well as the impact of temp., buffer salinity, and a small-mol. inhibitor. The low vol. capability of dynamic light scattering reduced the required sample to 426 pmol/expt., with detection limits for 150-kDa proteins anticipated to be in the low femtomole range.
- 18Berkowitz, S. A.; Philo, J. S. Characterizing Biopharmaceuticals using Analytical Ultracentrifugation. In Biophysical Characterization of Proteins in Developing Biopharmaceuticals; Houde, D. J., Berkowitz, S. A., Eds.; Elsevier: Amsterdam, 2015; pp 211– 260.There is no corresponding record for this reference.
- 19Uchiyama, S.; Noda, M.; Krayukhina, E. Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodies. Biophys. Rev. 2018, 10, 259– 269, DOI: 10.1007/s12551-017-0374-319https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1Gqtg%253D%253D&md5=eff4b1bdce19b9a66e0b35ee1982bf62Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodiesUchiyama, Susumu; Noda, Masanori; Krayukhina, ElenaBiophysical Reviews (2018), 10 (2), 259-269CODEN: BRIECG; ISSN:1867-2450. (Springer)A review. Sedimentation velocity anal. ultracentrifugation (SV-AUC) coupled with direct computational fitting of the obsd. concn. profiles (sedimentating boundary) have been developed and widely used for the characterization of macromols. and nanoparticles in soln. In particular, size distribution anal. by SV-AUC has become a reliable and essential approach for the characterization of biopharmaceuticals including therapeutic antibodies. In this review, we describe the importance and advantages of SV-AUC for studying biopharmaceuticals, with an emphasis on strategies for sample prepn., data acquisition, and data anal. Recent discoveries enabled by AUC with a fluorescence detection system and potential future applications are also discussed.
- 20Schuck, P. Analytical Ultracentrifugation as a Tool for Studying Protein Interactions. Biophys. Rev. 2013, 5, 159– 171, DOI: 10.1007/s12551-013-0106-220https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXntVWqsb0%253D&md5=088ec73746d699077e6b29317ea03974Analytical ultracentrifugation as a tool for studying protein interactionsSchuck, PeterBiophysical Reviews (2013), 5 (2), 159-171CODEN: BRIECG; ISSN:1867-2450. (Springer)A review. The last two decades have led to significant progress in the field of anal. ultracentrifugation driven by instrumental, theor., and computational methods. This review will highlight key developments in sedimentation equil. (SE) and sedimentation velocity (SV) anal. For SE, this includes the anal. of tracer sedimentation equil. at high concns. with strong thermodn. non-ideality, and for ideally interacting systems, the development of strategies for the anal. of heterogeneous interactions towards global multi-signal and multi-speed SE anal. with implicit mass conservation. For SV, this includes the development and applications of numerical solns. of the Lamm equation, noise decompn. techniques enabling direct boundary fitting, diffusion deconvoluted sedimentation coeff. distributions, and multi-signal sedimentation coeff. distributions. Recently, effective particle theory has uncovered simple phys. rules for the co-migration of rapidly exchanging systems of interacting components in SV. This has opened new possibilities for the robust interpretation of the boundary patterns of heterogeneous interacting systems. Together, these SE and SV techniques have led to new approaches to study macromol. interactions across the entire spectrum of affinities, including both attractive and repulsive interactions, in both dil. and highly concd. solns., which can be applied to single-component solns. of self-assocg. proteins as well as the study of multi-protein complex formation in multi-component solns.
- 21Gandhi, A. V.; Pothecary, M. R.; Bain, D. L.; Carpenter, J. F. Some Lessons Learned From a Comparison Between Sedimentation Velocity Analytical Ultracentrifugation and Size Exclusion Chromatography to Characterize and Quantify Protein Aggregates. J. Pharm. Sci. 2017, 106, 2178– 2186, DOI: 10.1016/j.xphs.2017.04.04821https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXptFClsL4%253D&md5=7b172c52f61236864ae6763d22439bbbSome Lessons Learned From a Comparison Between Sedimentation Velocity Analytical Ultracentrifugation and Size Exclusion Chromatography to Characterize and Quantify Protein AggregatesGandhi, Aditya V.; Pothecary, Mark R.; Bain, David L.; Carpenter, John F.Journal of Pharmaceutical Sciences (2017), 106 (8), 2178-2186CODEN: JPMSAE; ISSN:0022-3549. (Elsevier Inc.)There are numerous problems with size exclusion chromatog. (SEC), which often lead to inaccuracies in protein aggregate characterization. Hence, this study tested sedimentation velocity anal. ultracentrifugation (SV-AUC) as an orthogonal tool to SEC for quantifying the monomer and aggregates in i.v. Ig (IVIg) formulations. IVIg samples were subjected to agitation stress and analyzed using SEC mobile phases composed of 200 mM sodium phosphate (pH 7.0) with 0, 50, 100, 200, or 400 mM of NaCl. Surprisingly, 400 mM of NaCl was required in the mobile phase to attain complete protein recovery from the SEC column. Significant discrepancies between SEC and SV-AUC are reported when SEC anal. was performed using suboptimal concns. (e.g., 0, 50, 100, and 200 mM) of NaCl in the mobile phase. The continuous sedimentation coeff. distributions obtained with SV-AUC resolved the high mol. wt. species, whereas with SEC the high mol. wt. species eluted as a single peak. Only with the orthogonal use of SV-AUC, the authors were able to develop a robust SEC method for accurate quantitation of monomer and aggregates in unagitated and agitated IVIg samples. Addnl., this article describes a modification to an existing method of quantitating insol. aggregates from SV-AUC boundary data.
- 22Moser, A. C.; Trenhaile, S.; Frankenberg, K. Studies of antibody-antigen interactions by capillary electrophoresis: A review. Methods 2018, 146, 66– 75, DOI: 10.1016/j.ymeth.2018.03.00622https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXltFWhu7k%253D&md5=a420f715c049d1659561ef685c6dc05cStudies of antibody-antigen interactions by capillary electrophoresis: A reviewMoser, Annette C.; Trenhaile, Sidney; Frankenberg, KatiMethods (Amsterdam, Netherlands) (2018), 146 (), 66-75CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)Antibody-antigen interactions are vital in immunoassay development and can det. detection limits and anal. times. Capillary electrophoresis (CE) is a powerful technique that can be used to quantify antibody-antigen interactions. These CE methods range from simple sepns. of a premixed antibody and antigen sample applied as a short plug to allow for sepn. of complex, free antibody, and free antigen to more complex systems which inject complexed samples in the presence of antibody or antigen; or even injections of antibody and antigen sequentially. The objective of this review is to identify and describe various CE techniques which have been used to study antibody-antigen interactions. A brief discussion of linear and nonlinear curve fitting is also included.
- 23Kumar, R.; Guttman, A.; Rathore, A. S. Applications of capillary electrophoresis for biopharmaceutical product characterization. Electrophoresis 2021, DOI: 10.1002/elps.202100182There is no corresponding record for this reference.
- 24Leney, A. C.; Heck, A. J. R. Native Mass Spectrometry: What is in the Name?. J. Am. Soc. Mass Spectrom. 2017, 28, 5– 13, DOI: 10.1007/s13361-016-1545-324https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvFylsLzF&md5=09d56e0a888ad31a6f655c4e1d840775Native Mass Spectrometry: What is in the Name?Leney, Aneika C.; Heck, Albert J. R.Journal of the American Society for Mass Spectrometry (2017), 28 (1), 5-13CODEN: JAMSEF; ISSN:1044-0305. (Springer)A review. Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomol. mass spectrometry and proteomics. Advances in sample prepn. and mass analyzers have enabled researchers to ext. much more information from biol. samples than just the mol. wt. In particular, relevant for structural biol., noncovalent protein-protein and protein-ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomol. mass spectrometry, nowadays often referred to as "native MS," has come to maturation over the last two decades, with dozens of labs. using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure-function relations. In this perspective, the authors describe the origins of and (re)define the term native MS, portraying in detail what the authors meant by "native MS," when the term was coined and also describing what it does (according to the authors) not entail. Addnl., the authors describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technol. has in solving complex biol. questions.
- 25Tamara, S.; den Boer, M. A.; Heck, A. J. R. High-Resolution Native Mass Spectrometry. Chem. Rev. 2021, DOI: 10.1021/acs.chemrev.1c00212There is no corresponding record for this reference.
- 26Thompson, N. J.; Hendriks, L. J.; de Kruif, J.; Throsby, M.; Heck, A. J. Complex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometry. mAbs 2014, 6, 197– 203, DOI: 10.4161/mabs.2712626https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2c3nsF2rsQ%253D%253D&md5=00569249b017ebcc1944f5af74efc52cComplex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometryThompson Natalie J; Hendriks Linda J A; de Kruif John; Throsby Mark; Heck Albert J RmAbs (2014), 6 (1), 197-203 ISSN:.Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However, parallel development of analytical technologies is required to provide fast, thorough, accurate, and robust characterization of these mixtures. Here, we evaluate the utility of native mass spectrometry on an Orbitrap platform with high mass resolving power to characterize composite mixtures of up to 15 separate antibodies. With this technique, unambiguous identification of each antibody in the mixtures was achieved. Mass measurements of the intact antibodies varied 7 ppm on average, allowing highly reproducible identification and quantitation of each compound in these complex mixtures. We show that with the high mass-resolving power and robustness of this technology, high-resolution native mass spectrometry can be used efficiently even for batch-to batch characterization.
- 27Yang, Y.; Liu, F.; Franc, V.; Halim, L. A.; Schellekens, H.; Heck, A. J. R. Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity. Nat. Commun. 2016, 7, 13397, DOI: 10.1038/ncomms1339727https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvVGjsL%252FI&md5=30540791a6e2417ea0e4b9cb1dae2a59Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarityYang, Yang; Liu, Fan; Franc, Vojtech; Halim, Liem Andhyk; Schellekens, Huub; Heck, Albert J. R.Nature Communications (2016), 7 (), 13397CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Many biopharmaceutical products exhibit extensive structural micro-heterogeneity due to an array of co-occurring post-translational modifications. These modifications often effect the functionality of the product and therefore need to be characterized in detail. Here, we present an integrative approach, combining two advanced mass spectrometry-based methods, high-resoln. native mass spectrometry and middle-down proteomics, to analyze this micro-heterogeneity. Taking human erythropoietin and the human plasma properdin as model systems, we demonstrate that this strategy bridges the gap between peptide- and protein-based mass spectrometry platforms, providing the most complete profiling of glycoproteins. Integration of the two methods enabled the discovery of three undescribed C-glycosylation sites on properdin, and revealed in addn. unexpected heterogeneity in occupancies of C-mannosylation. Furthermore, using various sources of erythropoietin we define and demonstrate the usage of a biosimilarity score to quant. assess structural similarity, which would also be beneficial for profiling other therapeutic proteins and even plasma protein biomarkers.
- 28Valliere-Douglass, J. F.; McFee, W. A.; Salas-Solano, O. Native Intact Mass Determination of Antibodies Conjugated with Monomethyl Auristatin E and F at Interchain Cysteine Residues. Anal. Chem. 2012, 84, 2843– 2849, DOI: 10.1021/ac203346c28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xit1aktrk%253D&md5=8aa2b994ce97fdf12d66c83eb4bc870cNative Intact Mass Determination of Antibodies Conjugated with Monomethyl Auristatin E and F at Interchain Cysteine ResiduesValliere-Douglass, John F.; McFee, William A.; Salas-Solano, OscarAnalytical Chemistry (Washington, DC, United States) (2012), 84 (6), 2843-2849CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)We present here a method for the rapid detn. of the intact mass of noncovalently assocd. antibody heavy chains (HC) and light chains (LC) which result from the attachment of drug conjugates to interchain cysteine residues. By analyzing the antibody-drug conjugate (ADC) using native desalting conditions, we maintain the intact bivalent structure of the ADC, which ordinarily would decomp. as a consequence of denaturing chromatog. conditions typically used for liq. chromatog.-mass spectrometric (LC-MS) anal. The mass of the desalted ADC is subsequently detd. using std. desolvation and ionization conditions. Methods presented previously in the literature for analyzing interchain cysteinyl-linked ADCs are either not amenable to online mass spectrometry or result in the denaturing dissocn. of conjugated HC and LC during chromatog. sepn. and subsequent mass measurement. We have avoided this outcome with our method and have successfully and routinely obtained intact mass measurement of IgG1 mAbs conjugated with maleimidocaproyl-monomethyl Auristatin F (mcMMAF) and valine-citrulline-monomethyl Auristatin E (vcMMAE) at interchain cysteine residues. Our results thus represent the first reported direct measurement of the intact mass of an ADC conjugated at interchain cysteine residues.
- 29Hengel, S. M.; Sanderson, R.; Valliere-Douglass, J.; Nicholas, N.; Leiske, C.; Alley, S. C. Measurement of in Vivo Drug Load Distribution of Cysteine-Linked Antibody–Drug Conjugates Using Microscale Liquid Chromatography Mass Spectrometry. Anal. Chem. 2014, 86, 3420– 3425, DOI: 10.1021/ac403860c29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjt12qsrg%253D&md5=e60eaebc1ca27c3db4677eabcb55d178Measurement of in Vivo Drug Load Distribution of Cysteine-Linked Antibody-Drug Conjugates Using Microscale Liquid Chromatography Mass SpectrometryHengel, Shawna Mae; Sanderson, Russell; Valliere-Douglass, John; Nicholas, Nicole; Leiske, Chris; Alley, Stephen C.Analytical Chemistry (Washington, DC, United States) (2014), 86 (7), 3420-3425CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Anal. of samples contg. intact antibody-drug conjugates (ADC) using mass spectrometry provides a direct measurement of the drug-load distribution. Once dosed, the drug load distribution changes due to a combination of biol. and chem. factors. Liq. chromatog.-mass spectrometry (LC-MS) methods to measure the in vivo drug load distribution have been established for ADCs contg. native disulfide bonds (lysine-linked or cysteine-linked). However, because of an IgG redn. step in conjugation processes, using LC-MS to analyze intact cysteine-linked ADCs requires native conditions, thus limiting sensitivity. While this limitation has been overcome at the anal. scale, to date, these methods have not been translated to a smaller scale that is required for animal or clin. doses/sampling. In this manuscript, we describe the development of ADC specific affinity capture reagents for processing in vivo samples and optimization of native LC-MS methods at a microscale. These methods are then used to detect the changing drug load distribution over time from a set of in vivo samples, representing to our knowledge the first native mass spectra of cysteine-linked ADCs from an in vivo source.
- 30Dyachenko, A.; Wang, G.; Belov, M.; Makarov, A.; de Jong, R. N.; van den Bremer, E. T. J.; Parren, P. W. H. I.; Heck, A. J. R. Tandem Native Mass-Spectrometry on Antibody-Drug Conjugates and Submillion Da Antibody-Antigen Protein Assemblies on an Orbitrap EMR Equipped with a High-Mass Quadrupole Mass Selector. Anal. Chem. 2015, 87, 6095– 6102, DOI: 10.1021/acs.analchem.5b0078830https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXosVChtrY%253D&md5=43e2f71fd709b79be7b50804993f6ea3Tandem native mass-spectrometry on antibody-drug conjugates and submillion Da antibody-antigen protein assemblies on an Orbitrap EMR equipped with a high-mass quadrupole mass selectorDyachenko, Andrey; Wang, Guanbo; Belov, Mike; Makarov, Alexander; de Jong, Rob N.; van den Bremer, Ewald T. J.; Parren, Paul W. H. I.; Heck, Albert J. R.Analytical Chemistry (Washington, DC, United States) (2015), 87 (12), 6095-6102CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Native mass spectrometry is emerging as a powerful tool for the characterization of intact antibodies and antibody-based therapeutics. Here, we demonstrate new possibilities provided by the implementation of a high mass quadrupole mass selector on the recently introduced Orbitrap Exactive EMR mass spectrometer. This configuration allows precursor ion selection, and thus tandem mass spectrometry expts., even on analytes with masses in the hundreds of kilodaltons. We apply tandem mass spectrometry to localize the drug mols. in the therapeutic antibody-drug conjugate brentuximab vedotin, which displays a heterogeneous drug load. Our tandem MS data reveal that drug conjugation takes place nonhomogeneously to cysteine residues both on the light and heavy chains. Next, we analyzed how many antigens bind to IgG hexamers, based on a recently described antibody mutant IgG1-RGY that forms hexamers and activates complement in soln. The fully satd. IgG1-RGY-antigen complexes displayed a stoichiometry of IgG:CD38 of 6:12, possessing a mol. wt. of about 1.26 MDa and demonstrating that IgG assembly does not hamper antigen binding. Through tandem MS expts., we retrieve information about the spatial arrangement and stoichiometry of the subunits within this complex. These examples underscore the potential of this further modified Orbitrap-EMR instrument esp. for the in-depth characterization by native tandem mass spectrometry of antibodies and antibody-based constructs.
- 31Atmanene, C.; Wagner-Rousset, E.; Malissard, M.; Chol, B.; Robert, A.; Corvaïa, N.; Dorsselaer, A. V.; Beck, A.; Sanglier-Cianférani, S. Extending Mass Spectrometry Contribution to Therapeutic Monoclonal Antibody Lead Optimization: Characterization of Immune Complexes Using Noncovalent ESI-MS. Anal. Chem. 2009, 81, 6364– 6373, DOI: 10.1021/ac900755731https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXot12nsLg%253D&md5=e82cff344f0429b338753105c22c46c7Extending Mass Spectrometry Contribution to Therapeutic Monoclonal Antibody Lead Optimization: Characterization of Immune Complexes Using Noncovalent ESI-MSAtmanene, Cedric; Wagner-Rousset, Elsa; Malissard, Martine; Chol, Bertrand; Robert, Alain; Corvaia, Nathalie; Van Dorsselaer, Alain; Beck, Alain; Sanglier-Cianferani, SarahAnalytical Chemistry (Washington, DC, United States) (2009), 81 (15), 6364-6373CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases including cancers, immunol. disorders, and other pathologies. These large biomols. display specific structural features, which affect their efficiency and need, therefore, to be extensively characterized using sensitive and orthogonal anal. techniques. Among them, mass spectrometry (MS) has become the method of choice to study mAb amino acid sequences as well as their post-translational modifications. In the present work, recent noncovalent MS-technologies including automated chip-based nanoelectrospray MS and traveling wave ion mobility MS were used for the first time to characterize immune complexes involving both murine and humanized mAb 6F4 directed against human JAM-A, a newly identified antigenic protein (Ag) overexpressed in tumor cells. MS-based structural insights evidenced that heterogeneous disulfide bridge pairings of recombinant JAM-A alter neither its native structure nor mAbs 6F4 recognition properties. Investigations focused on mAb:Ag complexes revealed that, similarly to murine mAb, humanized mAb 6F4 binds selectively up to four antigen mols. with a similar affinity, confirming in this way the reliability of the humanization process. Noncovalent MS appears as an addnl. supporting technique for therapeutic mAbs lead characterization and development.
- 32Debaene, F.; Wagner-Rousset, E.; Colas, O.; Ayoub, D.; Corvaïa, N.; Van Dorsselaer, A.; Beck, A.; Cianférani, S. Time Resolved Native Ion-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and “Bispecific” Monoclonal Antibody Formation. Anal. Chem. 2013, 85, 9785– 9792, DOI: 10.1021/ac402237v32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhsVSit7bI&md5=8c98eab1931fb738c4a647ac61e26c64Time Resolved Native Ion-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and "Bispecific" Monoclonal Antibody FormationDebaene, Francois; Wagner-Rousset, Elsa; Colas, Olivier; Ayoub, Daniel; Corvaia, Nathalie; Van Dorsselaer, Alain; Beck, Alain; Cianferani, SarahAnalytical Chemistry (Washington, DC, United States) (2013), 85 (20), 9785-9792CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)Monoclonal antibodies (mAbs) and derivs. such as antibody-drug conjugates (ADC) and bispecific antibodies (bsAb), are the fastest growing class of human therapeutics. Most of the therapeutic antibodies currently on the market and in clin. trials are chimeric, humanized, and human IgG1. An increasing no. of IgG2s and IgG4s that have distinct structural and functional properties are also investigated to develop products that lack or have diminished antibody effector functions compared to IgG1. Importantly, wild type IgG4 has been shown to form half mols. (one heavy chain and one light chain) that lack interheavy chain disulfide bonds and form intrachain disulfide bonds. Moreover, IgG4 undergoes a process of Fab-arm exchange (FAE) in which the heavy chains of antibodies of different specificities can dissoc. and recombine in bispecific antibodies both in vitro and in vivo. Here, native mass spectrometry (MS) and time-resolved traveling wave ion mobility MS (TWIM-MS) were used for the first time for online monitoring of FAE and bsAb formation using Hz6F4-2v3 and natalizumab, two humanized IgG4s which bind to human Junctional Adhesion Mol.-A (JAM-A) and alpha4 integrin, resp. In addn., native MS anal. of bsAb/JAM-A immune complexes revealed that bsAb can bind up to two antigen mols., confirming that the Hz6F4 family preferentially binds dimeric JAM-A. Our results illustrate how IM-MS can rapidly assess bsAb structural heterogeneity and be easily implemented into MS workflows for bsAb prodn. follow up and bsAb/antigen complex characterization. Altogether, these results provide new MS-based methodologies for in-depth FAE and bsAb formation monitoring. Native MS and IM-MS will play an increasing role in next generation biopharmaceutical product characterization like bsAbs, antibody mixts., and antibody-drug conjugates (ADC) as well as for biosimilar and biobetter antibodies.
- 33Wang, G.; de Jong, R. N.; van den Bremer, E. T. J.; Parren, P. W. H. I.; Heck, A. J. R. Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry. Anal. Chem. 2017, 89, 4793– 4797, DOI: 10.1021/acs.analchem.6b0512933https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXls1Slurs%253D&md5=5b2d372e85bb05f5733d77e75fd21438Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass SpectrometryWang, Guanbo; de Jong, Rob N.; van den Bremer, Ewald T. J.; Parren, Paul W. H. I.; Heck, Albert J. R.Analytical Chemistry (Washington, DC, United States) (2017), 89 (9), 4793-4797CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)The detn. of mol. wts. (MWs) of heavily glycosylated proteins is seriously hampered by the physicochem. characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during SDS-PAGE and size-exclusion chromatog. (SEC) anal. Std. electrospray ionization (ESI)-mass spectrometry does not provide a direct soln. as this approach is hindered by extensive interference of ion signals caused by closely spaced charge states of broadly distributed glycoforms. Here, the authors introduce a native tandem MS-based approach, enabling charge-state resoln. and charge assignment of protein ions including those that escape mass anal. under std. MS conditions. Using this method, the authors detd. the MW of two model glycoproteins, the extra-cellular domains of the highly and heterogeneously glycosylated proteins CD38 and epidermal growth factor receptor (EGFR), as well as the overall MW and binding stoichiometries of these proteins in complex with a specific antibody.
- 34Wang, G.; de Jong, R. N.; van den Bremer, E. T. J.; Beurskens, F. J.; Labrijn, A. F.; Ugurlar, D.; Gros, P.; Schuurman, J.; Parren, P. W. H. I.; Heck, A. J. R. Molecular Basis of Assembly and Activation of Complement Component C1 in Complex with Immunoglobulin G1 and Antigen. Mol. Cell 2016, 63, 135– 145, DOI: 10.1016/j.molcel.2016.05.01634https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVWltrjN&md5=cf1f473fd61b1122af3e4f665f08110aMolecular Basis of Assembly and Activation of Complement Component C1 in Complex with Immunoglobulin G1 and AntigenWang, Guanbo; de Jong, Rob N.; van den Bremer, Ewald T. J.; Beurskens, Frank J.; Labrijn, Aran F.; Ugurlar, Deniz; Gros, Piet; Schuurman, Janine; Parren, Paul W. H. I.; Heck, Albert J. R.Molecular Cell (2016), 63 (1), 135-145CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)The classical complement pathway contributes to the natural immune defense against pathogens and tumors. IgG antibodies can assemble at the cell surface into hexamers via Fc:Fc interactions, which recruit complement component C1q and induce complement activation. Biophys. characterization of the C1:IgG complex has remained elusive primarily due to the low affinity of IgG-C1q binding. Using IgG variants that dynamically form hexamers efficient in C1q binding and complement activation, we could assess C1q binding in soln. by native mass spectrometry and size-exclusion chromatog. Fc-domain deglycosylation, described to abrogate complement activation, affected IgG hexamerization and C1q binding. Strikingly, antigen binding by IgG hexamers or deletion of the Fab arms substantially potentiated complement initiation, suggesting that Fab-mediated effects impact downstream Fc-mediated events. Finally, we characterized a reconstituted 2,045.3 ± 0.4-kDa complex of intact C1 bound to antigen-satd. IgG hexamer by native mass spectrometry, providing a clear visualization of a complete complement initiation complex.
- 35Wörner, T. P.; Snijder, J.; Bennett, A.; Agbandje-McKenna, M.; Makarov, A. A.; Heck, A. J. R. Resolving heterogeneous macromolecular assemblies by Orbitrap-based single-particle charge detection mass spectrometry. Nat. Methods 2020, 17, 395– 398, DOI: 10.1038/s41592-020-0770-735https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXks1Kgs7w%253D&md5=01b8a2d8e1d482367755ce3f107c6667Resolving heterogeneous macromolecular assemblies by Orbitrap-based single-particle charge detection mass spectrometryWorner, Tobias P.; Snijder, Joost; Bennett, Antonette; Agbandje-McKenna, Mavis; Makarov, Alexander A.; Heck, Albert J. R.Nature Methods (2020), 17 (4), 395-398CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)We demonstrate single-particle charge detection mass spectrometry on an Orbitrap for the anal. of megadalton biomol. assemblies. We establish that the signal amplitudes of individual ions scale linearly with their charge, which can be used to resolve mixed ion populations, det. charge states and thus also det. the masses of individual ions. This enables the ultrasensitive anal. of heterogeneous protein assemblies including Ig oligomers, ribosomes, proteinaceous nanocontainers and genome-packed adeno-assocd. viruses.
- 36Eschweiler, J. D.; Kerr, R.; Rabuck-Gibbons, J.; Ruotolo, B. T. Sizing Up Protein–Ligand Complexes: The Rise of Structural Mass Spectrometry Approaches in the Pharmaceutical Sciences. Annu. Rev. Anal. Chem. 2017, 10, 25– 44, DOI: 10.1146/annurev-anchem-061516-04541436https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXksVOksrY%253D&md5=15c08f3dc25938a1436d70d24401347fSizing Up Protein-Ligand Complexes: The Rise of Structural Mass Spectrometry Approaches in the Pharmaceutical SciencesEschweiler, Joseph D.; Kerr, Richard; Rabuck-Gibbons, Jessica; Ruotolo, Brandon T.Annual Review of Analytical Chemistry (2017), 10 (), 25-44CODEN: ARACFU; ISSN:1936-1327. (Annual Reviews)Capturing the dynamic interplay between proteins and their myriad interaction partners is critically important for advancing our understanding of almost every biochem. process and human disease. The importance of this general area has spawned many measurement methods capable of assaying such protein complexes, and the mass spectrometry-based structural biol. methods described in this review form an important part of that anal. arsenal. Here, we survey the basic principles of such measurements, cover recent applications of the technol. that have focused on protein-small-mol. complexes, and discuss the bright future awaiting this group of technologies.
- 37Mathur, S.; Badertscher, M.; Scott, M.; Zenobi, R. Critical evaluation of mass spectrometric measurement of dissociation constants: accuracy and cross-validation against surface plasmon resonance and circular dichroism for the calmodulin–melittin system. Phys. Chem. Chem. Phys. 2007, 9, 6187– 6198, DOI: 10.1039/b707946j37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlKht7rJ&md5=e9069d5a5561deca0039928297af8d55Critical evaluation of mass spectrometric measurement of dissociation constants: accuracy and cross-validation against surface plasmon resonance and circular dichroism for the calmodulin-melittin systemMathur, Sonal; Badertscher, Martin; Scott, Michael; Zenobi, RenatoPhysical Chemistry Chemical Physics (2007), 9 (47), 6187-6198CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)The authors present a comprehensive study for detg. the binding affinity of a protein-ligand complex, using mass spectrometric methods. Mass spectrometry has been used to study noncovalent interactions for a no. of years. However, the use of soft ionization mass spectrometry for quant. anal. of noncovalently bound complexes is not widely accepted. This paper reports a comparison of MS methods against established methods such as surface plasmon resonance (SPR) and CD whose suitability for the quant. assessment of noncovalent interactions is well known. ESI titrn. and MALDI-SUPREX were used as representative mass spectrometric methods for this work. The authors chose to study the calmodulin-melittin complex that presents three challenges: (i) it exhibits a high affinity (low nanomolar KD); (ii) complexes are formed only in the presence of a coactivator, calcium ions in this case; and (iii) the protein and the complex show a different ionization efficiency. Dissocn. consts. were obtained from each method for the selected system and compared thoroughly to elucidate pros and cons of the selected methodologies in terms of their ability for the detn. of binding consts. of protein-ligand complexes. ESI titrn., SPR, CD and MALDI-SUPREX yielded KD values in the low nanomolar range that are in general agreement with an older value reported in the literature. The authors also critically evaluated the limitations in particular of the MS methods and the assocd. data evaluation procedures. The authors present an improved evaluation of SUPREX data, as well as a detailed error anal. for all methods used.
- 38Kitova, E. N.; El-Hawiet, A.; Schnier, P. D.; Klassen, J. S. Reliable Determinations of Protein–Ligand Interactions by Direct ESI-MS Measurements. Are We There Yet?. J. Am. Soc. Mass Spectrom. 2012, 23, 431– 441, DOI: 10.1007/s13361-011-0311-938https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XltVahs7k%253D&md5=2059dccb131ddbff901a9e193ecde795Reliable determinations of protein-ligand interactions by direct ESI-MS measurements. Are we there yet?Kitova, Elena N.; El-Hawiet, Amr; Schnier, Paul D.; Klassen, John S.Journal of the American Society for Mass Spectrometry (2012), 23 (3), 431-441CODEN: JAMSEF; ISSN:1044-0305. (Springer)A review. The assocn.-dissocn. of noncovalent interactions between protein and ligands, such as other proteins, carbohydrates, lipids, DNA, or small mols., are crit. events in many biol. processes. The discovery and characterization of these interactions is essential to a complete understanding of biochem. reactions and pathways and to the design of novel therapeutic agents that may be used to treat a variety of diseases and infections. Over the last 20 y, electrospray ionization mass spectrometry (ESI-MS) has emerged as a versatile tool for the identification and quantification of protein-ligand interactions in vitro. Here, the authors describe the implementation of the direct ESI-MS assay for the detn. of protein-ligand binding stoichiometry and affinity. Addnl., the authors outline common sources of error encountered with these measurements and various strategies to overcome them. Finally, the authors comment on some of the outstanding challenges assocd. with the implementation of the assay and highlight new areas where direct ESI-MS measurements are expected to make significant contributions in the future.
- 39Daniel, J. M.; Friess, S. D.; Rajagopalan, S.; Wendt, S.; Zenobi, R. Quantitative determination of noncovalent binding interactions using soft ionization mass spectrometry. Int. J. Mass Spectrom. 2002, 216, 1– 27, DOI: 10.1016/s1387-3806(02)00585-739https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xis1enur4%253D&md5=2b46b1d642f0e21f6948ce0064a1ef25Quantitative determination of noncovalent binding interactions using soft ionization mass spectrometryDaniel, Jurg M.; Friess, Sebastian D.; Rajagopalan, Sudha; Wendt, Silke; Zenobi, RenatoInternational Journal of Mass Spectrometry (2002), 216 (1), 1-27CODEN: IMSPF8; ISSN:1387-3806. (Elsevier Science B.V.)A review. For a no. of years, soft ionization mass spectrometry was used for studying noncovalently bound complexes. An intriguing question in this context is whether MS expts. can be used to measure the interaction strength. A no. of recent studies have addressed this question. The results of these studies, as well as the methods employed are reviewed here. We distinguish between liq.-phase methods such as mass spectrometrically detected melting curves, titrn. expts., or competition expts., and gas-phase methods such as cone voltage-driven dissocn., collision-induced dissocn., blackbody IR radiative dissocn., or thermal dissocn. of gas-phase complex ions. With a few exceptions, no agreement exists between soln.-phase and gas-phase binding energies. The main reason is that electrostatic and dipolar noncovalent interactions are strengthened in the absence of solvent shielding, while other noncovalent interactions, in particular hydrophobic interactions, become less important in the absence of solvent. The possibility to quant. measure soln.-phase as well as gas-phase noncovalent interaction strengths by mass spectrometry opens fascinating perspectives for very high sensitivity screening assays as well as for improved fundamental understanding of the nature of noncovalent interactions.
- 40Rose, R. J.; Labrijn, A. F.; van den Bremer, E. T. J.; Loverix, S.; Lasters, I.; van Berkel, P. H. C.; van de Winkel, J. G. J.; Schuurman, J.; Parren, P. W. H. I.; Heck, A. J. R.; Schuurman, J.; Parren, P. W. H. I.; Heck, A. J. R. Quantitative Analysis of the Interaction Strength and Dynamics of Human IgG4 Half Molecules by Native Mass Spectrometry. Structure 2011, 19, 1274– 1282, DOI: 10.1016/j.str.2011.06.01640https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFaru7fL&md5=ecca0a3dd3826087d4881565fa423c4bQuantitative Analysis of the Interaction Strength and Dynamics of Human IgG4 Half Molecules by Native Mass SpectrometryRose, Rebecca J.; Labrijn, Aran F.; van den Bremer, Ewald T. J.; Loverix, Stefan; Lasters, Ignace; van Berkel, Patrick H. C.; van de Winkel, Jan G. J.; Schuurman, Janine; Parren, Paul W. H. I.; Heck, Albert J. R.Structure (Cambridge, MA, United States) (2011), 19 (9), 1274-1282CODEN: STRUE6; ISSN:0969-2126. (Cell Press)Native mass spectrometry (MS) is a powerful technique for studying noncovalent protein-protein interactions. Here, native MS was employed to examine the noncovalent interactions involved in homodimerization of antibody half mols. (HL) in hinge-deleted human IgG4 (IgG4Δhinge). By analyzing the concn. dependence of the relative distribution of monomer HL and dimer (HL)2 species, the apparent dissocn. const. (KD) for this interaction was detd. In combination with site-directed mutagenesis, the relative contributions of residues at the CH3-CH3 interface to this interaction could be characterized and corresponding KD values quantified over a range of 10-10-10-4 M. The crit. importance of this noncovalent interaction in maintaining the intact dimeric structure was also proven for the full-length IgG4 backbone. Using time-resolved MS, the kinetics of the interaction could be measured, reflecting the dynamics of IgG4 HL exchange. Hence, native MS has provided a quant. view of local structural features that define biol. properties of IgG4.
- 41Belov, A. M.; Viner, R.; Santos, M. R.; Horn, D. M.; Bern, M.; Karger, B. L.; Ivanov, A. R. Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2017, 28, 2614– 2634, DOI: 10.1007/s13361-017-1781-141https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVOjsLnP&md5=6c6178d79eaa15c6ae8eb8830d78de42Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass SpectrometryBelov, Arseniy M.; Viner, Rosa; Santos, Marcia R.; Horn, David M.; Bern, Marshall; Karger, Barry L.; Ivanov, Alexander R.Journal of the American Society for Mass Spectrometry (2017), 28 (12), 2614-2634CODEN: JAMSEF; ISSN:1044-0305. (Springer)Native mass spectrometry (MS) is a rapidly advancing field in the anal. of proteins, protein complexes, and macromol. species of various types. The majority of native MS expts. reported to-date has been conducted using direct infusion of purified analytes into a mass spectrometer. Capillary zone electrophoresis (CZE) was coupled online to Orbitrap mass spectrometers using a com. sheathless interface to enable high-performance sepn., identification, and structural characterization of limited amts. of purified proteins and protein complexes, the latter with preserved noncovalent assocns. under native conditions. The performance of both bare-fused silica and polyacrylamide-coated capillaries was assessed using mixts. of protein stds. known to form noncovalent protein-protein and protein-ligand complexes. High-efficiency sepn. of native complexes is demonstrated using both capillary types, while the polyacrylamide neutral-coated capillary showed better reproducibility and higher efficiency for more complex samples. The platform was then evaluated for the detn. of monoclonal antibody aggregation and for anal. of proteomes of limited complexity using a ribosomal isolate from E. coli. Native CZE-MS, using accurate single stage and tandem-MS measurements, enabled identification of proteoforms and noncovalent complexes at femtomole levels. Native CZE-MS can serve as an orthogonal and complementary technique to conventional native MS methodologies with the advantages of low sample consumption, minimal sample processing and losses, and high throughput and sensitivity. This study presents a novel platform for anal. of ribosomes and other macromol. complexes and organelles, with the potential for discovery of novel structural features defining cellular phenotypes (e.g., specialized ribosomes).
- 42VanAernum, Z. L.; Busch, F.; Jones, B. J.; Jia, M.; Chen, Z.; Boyken, S. E.; Sahasrabuddhe, A.; Baker, D.; Wysocki, V. H. Rapid online buffer exchange for screening of proteins, protein complexes and cell lysates by native mass spectrometry. Nat. Protoc. 2020, 15, 1132– 1157, DOI: 10.1038/s41596-019-0281-042https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB38%252FmvFWgug%253D%253D&md5=c646764d754642a085d18013fb986ae2Rapid online buffer exchange for screening of proteins, protein complexes and cell lysates by native mass spectrometryVanAernum Zachary L; Busch Florian; Jones Benjamin J; Jia Mengxuan; Sahasrabuddhe Aniruddha; Wysocki Vicki H; VanAernum Zachary L; Busch Florian; Jones Benjamin J; Jia Mengxuan; Wysocki Vicki H; Chen Zibo; Boyken Scott E; Baker David; Chen Zibo; Boyken Scott E; Baker David; Chen Zibo; Boyken Scott E; Sahasrabuddhe Aniruddha; Baker DavidNature protocols (2020), 15 (3), 1132-1157 ISSN:.It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.
- 43Keifer, D. Z.; Jarrold, M. F. Single-molecule mass spectrometry. Mass Spectrom. Rev. 2017, 36, 715– 733, DOI: 10.1002/mas.2149543https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhs1Wisb3I&md5=5f6d139ce05f4572c3717daee02d1f6dSingle-molecule mass spectrometryKeifer, David Z.; Jarrold, Martin F.Mass Spectrometry Reviews (2017), 36 (6), 715-733CODEN: MSRVD3; ISSN:0277-7037. (John Wiley & Sons, Inc.)A review. In single-mol. mass spectrometry, the mass of each ion is measured individually; making it suitable for the anal. of very large, heterogeneous objects that cannot be analyzed by conventional means. A range of single-mol. mass spectrometry techniques has been developed, including time-of-flight with cryogenic detectors, a quadrupole ion trap with optical detection, single-mol. Fourier transform ion cyclotron resonance, charge detection mass spectrometry, quadrupole ion traps coupled to charge detector plates, and nanomech. oscillators. In addn. to providing information on mass and heterogeneity, these techniques have been used to study impact craters from cosmic dust, monitor the assembly of viruses, elucidate the fluorescence dynamics of quantum dots, and much more. This review focuses on the merits of each of these technologies, their limitations, and their applications. Mass Spec Rev 36:715-733, 2017.
- 44Kafader, J. O.; Melani, R. D.; Durbin, K. R.; Ikwuagwu, B.; Early, B. P.; Fellers, R. T.; Beu, S. C.; Zabrouskov, V.; Makarov, A. A.; Maze, J. T.; Shinholt, D. L.; Yip, P. F.; Tullman-Ercek, D.; Senko, M. W.; Compton, P. D.; Kelleher, N. L. Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes. Nat. Methods 2020, 17, 391– 394, DOI: 10.1038/s41592-020-0764-544https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXktFelsro%253D&md5=8c506e953825257f71b5721adfa4d24cMultiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexesKafader, Jared O.; Melani, Rafael D.; Durbin, Kenneth R.; Ikwuagwu, Bon; Early, Bryan P.; Fellers, Ryan T.; Beu, Steven C.; Zabrouskov, Vlad; Makarov, Alexander A.; Maze, Joshua T.; Shinholt, Deven L.; Yip, Ping F.; Tullman-Ercek, Danielle; Senko, Michael W.; Compton, Philip D.; Kelleher, Neil L.Nature Methods (2020), 17 (4), 391-394CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)An Orbitrap-based ion anal. procedure dets. the direct charge for numerous individual protein ions to generate true mass spectra. This individual ion mass spectrometry (I2MS) method for charge detection enables the characterization of highly complicated mixts. of proteoforms and their complexes in both denatured and native modes of operation, revealing information not obtainable by typical measurements of ensembles of ions.
- 45Young, G.; Hundt, N.; Cole, D.; Fineberg, A.; Andrecka, J.; Tyler, A.; Olerinyova, A.; Ansari, A.; Marklund, E. G.; Collier, M. P.; Chandler, S. A.; Tkachenko, O.; Allen, J.; Crispin, M.; Billington, N.; Takagi, Y.; Sellers, J. R.; Eichmann, C.; Selenko, P.; Frey, L.; Riek, R.; Galpin, M. R.; Struwe, W. B.; Benesch, J. L. P.; Kukura, P. Quantitative mass imaging of single biological macromolecules. Science 2018, 360, 423– 427, DOI: 10.1126/science.aar583945https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXotFyku7k%253D&md5=38df454f60283833ea53eb4e1577e31dQuantitative mass imaging of single biological macromoleculesYoung, Gavin; Hundt, Nikolas; Cole, Daniel; Fineberg, Adam; Andrecka, Joanna; Tyler, Andrew; Olerinyova, Anna; Ansari, Ayla; Marklund, Erik G.; Collier, Miranda P.; Chandler, Shane A.; Tkachenko, Olga; Allen, Joel; Crispin, Max; Billington, Neil; Takagi, Yasuharu; Sellers, James R.; Eichmann, Cedric; Selenko, Philipp; Frey, Lukas; Riek, Roland; Galpin, Martin R.; Struwe, Weston B.; Benesch, Justin L. P.; Kukura, PhilippScience (Washington, DC, United States) (2018), 360 (6387), 423-427CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The cellular processes underpinning life are orchestrated by proteins and their interactions. The assocd. structural and dynamic heterogeneity, despite being key to function, poses a fundamental challenge to existing anal. and structural methodologies. The authors used interferometric scattering microscopy to quantify the mass of single biomols. in soln. with 2% sequence mass accuracy, up to 19-kDa resoln., and 1-kDa precision. The authors resolved oligomeric distributions at high dynamic range, detected small-mol. binding, and mass-imaged proteins with assocd. lipids and sugars. These capabilities enabled us to characterize the mol. dynamics of processes as diverse as glycoprotein crosslinking, amyloidogenic protein aggregation, and actin polymn. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomol. interactions, one mol. at a time.
- 46Ortega Arroyo, J.; Andrecka, J.; Spillane, K. M.; Billington, N.; Takagi, Y.; Sellers, J. R.; Kukura, P. Label-Free, All-Optical Detection, Imaging, and Tracking of a Single Protein. Nano Lett. 2014, 14, 2065– 2070, DOI: 10.1021/nl500234t46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXjsFOksbs%253D&md5=89e8424b8ccc5aa9a2aac0817cbcb8f1Label-Free, All-Optical Detection, Imaging, and Tracking of a Single ProteinOrtega Arroyo, J.; Andrecka, J.; Spillane, K. M.; Billington, N.; Takagi, Y.; Sellers, J. R.; Kukura, P.Nano Letters (2014), 14 (4), 2065-2070CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Optical detection of individual proteins requires fluorescent labeling. Cavity and plasmonic methodologies enhance single mol. signatures in the absence of any labels but have struggled to demonstrate routine and quant. single protein detection. Here, we used interferometric scattering microscopy not only to detect but also to image and nanometrically track the motion of single myosin 5a heavy meromyosin mols. without the use of labels or any nanoscopic amplification. Together with the simple exptl. arrangement, an intrinsic independence from strong electronic transition dipoles and a detection limit of <60 kDa, our approach paves the way toward nonresonant, label-free sensing and imaging of nanoscopic objects down to the single protein level.
- 47Piliarik, M.; Sandoghdar, V. Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites. Nat. Commun. 2014, 5, 4495, DOI: 10.1038/ncomms549547https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXksVChsbc%253D&md5=376e935af25d8584b338908df79e8b97Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sitesPiliarik, Marek; Sandoghdar, VahidNature Communications (2014), 5 (), 4495CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Detection of single analyte mols. without the use of any label would improve the sensitivity of current biosensors by orders of magnitude to the ultimate graininess of biol. matter. Over two decades, scientists have succeeded in pushing the limits of optical detection to single mols. using fluorescence. However, restrictions in photophysics and labeling protocols make this technique less attractive for biosensing. Recently, mechanisms based on vibrational spectroscopy, photothermal detection, plasmonics and microcavities have been explored for fluorescence-free detection of single biomols. Here, we show that interferometric detection of scattering (iSCAT) can achieve this goal in a direct and label-free fashion. In particular, we demonstrate detection of cancer marker proteins in buffer soln. and in the presence of other abundant proteins. Furthermore, we present super-resoln. imaging of protein binding with nanometer localization precision. The ease of iSCAT instrumentation promises a breakthrough for label-free studies of interactions involving proteins and other small biomols.
- 48Cole, D.; Young, G.; Weigel, A.; Sebesta, A.; Kukura, P. Label-Free Single-Molecule Imaging with Numerical-Aperture-Shaped Interferometric Scattering Microscopy. ACS Photonics 2017, 4, 211– 216, DOI: 10.1021/acsphotonics.6b0091248https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtFWntr4%253D&md5=d3d61d58c3f8de0f3f523c11cdc6265aLabel-Free Single-Molecule Imaging with Numerical-Aperture-Shaped Interferometric Scattering MicroscopyCole, Daniel; Young, Gavin; Weigel, Alexander; Sebesta, Aleksandar; Kukura, PhilippACS Photonics (2017), 4 (2), 211-216CODEN: APCHD5; ISSN:2330-4022. (American Chemical Society)Our ability to optically interrogate nanoscopic objects is controlled by the difference between their extinction cross sections and the diffraction-limited area to which light can be confined in the far field. We show that a partially transmissive spatial mask placed near the back focal plane of a high numerical aperture microscope objective enhances the extinction contrast of a scatterer near an interface by approx. T-1/2, where T is the transmissivity of the mask. Numerical-aperture-based differentiation of background from scattered light represents a general approach to increasing extinction contrast and enables routine label-free imaging down to the single-mol. level.
- 49Young, G.; Kukura, P. Interferometric Scattering Microscopy. Annu. Rev. Phys. Chem. 2019, 70, 301– 322, DOI: 10.1146/annurev-physchem-050317-02124749https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXnsVSlsLs%253D&md5=4aeb584a1b7322c88b48047395fb29daInterferometric Scattering MicroscopyYoung, Gavin; Kukura, PhilippAnnual Review of Physical Chemistry (2019), 70 (), 301-322CODEN: ARPLAP; ISSN:0066-426X. (Annual Reviews)Interferometric scattering microscopy (iSCAT) is an extremely sensitive imaging method based on the efficient detection of light scattered by nanoscopic objects. The ability to, at least in principle, maintain high imaging contrast independent of the exposure time or the scattering cross section of the object allows for unique applications in single-particle tracking, label-free imaging of nanoscopic (dis)assembly, and quant. single-mol. characterization. We illustrate these capabilities in areas as diverse as mechanistic studies of motor protein function, viral capsid assembly, and single-mol. mass measurement in soln. We anticipate that iSCAT will become a widely used approach to unravel previously hidden details of biomol. dynamics and interactions.
- 50Lai, S.-H.; Tamara, S.; Heck, A. J. R. Single-particle mass analysis of intact ribosomes by mass photometry and Orbitrap-based charge detection mass spectrometry. iScience 2021, 24, 103211, DOI: 10.1016/j.isci.2021.10321150https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xlt1Ortbc%253D&md5=51d6e5bbaac3478205ec360ba0c9369bSingle-particle mass analysis of intact ribosomes by mass photometry and Orbitrap-based charge detection mass spectrometryLai, Szu-Hsueh; Tamara, Sem; Heck, Albert J. R.iScience (2021), 24 (11), 103211CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)Std. methods for mass anal. measure ensembles of thousand to millions of mols. This approach enables anal. of monodisperse recombinant proteins, whereas some heterogeneous protein assemblies pose a significant challenge, whereby co-occurring stoichiometries, sub-complexes, and modifications hamper anal. using native mass spectrometry. To tackle the challenges posed by mass heterogeneity, single-particle methods may come to the rescue. Recently, two such approaches have been introduced, namely, mass photometry (MP) and Orbitrap-based charge detection mass spectrometry (CDMS). Both methods assess masses of individual mols., albeit adhering to distinct phys. principles. To evaluate these methods side by side, we analyzed a set of ribosomal particles, representing polydisperse ribonucleoprotein assemblies in the MDa range. MP and CDMS provide accurate masses for intact ribosomes and enable quant. anal. of concomitant distinct particles within each ribosome sample. Here, we discuss pros and cons of these single-mol. techniques, also in the context of other techniques used for mass anal.
- 51Liebel, M.; Hugall, J. T.; van Hulst, N. F. Ultrasensitive Label-Free Nanosensing and High-Speed Tracking of Single Proteins. Nano Lett. 2017, 17, 1277– 1281, DOI: 10.1021/acs.nanolett.6b0504051https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXps1eqtQ%253D%253D&md5=e1a54380192acc8d088626422814adccUltrasensitive Label-Free Nanosensing and High-Speed Tracking of Single ProteinsLiebel, Matz; Hugall, James T.; van Hulst, Niek F.Nano Letters (2017), 17 (2), 1277-1281CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)Label-free detection, anal., and rapid tracking of nanoparticles is crucial for future ultrasensitive sensing applications, ranging from understanding of biol. interactions to the study of size-dependent classical-quantum transitions. Yet optical techniques to distinguish nanoparticles directly among their background remain challenging. Here the authors present amplified interferometric scattering microscopy (a-iSCAT) as a new all-optical method capable of detecting individual nanoparticles as small as 15 kDa proteins that is equiv. to half a GFP. By balancing scattering and reflection amplitudes the interference contrast of the nanoparticle signal is amplified 1 to 2 orders of magnitude. Beyond high sensitivity, a-iSCAT allows high-speed image acquisition exceeding several hundreds of frames-per-second. The authors showcase the performance of their approach by detecting single Streptavidin binding events and by tracking single Ferritin proteins at 400 frames-per-second with 12 nm localization precision over seconds. Moreover, due to its extremely simple exptl. realization, this advancement finally enables a cheap and routine implementation of label-free all-optical single nanoparticle detection platforms with sensitivity operating at the single protein level.
- 52Soltermann, F.; Foley, E. D. B.; Pagnoni, V.; Galpin, M.; Benesch, J. L. P.; Kukura, P.; Struwe, W. B. Quantifying Protein–Protein Interactions by Molecular Counting with Mass Photometry. Angew. Chem., Int. Ed. 2020, 59, 10774– 10779, DOI: 10.1002/anie.20200157852https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmt1Gnsbw%253D&md5=d8a6c0cf086ae98d816a799c723b9f61Quantifying Protein-Protein Interactions by Molecular Counting with Mass PhotometrySoltermann, Fabian; Foley, Eric D. B.; Pagnoni, Veronica; Galpin, Martin; Benesch, Justin L. P.; Kukura, Philipp; Struwe, Weston B.Angewandte Chemie, International Edition (2020), 59 (27), 10774-10779CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Interactions between biomols. control the processes of life in health and their malfunction in disease, making their characterization and quantification essential. Immobilization- and label-free anal. techniques are desirable because of their simplicity and minimal invasiveness, but they struggle with quantifying tight interactions. Here, the authors show that mass photometry can accurately count, distinguish by mol. mass, and thereby reveal the relative abundance of different unlabeled biomols. and their complexes in mixts. at the single-mol. level. These measurements det. binding affinities over four orders of magnitude at equil. for both simple and complex stoichiometries within minutes, as well as the assocd. kinetics. These results introduce mass photometry as a rapid, simple and label-free method for studying sub-micromolar binding affinities, with potential for extension towards a universal approach for characterizing complex biomol. interactions.
- 53Wu, D.; Piszczek, G. Measuring the affinity of protein-protein interactions on a single-molecule level by mass photometry. Anal. Biochem. 2020, 592, 113575, DOI: 10.1016/j.ab.2020.11357553https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtFCgs7Y%253D&md5=86f67da91b8c3d84c6bef22c68f76dcdMeasuring the affinity of protein-protein interactions on a single-molecule level by mass photometryWu, Di; Piszczek, GrzegorzAnalytical Biochemistry (2020), 592 (), 113575CODEN: ANBCA2; ISSN:0003-2697. (Elsevier B.V.)Measurements of biomol. interactions are crucial to understand the mechanisms of the biol. processes they facilitate. Bulk-based methods such as ITC and SPR provide important information on binding affinities, stoichiometry, and kinetics of interactions. However, the ensemble averaging approaches are not able to probe the intrinsic heterogeneity often displayed by biol. systems. Interactions that involve cooperativity or gave multicomponent complexes pose addnl. exptl. challenges. Single-mol. techniques have previously been applied to solve these problems. However, single-mol. expts. are often tech. demanding and require labeling or immobilization of the mols. under study. A recently developed single-mol. method, mass photometry (MP), overcomes these limitations. Here the authors applied MP to measure the affinities of biomol. interactions. MP allows the user to study multivalent complexes and quantify the affinities of different binding sites in a single measurement. Results obtained from this single-mol. technique have been validated by ITC and BLI. The quality and information content of the MP data, combined with simple and fast measurements and low sample consumption makes MP a new preferred method for measuring strong protein-protein interactions.
- 54Bleeker, W. K.; Lammerts van Bueren, J. J.; van Ojik, H. H.; Gerritsen, A. F.; Pluyter, M.; Houtkamp, M.; Halk, E.; Goldstein, J.; Schuurman, J.; van Dijk, M. A.; van de Winkel, J. G. J.; Parren, P. W. H. I. Dual Mode of Action of a Human Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Cancer Therapy. J. Immunol. 2004, 173, 4699– 4707, DOI: 10.4049/jimmunol.173.7.469954https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXnslWmtbs%253D&md5=8fd2e530117b697dea49b8b97b0c3521Dual Mode of Action of a Human Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Cancer TherapyBleeker, Wim K.; Lammerts van Bueren, Jeroen J.; van Ojik, Heidi H.; Gerritsen, Arnout F.; Pluyter, Marielle; Houtkamp, Mischa; Halk, Ed; Goldstein, Joel; Schuurman, Janine; van Dijk, Marc A.; van de Winkel, Jan G. J.; Parren, Paul W. H. I.Journal of Immunology (2004), 173 (7), 4699-4707CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)Epidermal growth factor receptor (EGF-R) overexpression is common in a large no. of solid tumors and represents a neg. prognostic indicator. Overexpression of EGF-R is strongly tumor assocd., and this tyrosine kinase type receptor is considered an attractive target for Ab therapy. In this study, we describe the evaluation of mAb 2F8, a high avidity human mAb (IgG1κ) directed against EGF-R, developed using human Ig transgenic mice. MAb 2F8 effectively blocked binding of EGF and TGF-α to the EGF-R. At satg. concns., 2F8 completely blocked EGF-R signaling and inhibited the in vitro proliferation of EGF-R-overexpressing A431 cells. At much lower concns., assocd. with low receptor occupancy, 2F8 induced efficient Ab-dependent cell-mediated cytotoxicity (ADCC) in vitro. In vivo studies showed potent antitumor effects in models with A431 tumor xenografts in athymic mice. Ex vivo anal. of the EGF-R status in tumor xenografts in 2F8-treated mice revealed that there are two therapeutic mechanisms. First, blocking of EGF-R signaling, which is most effective at complete receptor satn. and therefore requires a relatively high Ab dose. Second, at very low 2F8 receptor occupancy, we obsd. potent antitumor effects in mice, which are likely based on the engagement of immune effector mechanisms, in particular ADCC. Taken together, our findings indicate that ADCC represents an important effector mechanism of this Ab, which is effective at relatively low dose.
- 55de Jong, R. N.; Beurskens, F. J.; Verploegen, S.; Strumane, K.; van Kampen, M. D.; Horstman, W.; Engelberts, P. J.; Oostindie, S. C.; Wang, G.; Heck, A. J. R.; Schuurman, J.; Parren, P. W. H. I. A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface. PLoS Biol. 2016, 14, e1002344 DOI: 10.1371/journal.pbio.100234455https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVKlurbI&md5=c0c494715c1bd4f6102429baf06bfaf9A novel platform for the potentiation of therapeutic antibodies based on antigen-dependent formation of IgG hexamers at the cell surfacede Jong, Rob N.; Beurskens, Frank J.; Verploegen, Sandra; Strumane, Kristin; van Kampen, Muriel D.; Voorhorst, Marleen; Horstman, Wendy; Engelberts, Patrick J.; Oostindie, Simone C.; Wang, Guanbo; Heck, Albert J. R.; Schuurman, Janine; Parren, Paul W. H. I.PLoS Biology (2016), 14 (1), e1002344/1-e1002344/24CODEN: PBLIBG; ISSN:1545-7885. (Public Library of Science)IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technol. platform (HexaBody technol.) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematol. and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell-expressed antigen.
- 56Wu, S.-L.; Taylor, A. D.; Lu, Q.; Hanash, S. M.; Im, H.; Snyder, M.; Hancock, W. S. Identification of Potential Glycan Cancer Markers with Sialic Acid Attached to Sialic Acid and Up-regulated Fucosylated Galactose Structures in Epidermal Growth Factor Receptor Secreted from A431 Cell Line. Mol. Cell. Proteomics 2013, 12, 1239– 1249, DOI: 10.1074/mcp.m112.02455456https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXntVGnur0%253D&md5=38f1d9b9ba4445dbcd6fd3853605ee0bIdentification of Potential Glycan Cancer Markers with Sialic Acid Attached to Sialic Acid and Up-regulated Fucosylated Galactose Structures in Epidermal Growth Factor Receptor Secreted from A431 Cell LineWu, Shiaw-Lin; Taylor, Allen D.; Lu, Qiaozhen; Hanash, Samir M.; Im, Hogune; Snyder, Michael; Hancock, William S.Molecular & Cellular Proteomics (2013), 12 (5), 1239-1249CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)We have used powerful HPLC-mass spectrometric approaches to characterize the secreted form of epidermal growth factor receptor (sEGFR). We demonstrated that the amino acid sequence lacked the cytoplasmic domain and was consistent with the primary sequence reported for EGFR purified from a human plasma pool. One of the sEGFR forms, attributed to the alternative RNA splicing, was also confirmed by transcriptional anal. (RNA sequencing). Two unusual types of glycan structures were obsd. in sEGFR as compared with membrane-bound EGFR from the A431 cell line. The unusual glycan structures were di-sialylated glycans (sialic acid attached to sialic acid) at Asn-151 and N-acetylhexosamine attached to a branched fucosylated galactose with N-acetylglucosamine moieties (HexNAc-(Fuc)Gal-GlcNAc) at Asn-420. These unusual glycans at specific sites were either present at a much lower level or were not observable in membrane-bound EGFR present in the A431 cell lysate. The observation of these di-sialylated glycan structures was consistent with the obsd. expression of the corresponding α-N-acetylneuraminide α-2,8-sialyltransferase 2 (ST8SiA2) and α-N-acetylneuraminide α-2,8-sialyltransferase 4 (ST8SiA4), by quant. real time RT-PCR. The connectivity present at the branched fucosylated galactose was also confirmed by methylation of the glycans followed by anal. with sequential fragmentation in mass spectrometry. We hypothesize that the presence of such glycan structures could promote secretion via anionic or steric repulsion mechanisms and thus facilitate the observation of these glycan forms in the secreted fractions. We plan to use this model system to facilitate the search for novel glycan structures present at specific sites in sEGFR as well as other secreted oncoproteins such as Erbb2 as markers of disease progression in blood samples from cancer patients.
- 57Cruz, A. R.; Boer, M. A. d.; Strasser, J.; Zwarthoff, S. A.; Beurskens, F. J.; de Haas, C. J. C.; Aerts, P. C.; Wang, G.; de Jong, R. N.; Bagnoli, F.; van Strijp, J. A. G.; van Kessel, K. P. M.; Schuurman, J.; Preiner, J.; Heck, A. J. R.; Rooijakkers, S. H. M. Staphylococcal protein A inhibits complement activation by interfering with IgG hexamer formation. Proc. Natl. Acad. Sci. U.S.A. 2021, 118, e2016772118 DOI: 10.1073/pnas.201677211857https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXktlGju7g%253D&md5=f679d81dba72a6829863ce0ef89d4cabStaphylococcal protein A inhibits complement activation by interfering with IgG hexamer formationCruz, Ana Rita; den Boer, Maurits A.; Strasser, Juergen; Zwarthoff, Seline A.; Beurskens, Frank J.; de Haas, Carla J. C.; Aerts, Piet C.; Wang, Guanbo; de Jong, Rob N.; Bagnoli, Fabio; van Strijp, Jos A. G.; van Kessel, Kok P. M.; Schuurman, Janine; Preiner, Johannes; Heck, Albert J. R.; Rooijakkers, Suzan H. M.Proceedings of the National Academy of Sciences of the United States of America (2021), 118 (7), e2016772118CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Ig (Ig) G mols. are essential players in the human immune response against bacterial infections. An important effector of IgG-dependent immunity is the induction of complement activation, a reaction that triggers a variety of responses that help kill bacteria. Antibody-dependent complement activation is promoted by the organization of target-bound IgGs into hexamers that are held together via noncovalent Fc-Fc interactions. Here we show that staphylococcal protein A (SpA), an important virulence factor and vaccine candidate of Staphylococcus aureus, effectively blocks IgG hexamerization and subsequent complement activation. Using native mass spectrometry and high-speed at. force microscopy, we demonstrate that SpA blocks IgG hexamerization through competitive binding to the Fc-Fc interaction interface on IgG monomers. In concordance, we show that SpA interferes with the formation of (IgG)6:C1q complexes and prevents downstream complement activation on the surface of S. aureus. Finally, we demonstrate that IgG3 antibodies against S. aureus can potently induce complement activation and opsonophagocytic killing even in the presence of SpA. Together, our findings identify SpA as an immune evasion protein that specifically blocks IgG hexamerization.
- 58van Kampen, M. D.; Kuipers-De Wilt, L. H. A. M.; van Egmond, M. L.; Reinders-Blankert, P.; van den Bremer, E. T. J.; Wang, G.; Heck, A. J. R.; Parren, P. W. H. I.; Beurskens, F. J.; Schuurman, J.; de Jong, R. N. Biophysical Characterization and Stability of IgG1 Variants with Different Hexamerization Propensities; Genmab: Utrecht, the Netherlands, 2021, Unpublished work.There is no corresponding record for this reference.
- 59Gaboriaud, C.; Thielens, N. M.; Gregory, L. A.; Rossi, V.; Fontecilla-Camps, J. C.; Arlaud, G. J. Structure and activation of the C1 complex of complement: unraveling the puzzle. Trends Immunol. 2004, 25, 368– 373, DOI: 10.1016/j.it.2004.04.00859https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXltVKjtb4%253D&md5=7034d86f62bf41ce7444e21125208a19Structure and activation of the C1 complex of complement: unraveling the puzzleGaboriaud, Christine; Thielens, Nicole M.; Gregory, Lynn A.; Rossi, Veronique; Fontecilla-Camps, Juan C.; Arlaud, Gerard J.Trends in Immunology (2004), 25 (7), 368-373CODEN: TIRMAE; ISSN:1471-4906. (Elsevier Science Ltd.)A review. C1, the multimol. protease that triggers the classical pathway of complement, has a major role in the host defense against pathogens. It also participates in other biol. functions, such as immune tolerance, owing to the ability of its binding subunit, C1q, to recognize abnormal structures from self, including apoptotic cells. Structural biol. has been used over the past few years to elucidate the structure of its three subunits: C1q, C1r and C1s. These new advances have led to a comprehensive, three-dimensional model of C1 and provide insights into the mechanisms underlying its activation and the extraordinarily versatile recognition properties of its C1q subunit.
- 60Mortensen, S. A.; Sander, B.; Jensen, R. K.; Pedersen, J. S.; Golas, M. M.; Jensenius, J. C.; Hansen, A. G.; Thiel, S.; Andersen, G. R. Structure and activation of C1, the complex initiating the classical pathway of the complement cascade. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 986– 991, DOI: 10.1073/pnas.161699811460https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXht1Crsbk%253D&md5=e060af7ce5c8b8417192cf22d0067e52Structure and activation of C1, the complex initiating the classical pathway of the complement cascadeMortensen, Simon A.; Sander, Bjoern; Jensen, Rasmus K.; Pedersen, Jan Skov; Golas, Monika M.; Jensenius, Jens C.; Hansen, Annette G.; Thiel, Steffen; Andersen, Gregers R.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (5), 986-991CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The complement system is an important antimicrobial and inflammation-generating component of the innate immune system. The classical pathway of complement is activated upon binding of the 774-kDa C1 complex, consisting of the recognition mol. C1q and the tetrameric protease complex C1r2s2, to a variety of activators presenting specific mol. patterns such as IgG- and IgM-contg. immune complexes. A canonical model entails a C1r2s2 with its serine protease domains tightly packed together in the center of C1 and an intricate intramol. reaction mechanism for activation of C1r and C1s, induced upon C1 binding to the activator. Here, the authors show that the serine protease domains of C1r and C1s are located at the periphery of the C1r2s2 tetramer both when alone or within the nonactivated C1 complex. The authors' structural studies indicate that the C1 complex adopts a conformation incompatible with intramol. activation of C1, suggesting instead that intermol. proteolytic activation between neighboring C1 complexes bound to a complement activating surface occurs. The authors' results rationalize how a multitude of structurally unrelated mol. patterns can activate C1 and suggests a conserved mechanism for complement activation through the classical and the related lectin pathway.
- 61Hughes-Jones, N. C.; Gardner, B. Reaction between the isolated globular sub-units of the complement component Clq and IgG-complexes. Mol. Immunol. 1979, 16, 697– 701, DOI: 10.1016/0161-5890(79)90010-561https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3cXpt1GhtQ%253D%253D&md5=aa3f9f8733467c26e8a89d46e38b8a48Reaction between the isolated globular subunits of the complement component C1q and IgG-complexesHughes-Jones, N. C.; Gardner, BrigitteMolecular Immunology (1979), 16 (9), 697-701CODEN: MOIMD5; ISSN:0161-5890.The globular subunits of the C1q complement component isolated by collagenase digestion of purified C1q followed by gel filtration had mol. wt. 37,000. The globular subunits reacted with IgG immune complexes as shown by their ability to inhibit the reaction of the latter with C1q-125I; the functional affinity const. for the subunit IgG complex reaction was estd. to be 1.8-5.8 × 104M-1, values similar to those previously found for the reaction between monomeric IgG and intact C1q. C1q thus binds to immune complexes through the peripheral globular subunits.
- 62Feinstein, A.; Richardson, N.; Taussig, M. I. Immunoglobulin flexibility in complement activation. Immunol. Today 1986, 7, 169– 174, DOI: 10.1016/0167-5699(86)90168-462https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL28XksF2jt7s%253D&md5=3b8e73d0c6242c42f8a122e1a17b94c6Immunoglobulin flexibility in complement activationFeinstein, Arnold; Richardson, Neil; Taussig, Michael J.Immunology Today (1986), 7 (6), 169-74CODEN: IMTOD8; ISSN:0167-4919.A review with 40 refs. of the activation of complement C1 after it has bound to a site on the Fc portion of an antibody mol. and how this site is made available in different antibody classes.
- 63Burton, D. R. Immunoglobulin G: Functional sites. Mol. Immunol. 1985, 22, 161– 206, DOI: 10.1016/0161-5890(85)90151-863https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXhs1Cqs7k%253D&md5=77689b7b3b61fcb16eee95f1dde8d923Immunoglobulin G: functional sitesBurton, Dennis R.Molecular Immunology (1985), 22 (3), 161-206CODEN: MOIMD5; ISSN:0161-5890.A review with ∼240 refs. on the functional binding sites of the IgG mol. Data from crystallog. studies and of IgG interactions are considered with respect to the localization of the binding sites.
- 64Sharp, T. H.; Boyle, A. L.; Diebolder, C. A.; Kros, A.; Koster, A. J.; Gros, P. Insights into IgM-mediated complement activation based on in situ structures of IgM-C1-C4b. Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 11900– 11905, DOI: 10.1073/pnas.190184111664https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtFeqsLjP&md5=a295722047ff0a169ab2ec96e15d375bInsights into IgM-mediated complement activation based on in situ structures of IgM-C1-C4bSharp, Thomas H.; Boyle, Aimee L.; Diebolder, Christoph A.; Kros, Alexander; Koster, Abraham J.; Gros, PietProceedings of the National Academy of Sciences of the United States of America (2019), 116 (24), 11900-11905CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Antigen binding by serum Ig-M (IgM) protects against microbial infections and helps to prevent autoimmunity, but causes life-threatening diseases when mistargeted. How antigen-bound IgM activates complement-immune responses remains unclear. We present cryoelectron tomog. structures of IgM, C1, and C4b complexes formed on antigen-bearing lipid membranes by normal human serum at 4°C. The IgM-C1-C4b complexes revealed C4b product release as the temp.-limiting step in complement activation. Both IgM hexamers and pentamers adopted hexagonal, dome-shaped structures with Fab pairs, dimerized by hinge domains, bound to surface antigens that support a platform of Fc regions. C1 binds IgM through widely spread C1q-collagen helixes, with C1r proteases pointing outward and C1s bending downward and interacting with surface-attached C4b, which further interacts with the adjacent IgM-Fab2 and globular C1q-recognition unit. Based on these data, we present mechanistic models for antibody-mediated, C1q-transmitted activation of C1 and for C4b deposition, while further conformational rearrangements are required to form C3 convertases.
Supporting Information
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.1c03656.
Supplementary methods, koff and Kd determination for IgG4Δhinge variants, SEC-MALS of IgG1 and sEGFR, native MS gas-phase dissociation of IgG1 hexamers, IgG1 hexamer dissociation upon jump dilution MP, SEC-MALS, native MS and MP characterization of C1q, SEC-MALS, native (CD) MS and MP characterization of (IgG1)6:(sEGFR)12 antibody–antigen complexes, comparison of masses measured by the assessed techniques, MP-derived kinetic rates and Kd values for IgG4Δhinge variants, comparison of advantages and disadvantages of the assessed techniques, and supplementary references (PDF)
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