Transforming Chemical Proteomics Enrichment into a High-Throughput Method Using an SP2E WorkflowClick to copy article linkArticle link copied!
- Tobias BeckerTobias BeckerInstitute for Chemical Epigenetics Munich, LMU Munich, 81375 Munich, GermanyMore by Tobias Becker
- Andreas WiestAndreas WiestInstitute for Chemical Epigenetics Munich, LMU Munich, 81375 Munich, GermanyMore by Andreas Wiest
- András TelekAndrás TelekInstitute for Chemical Epigenetics Munich, LMU Munich, 81375 Munich, GermanyMore by András Telek
- Daniel BejkoDaniel BejkoInstitute for Chemical Epigenetics Munich, LMU Munich, 81375 Munich, GermanyMore by Daniel Bejko
- Anja Hoffmann-RöderAnja Hoffmann-RöderDepartment of Chemistry, LMU Munich, 81377 Munich, GermanyMore by Anja Hoffmann-Röder
- Pavel Kielkowski*Pavel Kielkowski*Email: [email protected]Institute for Chemical Epigenetics Munich, LMU Munich, 81375 Munich, GermanyMore by Pavel Kielkowski
Abstract
Protein post-translational modifications (PTMs) play a critical role in the regulation of protein catalytic activity, localization, and protein–protein interactions. Attachment of PTMs onto proteins significantly diversifies their structure and function, resulting in proteoforms. However, the sole identification of post-translationally modified proteins, which are often cell type and disease-specific, is still a highly challenging task. Substoichiometric amounts and modifications of low abundant proteins necessitate the purification or enrichment of the modified proteins. Although the introduction of mass spectrometry-based chemical proteomic strategies has enabled the screening of protein PTMs with increased throughput, sample preparation remains highly time-consuming and tedious. Here, we report an optimized workflow for the enrichment of PTM proteins in a 96-well plate format, which could be extended to robotic automation. This platform allows us to significantly lower the input of total protein, which opens up the opportunity to screen specialized and difficult-to-culture cell lines in a high-throughput manner. The presented SP2E protocol is robust and time- and cost-effective, as well as suitable for large-scale screening of proteoforms. The application of the SP2E protocol will thus enable the characterization of proteoforms in various processes such as neurodevelopment, neurodegeneration, and cancer. This may contribute to an overall acceleration of the recently launched Human Proteoform Project.
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Introduction
Figure 1
Figure 1. Schematic overview of the chemical proteomic workflow. (A) Key steps of the standard chemical proteomic workflow and (B) schematic characterization of the SP2E workflow and basic parameters of the procedure in comparison to the previously used workflow with avidin-coated agarose beads. In the table below the SP2E workflow, the typical times required to proceed with 8 samples (large scale) or 24 samples (small scale) are shown. For comparison, the previously used workflow would typically take about 8 h for eight samples.
Results
Development of the SP2E Workflow for Chemical Proteomics
Figure 2
Figure 2. Development and optimization of the SP2E workflow using the AMPylation probe. (A) Pro-N6pA probe structure and the workflow used for the optimization of the SP2E method. (B) Optimization of the lysis buffer based on the efficiency of the CuAAC click chemistry. Lysis buffer compositions: line 1 (control cells treated with plain dimethyl sulfoxide (DMSO) and lysed in 1% NP-40, 0.2% SDS in 20 mM HEPES), line 2 (1% NP-40 in PBS), line 3 (1% NP-40, 0.2% SDS in PBS), line 4 (0.5% Triton in PBS), line 5 (0.5% Triton, 0.2% SDS in PBS), line 6 (1% NP-40 in 20 mM HEPES), line 7 (1% NP-40, 0.2% SDS in 20 mM HEPES), line 8 (0.5% Triton in 20 mM Hepes), line 9 (0.5% Triton, 0.2% SDS in 20 mM Hepes), and line 10 (8 M urea in 0.1 M Tris/HCl). (C) In-gel fluorescence showing the click reaction time optimization. In the control C, cells were treated with plain DMSO and the lysate was incubated with the click reaction mixture for 90 min. (D) Heatmap visualizing the SP2E workflow optimization based on fold enrichment of six marker proteins. Condition 1 (without added urea to the click reaction mixture before protein loading onto carboxylate magnetic beads), condition 2 (with added urea to the click reaction before protein loading onto carboxylate beads and one pot clean up and enrichment of modified proteins), and condition 3 (with added urea into the click reaction, but the spatial separation of the protein clean up and enrichment). The numbers in boxes represent fold enrichments. (E) Volcano plot showing significantly enriched proteins (red dots) using the pro-N6pA AMPylation probe with highlighted marker proteins (green dots) using the optimized SP2E workflow (condition 3 from Figure 2D); n = 4, cutoff lines at p-value >0.05 and 2-fold enrichment. (F) Plot displaying total fluorescence intensity from the in-gel analysis of the time optimization of biotin–streptavidin complex formation.
Application of the SP2E Workflow for Analysis of Protein AMPylation
Figure 3
Figure 3. Analysis of protein AMPylation under different stress conditions using the SP2E workflow. (A) Design of the experiment to test the impact of various inhibitors on protein AMPylation. (B) Volcano plot showing the enrichment of AMPylated proteins (pro-N6pA vs DMSO) from SH-SY5Y cells using the SP2E protocol; n = 4, cutoff lines at p-value >0.05 and 2-fold enrichment. (C) PCA of the inhibitor-treated cells and controls displaying separation of the monensin and bafilomycin as well as pro-N6pA-treated cells. Samples that were treated with DMSO and either rapamycin, 2-deoxy-d-glucose, TTFA, or without any inhibitor are depicted in red. Samples that were treated with pro-N6pA and either rapamycin, 2-deoxy-d-glucose, TTFA, or without any inhibitor are depicted in purple. (D) Representative heatmaps visualizing the Pearson correlation coefficients of LFQ intensities of DMSO and pro-N6pA replicates. (E) Profile plot displays the APP LFQ intensities under various conditions. The APP was not found in any other conditions, for example, in cells only treated with DMSO or some other inhibitors. (F) Profile plot displays the PLD3 LFQ intensities under various conditions.
Figure 4
Figure 4. Monensin concentration-dependent increase in APP and PLD3 modification. (A) PCA displays distinct changes in the enriched proteins with increasing monensin concentration. (B) Profile plot of the PLD3 LFQ intensities shows a rapid increase in the PLD3 modification with a 2 nM monensin concentration. (C) Monensin concentration-dependent enrichment of the modified PLD3. For the enrichment, the SP2E protocol was used but the proteins were released from the streptavidin beads by the loading buffer, separated by SDS-PAGE, and analyzed via western blotting with the anti-PLD3 antibody. (D) Enrichment of the modified PLD3 after the treatment with bafilomycin (100 nM) and monensin (2 μM). For the enrichment, the SP2E protocol was used but the proteins were released from the streptavidin beads by loading buffer, separated by SDS-PAGE, and analyzed via western blotting with the anti-PLD3 antibody. (E) Western blotting of the whole proteome from cells treated with bafilomycin (100 nM) and monensin (2 μM) stained with the anti-PLD3 antibody. (F) In contrast to PLD3, the profile plot of the APP LFQ intensity reveals that APP is only enriched with the pro-N6pA probe with 1 and 2 μM monensin in cell culture media.
Application of the SP2E Workflow for Analysis of Protein O-GlcNAcylation
Figure 5
Figure 5. Analysis of O-GlcNAcylation by the Ac34dGlcNAz probe, SPAAC, and SP2E workflow. (A) Chemical structure of the Ac34dGlcNAz probe for metabolic labeling of O-GlcNAcylated proteins and the DBCO–biotin reagent to functionalize the probe-modified proteins by SPAAC. (B) Volcano plot visualizing the enrichment of the O-GlcNAcylated proteins; n = 4, cutoff lines at p-value >0.05 and 2-fold enrichment. Red dots are significantly enriched proteins. (C) PCA graph points to a clear separation of the control and probe-treated samples. Of note, component 1 possesses a high value of 81.7%. (D) Box plot shows the total number of imputed values across the replicates in DMSO and Ac34dGlcNAz-treated cells. The number of imputed values indicates how many proteins were not identified in the sample but were found in at least one other sample/replicate. The increased number of imputed values in the DMSO controls demonstrates the efficiency of washing steps removing the nonspecific biding proteins. (E) Diagram showing the overlap between all significantly enriched proteins using the Ac34dGlcNAz probe and previously described O-GlcNAcylated proteins. (F) Enrichment of the modified NUP62 with the agarose-based and the SP2E protocol. In addition, the SP2E enrichment was performed with 400 μg of the protein input. Enriched proteins were released from the streptavidin beads by loading buffer, separated by SDS-PAGE, and analyzed via western blotting with the anti-NUP62 antibody.
Scale-Down of the SP2E Workflow into a 96-Well Plate Format
Figure 6
Figure 6. Scale-down of the SP2E workflow into a 96-well plate format. (A) SP2E protocol with 100 μg of the input protein performed in 1.5 mL tubes visualized in the volcano plot. (B) Optimization of the LC-MS/MS measurement with 100 μg protein input using the 96-well plate format SP2E protocol. (C) Heatmaps representing the Pearson correlation coefficients between the replicates. (D) Volcano plot showing the enrichment of O-GlcNAcylated proteins starting from 100 μg of the input protein in a 96-well plate format. (E) PCA of the small-scale Ac34dGlcNAz enrichment shows very good separation of controls from probe-treated samples, with component 1 value corresponding to 74%. All volcano plots, n = 4, cutoff lines at p-value >0.05 and 2-fold enrichment. Red dots are significantly enriched proteins.
Discussion
Methods
Culturing of HeLa and SH-SY5Y Cells
Probe Treatments
Cell Lysis
Measurement of Protein Concentrations
Large-Scale SP2E Workflow
Small-Scale SP2E Workflow
Large-Scale SPAAC Protocol
Small-Scale SPAAC Protocol
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.2c00284.
Supporting figures, cell culture conditions, SP2E workflow, LC-MS/MS acquisition conditions, data analysis, and list of reagents (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
This research project was supported by Liebig fellowship from VCI to P.K. and T.B., LMUexcellence Junior Fund to P.K., and generous support from SFB1309 by DFG. The authors thank Stefan Marchner (LMU Munich) for the preparation of the Ac34dGlcNAz probe and Dietrich Mostert (Technical University of Munich) for proofreading.
References
This article references 55 other publications.
- 1Becker, T.; Cappel, C.; Matteo, F. D.; Sonsalla, G.; Kaminska, E.; Spada, F.; Cappello, S.; Damme, M.; Kielkowski, P. AMPylation Profiling during Neuronal Differentiation Reveals Extensive Variation on Lysosomal Proteins. iScience 2021, 24, 103521 DOI: 10.1016/j.isci.2021.103521Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XptFGnsLY%253D&md5=6913a9e390099fa9b4960841ff07a1fdAMPylation profiling during neuronal differentiation reveals extensive variation on lysosomal proteinsBecker, Tobias; Cappel, Cedric; Di Matteo, Francesco; Sonsalla, Giovanna; Kaminska, Ewelina; Spada, Fabio; Cappello, Silvia; Damme, Markus; Kielkowski, PaveliScience (2021), 24 (12), 103521CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)Protein AMPylation is a posttranslational modification with an emerging role in neurodevelopment. In metazoans two highly conserved protein AMP-transferases together with a diverse group of AMPylated proteins have been identified using chem. proteomics and biochem. techniques. However, the function of AMPylation remains largely unknown. Particularly problematic is the localization of thus far identified AMPylated proteins and putative AMP-transferases. We show that protein AMPylation is likely a posttranslational modification of luminal lysosomal proteins characteristic in differentiating neurons. Through a combination of chem. proteomics, gel-based sepn. of modified and unmodified proteins, and an activity assay, we det. that the modified, lysosomal sol. form of exonuclease PLD3 increases dramatically during neuronal maturation and that AMPylation correlates with its catalytic activity. Together, our findings indicate that AMPylation is a so far unknown lysosomal posttranslational modification connected to neuronal differentiation and it may provide a mol. rationale behind lysosomal storage diseases and neurodegeneration.
- 2Mansfield, S. G.; Gordon-Weeks, P. R. Dynamic Post-Translational Modification of Tubulin in Rat Cerebral Cortical Neurons Extending Neurites in Culture: Effects of Taxol. J. Neurocytol. 1991, 20, 654– 666, DOI: 10.1007/bf01187067Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XhslWjurw%253D&md5=bd2a37c3b87c4b50742676042356ca01Dynamic post-translational modification of tubulin in rat cerebral cortical neurons extending neurites in culture: effects of taxolMansfield, S. G.; Gordon-Weeks, P. R.Journal of Neurocytology (1991), 20 (8), 654-66CODEN: JNCYA2; ISSN:0300-4864.Dissocd. embryonic (E18-E20) rat cortical neurons were grown in culture and double-labeled by immunofluorescence with antibodies directed against tyrosinated (YL 1/2), detyrosinated (SUP GLU), and acetylated (6-11B-1) α-tubulin. Within 90 min of plating, neurons extended growth cones that were YL 1/2+ but SUP GLU- and 6-11B-1-. The neurite that forms behind the advancing growth cone is also, initially, YL 1/2+ and SUP GLU-/6-11B-1-. However, when it has attained a length of about half the cell body diam., it becomes SUP GLU+ and 6-11B-1+. The effects of the microtubule polymg. agent taxol (15 μM) on growth cone and neurite α-tubulins was investigated. Taxol, as reported previously, caused the formation of microtubule loops in the central domain of the growth cone, a loss of filopodia, and the collapse of the growth cone onto the loops. The taxol effects peaked at 60 min, when >85% of neurites showed microtubule loops, and declined thereafter, so that at 420 min in taxol, only ∼23% of neurites had microtubule loops. Over this period there was an inverse correlation between the presence of microtubule loops and growth cones. Taxol had striking effects on the intensity of SUP GLU and 6-11B-1 staining in neurons. In 48 h cultures, a 30 min exposure to taxol enhanced the SUP GLU and 6-11B-1 staining of dendrites and axons and produced a loss of YL 1/2 staining in axons. Immunoblotting expts. confirmed that there was an overall redn. in YL 1/2 immunoreactivity and an increase in SUP GLU immunoreactivity. These observations support previous suggestions that the neurite microtubules are assembled in the growth cone and post-translationally modified in the neurite and, in addn., imply that growth cones can overcome the effects of taxol in the continued presence of the compd.
- 3Zheng, P.; Obara, C. J.; Szczesna, E.; Nixon-Abell, J.; Mahalingan, K. K.; Roll-Mecak, A.; Lippincott-Schwartz, J.; Blackstone, C. ER Proteins Decipher the Tubulin Code to Regulate Organelle Distribution. Nature 2022, 132– 138, DOI: 10.1038/s41586-021-04204-9Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXislGntbnI&md5=f7c86f932739a0be1fd09eaa4ef504deER proteins decipher the tubulin code to regulate organelle distributionZheng, Pengli; Obara, Christopher J.; Szczesna, Ewa; Nixon-Abell, Jonathon; Mahalingan, Kishore K.; Roll-Mecak, Antonina; Lippincott-Schwartz, Jennifer; Blackstone, CraigNature (London, United Kingdom) (2022), 601 (7891), 132-138CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions1,2. However, how cells interpret these post-translational microtubule modification codes to selectively regulate organelle positioning remains largely unknown. The endoplasmic reticulum (ER) is an interconnected network of diverse morphologies that extends promiscuously throughout the cytoplasm3, forming abundant contacts with other organelles4. Dysregulation of endoplasmic reticulum morphol. is tightly linked to neurol. disorders and cancer5,6. Here we demonstrate that three membrane-bound endoplasmic reticulum proteins preferentially interact with different microtubule populations, with CLIMP63 binding centrosome microtubules, kinectin (KTN1) binding perinuclear polyglutamylated microtubules, and p180 binding glutamylated microtubules. Knockout of these proteins or manipulation of microtubule populations and glutamylation status results in marked changes in endoplasmic reticulum positioning, leading to similar redistributions of other organelles. During nutrient starvation, cells modulate CLIMP63 protein levels and p180-microtubule binding to bidirectionally move endoplasmic reticulum and lysosomes for proper autophagic responses.
- 4Aebersold, R.; Agar, J. N.; Amster, I. J.; Baker, M. S.; Bertozzi, C. R.; Boja, E. S.; Costello, C. E.; Cravatt, B. F.; Fenselau, C.; Garcia, B. A.; Ge, Y.; Gunawardena, J.; Hendrickson, R. C.; Hergenrother, P. J.; Huber, C. G.; Ivanov, A. R.; Jensen, O. N.; Jewett, M. C.; Kelleher, N. L.; Kiessling, L. L.; Krogan, N. J.; Larsen, M. R.; Loo, J. A.; Loo, R. R. O.; Lundberg, E.; MacCoss, M. J.; Mallick, P.; Mootha, V. K.; Mrksich, M.; Muir, T. W.; Patrie, S. M.; Pesavento, J. J.; Pitteri, S. J.; Rodriguez, H.; Saghatelian, A.; Sandoval, W.; Schlüter, H.; Sechi, S.; Slavoff, S. A.; Smith, L. M.; Snyder, M. P.; Thomas, P. M.; Uhlén, M.; Eyk, J. E. V.; Vidal, M.; Walt, D. R.; White, F. M.; Williams, E. R.; Wohlschlager, T.; Wysocki, V. H.; Yates, N. A.; Young, N. L.; Zhang, B. How Many Human Proteoforms Are There?. Nat. Chem. Biol. 2018, 14, 206– 214, DOI: 10.1038/nchembio.2576Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXislSntLs%253D&md5=ba124ec3e824ec106295bc127a289e57How many human proteoforms are there?Aebersold, Ruedi; Agar, Jeffrey N.; Amster, I. Jonathan; Baker, Mark S.; Bertozzi, Carolyn R.; Boja, Emily S.; Costello, Catherine E.; Cravatt, Benjamin F.; Fenselau, Catherine; Garcia, Benjamin A.; Ge, Ying; Gunawardena, Jeremy; Hendrickson, Ronald C.; Hergenrother, Paul J.; Huber, Christian G.; Ivanov, Alexander R.; Jensen, Ole N.; Jewett, Michael C.; Kelleher, Neil L.; Kiessling, Laura L.; Krogan, Nevan J.; Larsen, Martin R.; Loo, Joseph A.; Ogorzalek Loo, Rachel R.; Lundberg, Emma; MacCoss, Michael J.; Mallick, Parag; Mootha, Vamsi K.; Mrksich, Milan; Muir, Tom W.; Patrie, Steven M.; Pesavento, James J.; Pitteri, Sharon J.; Rodriguez, Henry; Saghatelian, Alan; Sandoval, Wendy; Schluter, Hartmut; Sechi, Salvatore; Slavoff, Sarah A.; Smith, Lloyd M.; Snyder, Michael P.; Thomas, Paul M.; Uhlen, Mathias; Van Eyk, Jennifer E.; Vidal, Marc; Walt, David R.; White, Forest M.; Williams, Evan R.; Wohlschlager, Therese; Wysocki, Vicki H.; Yates, Nathan A.; Young, Nicolas L.; Zhang, BingNature Chemical Biology (2018), 14 (3), 206-214CODEN: NCBABT; ISSN:1552-4450. (Nature Research)A review. Despite decades of accumulated knowledge about proteins and their posttranslational modifications (PTMs), numerous questions remain regarding their mol. compn. and biol. function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, the authors outline what the authors know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, the authors examine prevailing notions about the no. of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. The authors frame central issues regarding detn. of protein-level variation and PTMs, including some paradoxes present in the field today. The authors use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes". The authors also explore prospects for improving measurements to better regularize protein-level biol. and efficiently assoc. PTMs to function and phenotype.
- 5Brüning, F.; Noya, S. B.; Bange, T.; Koutsouli, S.; Rudolph, J. D.; Tyagarajan, S. K.; Cox, J.; Mann, M.; Brown, S. A.; Robles, M. S. Sleep-Wake Cycles Drive Daily Dynamics of Synaptic Phosphorylation. Science 2019, 366, eaav3617 DOI: 10.1126/science.aav3617Google ScholarThere is no corresponding record for this reference.
- 6Truttmann, M. C.; Pincus, D.; Ploegh, H. L. Chaperone AMPylation Modulates Aggregation and Toxicity of Neurodegenerative Disease-Associated Polypeptides. Proc. Natl. Acad. Sci. U.S.A. 2018, 115, 201801989 DOI: 10.1073/pnas.1801989115Google ScholarThere is no corresponding record for this reference.
- 7Rogowski, K.; van Dijk, J.; Magiera, M. M.; Bosc, C.; Deloulme, J.-C.; Bosson, A.; Peris, L.; Gold, N. D.; Lacroix, B.; Grau, M. B.; Bec, N.; Larroque, C.; Desagher, S.; Holzer, M.; Andrieux, A.; Moutin, M.-J.; Janke, C. A Family of Protein-Deglutamylating Enzymes Associated with Neurodegeneration. Cell 2010, 143, 564– 578, DOI: 10.1016/j.cell.2010.10.014Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVahtbvN&md5=b6bf533a3dbf587ae1097bbac7284b30A Family of Protein-Deglutamylating Enzymes Associated with NeurodegenerationRogowski, Krzysztof; van Dijk, Juliette; Magiera, Maria M.; Bosc, Christophe; Deloulme, Jean-Christophe; Bosson, Anouk; Peris, Leticia; Gold, Nicholas D.; Lacroix, Benjamin; Grau, Montserrat Bosch; Bec, Nicole; Larroque, Christian; Desagher, Solange; Holzer, Max; Andrieux, Annie; Moutin, Marie-Jo; Janke, CarstenCell (Cambridge, MA, United States) (2010), 143 (4), 564-578CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Polyglutamylation is a posttranslational modification that generates glutamate side chains on tubulins and other proteins. Although this modification has been shown to be reversible, little is known about the enzymes catalyzing deglutamylation. Here we describe the enzymic mechanism of protein deglutamylation by members of the cytosolic carboxypeptidase (CCP) family. Three enzymes (CCP1, CCP4, and CCP6) catalyze the shortening of polyglutamate chains and a fourth (CCP5) specifically removes the branching point glutamates. In addn., CCP1, CCP4, and CCP6 also remove gene-encoded glutamates from the carboxyl termini of proteins. Accordingly, we show that these enzymes convert detyrosinated tubulin into Δ2-tubulin and also modify other substrates, including myosin light chain kinase 1. We further analyze Purkinje cell degeneration (pcd) mice that lack functional CCP1 and show that microtubule hyperglutamylation is directly linked to neurodegeneration. Taken together, our results reveal that controlling the length of the polyglutamate side chains on tubulin is crit. for neuronal survival.
- 8Hoch, N. C.; Polo, L. M. ADP-Ribosylation: From Molecular Mechanisms to Human Disease. Genet. Mol. Biol. 2020, 43, e20190075 DOI: 10.1590/1678-4685-gmb-2019-0075Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlamsLvP&md5=5f55dfa4e564a5fb84ee2e8d2613f968ADP-ribosylation: from molecular mechanisms to human diseaseHoch, Nicolas C.; Polo, Luis M.Genetics and Molecular Biology (2020), 43 (1Suppl.1), e20190075CODEN: GMBIFG; ISSN:1415-4757. (Sociedade Brasileira de Genetica)Post-translational modification of proteins by ADP-ribosylation, catalyzed by poly (ADP-ribose) polymerases (PARPs) using NAD+ as a substrate, plays central roles in DNA damage signalling and repair, modulates a range of cellular signalling cascades and initiates programmed cell death by parthanatos. Here, we present mechanistic aspects of ADP-ribose modification, PARP activation and the cellular functions of ADP-ribose signalling, and discuss how this knowledge is uncovering therapeutic avenues for the treatment of increasingly prevalent human diseases such as cancer, ischemic damage and neurodegeneration.
- 9Kam, T.-I.; Mao, X.; Park, H.; Chou, S.-C.; Karuppagounder, S. S.; Umanah, G.; Yun, S.; Brahmachari, S.; Panicker, N.; Chen, R.; Andrabi, S. A.; Qi, C.; Poirier, G. G.; Pletnikova, O.; Troncoso, J. C.; Bekris, L. M.; Leverenz, J. B.; Pantelyat, A.; Ko, H.; Rosenthal, L. S.; Dawson, T. M.; Dawson, V. L. Poly(ADP-Ribose) Drives Pathologic α-Synuclein Neurodegeneration in Parkinson’s Disease. Science 2018, 362, eaat8407 DOI: 10.1126/science.aat8407Google ScholarThere is no corresponding record for this reference.
- 10Smith, L. M.; Thomas, P. M.; Shortreed, M. R.; Schaffer, L. V.; Fellers, R. T.; LeDuc, R. D.; Tucholski, T.; Ge, Y.; Agar, J. N.; Anderson, L. C.; Chamot-Rooke, J.; Gault, J.; Loo, J. A.; Paša-Tolić, L.; Robinson, C. V.; Schlüter, H.; Tsybin, Y. O.; Vilaseca, M.; Vizcaíno, J. A.; Danis, P. O.; Kelleher, N. L. A Five-Level Classification System for Proteoform Identifications. Nat. Method 2019, 16, 939– 940, DOI: 10.1038/s41592-019-0573-xGoogle Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1Citb7L&md5=1a45637ecf9d919d47da51c1993c392bA five-level classification system for proteoform identificationsSmith, Lloyd M.; Thomas, Paul M.; Shortreed, Michael R.; Schaffer, Leah V.; Fellers, Ryan T.; LeDuc, Richard D.; Tucholski, Trisha; Ge, Ying; Agar, Jeffrey N.; Anderson, Lissa C.; Chamot-Rooke, Julia; Gault, Joseph; Loo, Joseph A.; Pasa-Tolic, Ljiljana; Robinson, Carol V.; Schluter, Hartmut; Tsybin, Yury O.; Vilaseca, Marta; Vizcaino, Juan Antonio; Danis, Paul O.; Kelleher, Neil L.Nature Methods (2019), 16 (10), 939-940CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)There is no expanded citation for this reference.
- 11Smith, L. M.; Agar, J. N.; Chamot-Rooke, J.; Danis, P. O.; Ge, Y.; Loo, J. A.; Paša-Tolić, L.; Tsybin, Y. O.; Kelleher, N. L.; The Consortium for Top-Down Proteomics The Human Proteoform Project: Defining the Human Proteome. Sci. Adv. 2021, 7, eabk0734 DOI: 10.1126/sciadv.abk0734Google ScholarThere is no corresponding record for this reference.
- 12Laughlin, S. T.; Bertozzi, C. R. Metabolic Labeling of Glycans with Azido Sugars and Subsequent Glycan-Profiling and Visualization via Staudinger Ligation. Nat. Protoc. 2007, 2, 2930– 2944, DOI: 10.1038/nprot.2007.422Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlSlsr%252FJ&md5=cc811cc5d3612b077dc95fde283fcc1eMetabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger ligationLaughlin, Scott T.; Bertozzi, Carolyn R.Nature Protocols (2007), 2 (11), 2930-2944CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Metabolic labeling of glycans with a bioorthogonal chem. reporter such as the azide enables their visualization in cells and organisms as well as the enrichment of specific glycoprotein types for proteomic anal. This process involves two steps. Azido sugars are fed to cells or organisms and integrated by the glycan biosynthetic machinery into various glycoconjugates. The azido sugars are then covalently tagged with imaging probes or epitope tags, either ex vivo or in vivo, using an azide-specific reaction. This protocol details the syntheses of the azido sugars N-azidoacetylmannosamine (ManNAz), N-azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine (GlcNAz) and 6-azidofucose (6AzFuc), and the detection reagents phosphine-FLAG and phosphine-FLAG-His6. Applications to the visualization of cellular glycans and enrichment of glycoproteins for proteomic anal. are described. The synthesis of the azido sugars (ManNAz, GalNAz, GlcNAz or 6AzFuc) or detection reagents (phosphine-FLAG or phosphine-FLAG-His6) can be completed in approx. 1 wk. A cell metabolic labeling expt. can be completed in approx. 4 d.
- 13Kallemeijn, W. W.; Lanyon-Hogg, T.; Panyain, N.; Grocin, A. G.; Ciepla, P.; Morales-Sanfrutos, J.; Tate, E. W. Proteome-Wide Analysis of Protein Lipidation Using Chemical Probes: In-Gel Fluorescence Visualization, Identification and Quantification of N-Myristoylation, N- and S-Acylation, O-Cholesterylation, S-Farnesylation and S-Geranylgeranylation. Nat. Protoc. 2021, 16, 5083– 5122, DOI: 10.1038/s41596-021-00601-6Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisVSht7bI&md5=879b68d06e6accf4fa072a36791b562cProteome-wide analysis of protein lipidation using chemical probes: in-gel fluorescence visualization, identification and quantification of N-myristoylation, N- and S-acylation, O-cholesterylation, S-farnesylation and S-geranylgeranylationKallemeijn, Wouter W.; Lanyon-Hogg, Thomas; Panyain, Nattawadee; Goya Grocin, Andrea; Ciepla, Paulina; Morales-Sanfrutos, Julia; Tate, Edward W.Nature Protocols (2021), 16 (11), 5083-5122CODEN: NPARDW; ISSN:1750-2799. (Nature Portfolio)Protein lipidation is one of the most widespread post-translational modifications (PTMs) found in nature, regulating protein function, structure and subcellular localization. Lipid transferases and their substrate proteins are also attracting increasing interest as drug targets because of their dysregulation in many disease states. However, the inherent hydrophobicity and potential dynamic nature of lipid modifications makes them notoriously challenging to detect by many anal. methods. Chem. proteomics provides a powerful approach to identify and quantify these diverse protein modifications by combining bespoke chem. tools for lipidated protein enrichment with quant. mass spectrometry-based proteomics. Here, we report a robust and proteome-wide approach for the exploration of five major classes of protein lipidation in living cells, through the use of specific chem. probes for each lipid PTM. In-cell labeling of lipidated proteins is achieved by the metabolic incorporation of a lipid probe that mimics the specific natural lipid, concomitantly wielding an alkyne as a bio-orthogonal labeling tag. After incorporation, the chem. tagged proteins can be coupled to multifunctional capture reagents by using click chem., allowing in-gel fluorescence visualization or enrichment via affinity handles for quant. chem. proteomics based on label-free quantification (LFQ) or tandem mass-tag (TMT) approaches. In this protocol, we describe the application of lipid probes for N-myristoylation, N- and S-acylation, O-cholesterylation, S-farnesylation and S-geranylgeranylation in multiple cell lines to illustrate both the workflow and data obtained in these expts. We provide detailed workflows for method optimization, sample prepn. for chem. proteomics and data processing. A properly trained researcher (e.g., technician, graduate student or postdoc) can complete all steps from optimizing metabolic labeling to data processing within 3 wk. This protocol enables sensitive and quant. anal. of lipidated proteins at a proteome-wide scale at native expression levels, which is crit. to understanding the role of lipid PTMs in health and disease.
- 14Martin, B. R.; Wang, C.; Adibekian, A.; Tully, S. E.; Cravatt, B. F. Global Profiling of Dynamic Protein Palmitoylation. Nat. Method 2012, 9, 84– 89, DOI: 10.1038/nmeth.1769Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVagtL3F&md5=d2e067d1896d6cfb00f87a2d2c06c9d8Global profiling of dynamic protein palmitoylationMartin, Brent R.; Wang, Chu; Adibekian, Alexander; Tully, Sarah E.; Cravatt, Benjamin F.Nature Methods (2012), 9 (1), 84-89CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The reversible thioester linkage of palmitic acid on cysteines, known as protein S-palmitoylation, facilitates the membrane assocn. and proper subcellular localization of proteins. Here we report the metabolic incorporation of the palmitic acid analog 17-octadecynoic acid (17-ODYA) in combination with stable-isotope labeling with amino acids in cell culture (SILAC) and pulse-chase methods to generate a global quant. map of dynamic protein palmitoylation events in cells. We distinguished stably palmitoylated proteins from those that turn over rapidly. Treatment with a serine lipase-selective inhibitor identified a pool of dynamically palmitoylated proteins regulated by palmitoyl-protein thioesterases. This subset was enriched in oncoproteins and other proteins linked to aberrant cell growth, migration and cancer. Our method provides a straightforward way to characterize global palmitoylation dynamics in cells and confirms enzyme-mediated depalmitoylation as a crit. regulatory mechanism for a specific subset of rapidly cycling palmitoylated proteins.
- 15Parker, C. G.; Pratt, M. R. Click Chemistry in Proteomic Investigations. Cell 2020, 180, 605– 632, DOI: 10.1016/j.cell.2020.01.025Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjsVOlsr8%253D&md5=5960886d7bd84bf58863f59f75de62a7Click Chemistry in Proteomic InvestigationsParker, Christopher G.; Pratt, Matthew R.Cell (Cambridge, MA, United States) (2020), 180 (4), 605-632CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A review. Despite advances in genetic and proteomic techniques, a complete portrait of the proteome and its complement of dynamic interactions and modifications remains a lofty, and as of yet, unrealized, objective. Specifically, traditional biol. and anal. approaches have not been able to address key questions relating to the interactions of proteins with small mols., including drugs, drug candidates, metabolites, or protein post-translational modifications (PTMs). Fortunately, chemists have bridged this exptl. gap through the creation of bioorthogonal reactions. These reactions allow for the incorporation of chem. groups with highly selective reactivity into small mols. or protein modifications without perturbing their biol. function, enabling the selective installation of an anal. tag for downstream investigations. The introduction of chem. strategies to parse and enrich subsets of the "functional" proteome has empowered mass spectrometry (MS)-based methods to delve more deeply and precisely into the biochem. state of cells and its perturbations by small mols. In this Primer, we discuss how one of the most versatile bioorthogonal reactions, "click chem.", has been exploited to overcome limitations of biol. approaches to enable the selective marking and functional investigation of crit. protein-small-mol. interactions and PTMs in native biol. environments.
- 16Kielkowski, P.; Buchsbaum, I. Y.; Becker, T.; Bach, K.; Cappello, S.; Sieber, S. A. A Pronucleotide Probe for Live-Cell Imaging of Protein AMPylation. ChemBioChem 2020, 21, 1285– 1287, DOI: 10.1002/cbic.201900716Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1yis7c%253D&md5=381685eedb002cfdfd5ad0f86a942349A Pronucleotide Probe for Live-Cell Imaging of Protein AMPylationKielkowski, Pavel; Buchsbaum, Isabel Y.; Becker, Tobias; Bach, Kathrin; Cappello, Silvia; Sieber, Stephan A.ChemBioChem (2020), 21 (9), 1285-1287CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Conjugation of proteins to AMP (AMPylation) is a prevalent post-translational modification (PTM) in human cells, involved in the regulation of unfolded protein response and neural development. Here we present a tailored pronucleotide probe suitable for in situ imaging and chem. proteomics profiling of AMPylated proteins. Using straightforward strain-promoted azide-alkyne click chem., the probe provides stable fluorescence labeling in living cells.
- 17Kielkowski, P.; Buchsbaum, I. Y.; Kirsch, V. C.; Bach, N. C.; Drukker, M.; Cappello, S.; Sieber, S. A. FICD Activity and AMPylation Remodelling Modulate Human Neurogenesis. Nat. Commun. 2020, 11, 517 DOI: 10.1038/s41467-019-14235-6Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXksFanurs%253D&md5=a1eddc8f5fa51409da12fe58890cb2cdFICD activity and AMPylation remodelling modulate human neurogenesisKielkowski, Pavel; Buchsbaum, Isabel Y.; Kirsch, Volker C.; Bach, Nina C.; Drukker, Micha; Cappello, Silvia; Sieber, Stephan A.Nature Communications (2020), 11 (1), 517CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Posttranslational modification (PTM) of proteins represents an important cellular mechanism for controlling diverse functions such as signaling, localization or protein-protein interactions. AMPylation (also termed adenylylation) has recently been discovered as a prevalent PTM for regulating protein activity. In human cells AMPylation has been exclusively studied with the FICD protein. Here we investigate the role of AMPylation in human neurogenesis by introducing a cell-permeable propargyl adenosine pronucleotide probe to infiltrate cellular AMPylation pathways and report distinct modifications in intact cancer cell lines, human-derived stem cells, neural progenitor cells (NPCs), neurons and cerebral organoids (COs) via LC-MS/MS as well as imaging methods. A total of 162 AMP modified proteins were identified. FICD-dependent AMPylation remodeling accelerates differentiation of neural progenitor cells into mature neurons in COs, demonstrating a so far unknown trigger of human neurogenesis.
- 18Sinha, A.; Mann, M. A Beginner’s Guide to Mass Spectrometry–Based Proteomics. Biochemist 2020, 42, 64– 69, DOI: 10.1042/bio20200057Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitlalsrfM&md5=d2b1b3b39f4d66e6a1b90d6e4f360787A beginner's guide to mass spectrometry-based proteomicsSinha, Ankit; Mann, MatthiasBiochemist (2020), 42 (5), 64-69CODEN: BCHMFZ; ISSN:1740-1194. (Portland Press Ltd.)Mass spectrometry (MS)-based proteomics is the most comprehensive approach for the quant. profiling of proteins, their interactions and modifications. It is a challenging topic as a firm grasp requires expertise in biochem. for sample prepn., anal. chem. for instrumentation and computational biol. for data anal. In this short guide, we highlight the various components of a mass spectrometer, the sample prepn. process for conversion of proteins into peptides, and quantification and anal. strategies. The advancing technol. of MS-based proteomics now opens up opportunities in clin. applications and single-cell anal.
- 19Cox, J.; Mann, M. MaxQuant Enables High Peptide Identification Rates, Individualized p.p.b.-Range Mass Accuracies and Proteome-Wide Protein Quantification. Nat. Biotechol. 2008, 26, 1367– 1372, DOI: 10.1038/nbt.1511Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVWjtLzJ&md5=675d31ca84e3a7e4fb9bdd601d8075eaMaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantificationCox, Juergen; Mann, MatthiasNature Biotechnology (2008), 26 (12), 1367-1372CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Efficient anal. of very large amts. of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resoln., quant. MS data. Using correlation anal. and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over std. techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome expt. and allows statistically robust identification and quantification of >4000 proteins in mammalian cell lysates.
- 20Tyanova, S.; Temu, T.; Sinitcyn, P.; Carlson, A.; Hein, M. Y.; Geiger, T.; Mann, M.; Cox, J. The Perseus Computational Platform for Comprehensive Analysis of (Prote)Omics Data. Nat. Method 2016, 13, 731– 740, DOI: 10.1038/nmeth.3901Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVKntbnN&md5=f8c3e2876e4d724518054bb1a2d1e6eeThe Perseus computational platform for comprehensive analysis of (prote)omics dataTyanova, Stefka; Temu, Tikira; Sinitcyn, Pavel; Carlson, Arthur; Hein, Marco Y.; Geiger, Tamar; Mann, Matthias; Cox, JuergenNature Methods (2016), 13 (9), 731-740CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A main bottleneck in proteomics is the downstream biol. anal. of highly multivariate quant. protein abundance data generated using mass-spectrometry-based anal. We developed the Perseus software platform (http://www.perseus-framework.org) to support biol. and biomedical researchers in interpreting protein quantification, interaction and post-translational modification data. Perseus contains a comprehensive portfolio of statistical tools for high-dimensional omics data anal. covering normalization, pattern recognition, time-series anal., cross-omics comparisons and multiple-hypothesis testing. A machine learning module supports the classification and validation of patient groups for diagnosis and prognosis, and it also detects predictive protein signatures. Central to Perseus is a user-friendly, interactive workflow environment that provides complete documentation of computational methods used in a publication. All activities in Perseus are realized as plugins, and users can extend the software by programming their own, which can be shared through a plugin store. We anticipate that Perseus's arsenal of algorithms and its intuitive usability will empower interdisciplinary anal. of complex large data sets.
- 21Yu, F.; Teo, G. C.; Kong, A. T.; Haynes, S. E.; Avtonomov, D. M.; Geiszler, D. J.; Nesvizhskii, A. I. Identification of Modified Peptides Using Localization-Aware Open Search. Nat. Commun. 2020, 11, 4065 DOI: 10.1038/s41467-020-17921-yGoogle Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1Wrtr7J&md5=f15cad0240097b55bd077ad641788807Identification of modified peptides using localization-aware open searchYu, Fengchao; Teo, Guo Ci; Kong, Andy T.; Haynes, Sarah E.; Avtonomov, Dmitry M.; Geiszler, Daniel J.; Nesvizhskii, Alexey I.Nature Communications (2020), 11 (1), 4065CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Abstr.: Identification of post-translationally or chem. modified peptides in mass spectrometry-based proteomics expts. is a crucial yet challenging task. We have recently introduced a fragment ion indexing method and the MSFragger search engine to empower an open search strategy for comprehensive anal. of modified peptides. However, this strategy does not consider fragment ions shifted by unknown modifications, preventing modification localization and limiting the sensitivity of the search. Here we present a localization-aware open search method, in which both modification-contg. (shifted) and regular fragment ions are indexed and used in scoring. We also implement a fast mass calibration and optimization method, allowing optimization of the mass tolerances and other key search parameters. We demonstrate that MSFragger with mass calibration and localization-aware open search identifies modified peptides with significantly higher sensitivity and accuracy. Comparing MSFragger to other modification-focused tools (pFind3, MetaMorpheus, and TagGraph) shows that MSFragger remains an excellent option for fast, comprehensive, and sensitive searches for modified peptides in shotgun proteomics data.
- 22Grammel, M.; Luong, P.; Orth, K.; Hang, H. C. A Chemical Reporter for Protein AMPylation. J. Am. Chem. Soc. 2011, 133, 17103– 17105, DOI: 10.1021/ja205137dGoogle Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1ymsr7F&md5=9e83c16371afb9fba2a17d5ea4a763ebA Chemical Reporter for Protein AMPylationGrammel, Markus; Luong, Phi; Orth, Kim; Hang, Howard C.Journal of the American Chemical Society (2011), 133 (43), 17103-17105CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein AMPylation is an emerging post-translational modification, which plays key roles in bacterial pathogenesis and cell biol. Enzymes with AMPylation activity, referred to as AMPylators, have been identified in several bacterial pathogens and eukaryotes. To facilitate the study of this unique modification, the authors developed an alkynyl chem. reporter for detection and identification of protein AMPylation substrates. Covalent functionalization of AMPylation substrates with the alkynyl reporter in lieu of adenylyl 5'-monophosphate (AMP) allows their subsequent bioorthogonal ligation with azide-fluorescent dyes or affinity enrichment tags. This chem. reporter is transferred by a range of AMPylators onto their cognate protein substrates and allows rapid detection and identification of AMPylated substrates.
- 23Kliza, K. W.; Liu, Q.; Roosenboom, L. W. M.; Jansen, P. W. T. C.; Filippov, D. V.; Vermeulen, M. Reading ADP-Ribosylation Signaling Using Chemical Biology and Interaction Proteomics. Mol. Cell 2021, 81, 4552.e8– 4567.e8, DOI: 10.1016/j.molcel.2021.08.037Google ScholarThere is no corresponding record for this reference.
- 24Yang, Y.-Y.; Ascano, J. M.; Hang, H. C. Bioorthogonal Chemical Reporters for Monitoring Protein Acetylation. J. Am. Chem. Soc. 2010, 132, 3640– 3641, DOI: 10.1021/ja908871tGoogle Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXisFGltrw%253D&md5=e7584db37fc5210158c281f7f85e23cbBioorthogonal Chemical Reporters for Monitoring Protein AcetylationYang, Yu-Ying; Ascano, Janice M.; Hang, Howard C.Journal of the American Chemical Society (2010), 132 (11), 3640-3641CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein acetylation is a key post-translational modification that regulates diverse biol. activities in eukaryotes. Bioorthogonal chem. reporters that enable direct in-gel fluorescent visualization and proteome-wide identification of acetylated proteins via copper-catalyzed azide-alkyne cycloaddn. are prepd. 3-Butynoyl, 4-pentynoyl, and 5-hexynoyl-CoA thioesters are prepd. as alkyne-contg. acetyl-CoA analogs; the sodium salts of 3-butynoic, 4-pentynoic, and 5-hexynoic acids are prepd. as alkyne-contg. acetate analogs. 4-Pentynoyl-CoA and 5-hexynoyl-CoA function as efficient substrates of the lysine acetyltransferase p300 and serve as sensitive reagents for monitoring p300-catalyzed protein acetylation in vitro. Sodium 3-butynoate, sodium 4-pentynoate, and sodium 5-hexynoate are metabolically incorporated onto cellular proteins through biosynthetic mechanisms for profiling of acetylated proteins in diverse cell types. Mass spectrometric anal. of the enriched 4-pentynoate-labeled proteins revealed many reported acetylated proteins as well as new candidate acetylated proteins from Jurkat T cells and also specific sites of lysine acetylation.
- 25Sieber, S. A.; Cappello, S.; Kielkowski, P. From Young to Old: AMPylation Hits the Brain. Cell. Chem. Biol. 2020, 27, 773– 779, DOI: 10.1016/j.chembiol.2020.05.009Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtFGhs7fJ&md5=eeaffb75cfd1779ad287f0971bb51e95From Young to Old: AMPylation Hits the BrainSieber, Stephan A.; Cappello, Silvia; Kielkowski, PavelCell Chemical Biology (2020), 27 (7), 773-779CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)Protein post-translational modifications (PTMs) are implicated in numerous physiol. processes and significantly contribute to complex regulatory networks of protein functions. Recently, a protein PTM called AMPylation was found to play a role in modulation of neurodevelopment and neurodegeneration. Combination of biochem. and chem. proteomic studies has uncovered the prevalence of this PTM in regulation of diverse metabolic pathways. In metazoans, thus far two protein AMP transferases have been identified to introduce AMPylation: FICD and SELO. These two proteins were found to be involved in unfolded protein response and redox homeostasis on the cellular level and in the case of FICD to adjust the development of glial cells and neurons in Drosophila and cerebral organoids, resp. Together with findings on AMPylation and its assocn. with toxic protein aggregation, we summarize in this Perspective the knowledge and putative future directions of protein AMPylation research.
- 26Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. A Strain-Promoted [3 + 2] Azide–Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems. J. Am. Chem. Soc. 2004, 126, 15046– 15047, DOI: 10.1021/ja044996fGoogle Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXpt1Sks7s%253D&md5=37af3dbaa89ae4cffaba2dee30e50ec0A strain-promoted [3+2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systemsAgard, Nicholas J.; Prescher, Jennifer A.; Bertozzi, Carolyn R.Journal of the American Chemical Society (2004), 126 (46), 15046-15047CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Selective chem. reactions that are orthogonal to the diverse functionality of biol. systems have become important tools in the field of chem. biol. Two notable examples are the Staudinger ligation of azides and phosphines and the Cu(I)-catalyzed [3+2] cycloaddn. of azides and alkynes ("click chem."). The Staudinger ligation has sufficient biocompatibility for performance in living animals but suffers from phosphine oxidn. and synthetic challenges. Click chem. obviates the requirement of phosphines, but the Cu(I) catalyst is toxic to cells, thereby precluding in vivo applications. Here we present a strain-promoted [3+2] cycloaddn. between cyclooctynes and azides that proceeds under physiol. conditions without the need for a catalyst. The utility of the reaction was demonstrated by selective modification of biomols. in vitro and on living cells, with no apparent toxicity.
- 27Zecha, J.; Satpathy, S.; Kanashova, T.; Avanessian, S. C.; Kane, M. H.; Clauser, K. R.; Mertins, P.; Carr, S. A.; Kuster, B. TMT Labeling for the Masses: A Robust and Cost-Efficient, In-Solution Labeling Approach* [S]. Mol. Cell. Proteomics 2019, 18, 1468– 1478, DOI: 10.1074/mcp.tir119.001385Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvFWms7fP&md5=230b43b5f8d2584131c63edc7ea87fe7TMT labeling for the masses: a robust and cost-efficient, in-solution labeling approachZecha, Jana; Satpathy, Shankha; Kanashova, Tamara; Avanessian, Shayan C.; Kane, M. Harry; Clauser, Karl R.; Mertins, Philipp; Carr, Steven A.; Kuster, BernhardMolecular & Cellular Proteomics (2019), 18 (7), 1468-1478CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)Isobaric stable isotope labeling using, for example, tandem mass tags (TMTs) is increasingly being applied for large-scale proteomic studies. Expts. focusing on proteoform anal. in drug time course or perturbation studies or in large patient cohorts greatly benefit from the reproducible quantification of single peptides across samples. However, such studies often require labeling of hundreds of micrograms of peptides such that the cost for labeling reagents represents a major contribution to the overall cost of an expt. Here, we describe and evaluate a robust and cost-effective protocol for TMT labeling that reduces the quantity of required labeling reagent by a factor of eight and achieves complete labeling. Under- and overlabeling of peptides derived from complex digests of tissues and cell lines were systematically evaluated using peptide quantities of between 12.5 and 800μg and TMT-to-peptide ratios (wt/wt) ranging from 8:1 to 1:2 at different TMT and peptide concns. When reaction vols. were reduced to maintain TMT and peptide concns. of at least 10 mM and 2 g/l, resp., TMT-to-peptide ratios as low as 1:1 (wt/wt) resulted in labeling efficiencies of > 99% and excellent intra- and interlab. reproducibility. The utility of the optimized protocol was further demonstrated in a deepscale proteome and phosphoproteome anal. of patientderived xenograft tumor tissue benchmarked against the labeling procedure recommended by the TMT vendor. Finally, we discuss the impact of labeling reaction parameters for N-hydroxysuccinimide ester-based chem. and provide guidance on adopting efficient labeling protocols for different peptide quantities.
- 28Cox, J.; Hein, M. Y.; Luber, C. A.; Paron, I.; Nagaraj, N.; Mann, M. Accurate Proteome-Wide Label-Free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ. Mol. Cell. Proteomics 2014, 13, 2513– 2526, DOI: 10.1074/mcp.M113.031591Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVynurrI&md5=f3f1c7dc8fbf729c568446968b89f37cAccurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQCox, Juergen; Hein, Marco Y.; Luber, Christian A.; Paron, Igor; Nagaraj, Nagarjuna; Mann, MatthiasMolecular & Cellular Proteomics (2014), 13 (9), 2513-2526CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)Protein quantification without isotopic labels has been a long-standing interest in the proteomics field. However, accurate and robust proteome-wide quantification with label-free approaches remains a challenge. We developed a new intensity detn. and normalization procedure called MaxLFQ that is fully compatible with any peptide or protein sepn. prior to LC-MS anal. Protein abundance profiles are assembled using the max. possible information from MS signals, given that the presence of quantifiable peptides varies from sample to sample. For a benchmark dataset with two proteomes mixed at known ratios, we accurately detected the mixing ratio over the entire protein expression range, with greater precision for abundant proteins. The significance of individual label-free quantifications was obtained via a t test approach. For a second benchmark dataset, we accurately quantify fold changes over several orders of magnitude, a task that is challenging with label-based methods. MaxLFQ is a generic label-free quantification technol. that is readily applicable to many biol. questions; it is compatible with std. statistical anal. workflows, and it has been validated in many and diverse biol. projects. Our algorithms can handle very large expts. of 500+ samples in a manageable computing time. It is implemented in the freely available MaxQuant computational proteomics platform and works completely seamlessly at the click of a button.
- 29Wiśniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M. Universal Sample Preparation Method for Proteome Analysis. Nat. Method 2009, 6, 359– 362, DOI: 10.1038/nmeth.1322Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXks12ksb0%253D&md5=f34cb14143462984852497dc1f9ee5c2Universal sample preparation method for proteome analysisWisniewski, Jacek R.; Zougman, Alexandre; Nagaraj, Nagarjuna; Mann, MatthiasNature Methods (2009), 6 (5), 359-362CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The authors describe a method, filter-aided sample prepn. (FASP), which combines the advantages of in-gel and in-soln. digestion for mass spectrometry-based proteomics. The authors completely solubilized the proteome in SDS, which the authors then exchanged by urea on a std. filtration device. Peptides eluted after digestion on the filter were pure, allowing single-run analyses of organelles and an unprecedented depth of proteome coverage.
- 30Müller, T.; Kalxdorf, M.; Longuespée, R.; Kazdal, D. N.; Stenzinger, A.; Krijgsveld, J. Automated Sample Preparation with SP3 for Low-input Clinical Proteomics. Mol. Syst. Biol. 2020, 16, e9111 DOI: 10.15252/msb.20199111Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFOis7o%253D&md5=5bf4201cc5b52478697c68c60cae6ee0Automated sample preparation with SP3 for low-input clinical proteomicsMueller, Torsten; Kalxdorf, Mathias; Longuespee, Remi; Kazdal, Daniel N.; Stenzinger, Albrecht; Krijgsveld, JeroenMolecular Systems Biology (2020), 16 (1), e9111CODEN: MSBOC3; ISSN:1744-4292. (Wiley-VCH Verlag GmbH & Co. KGaA)High-throughput and streamlined workflows are essential in clin. proteomics for standardized processing of samples from a variety of sources, including fresh-frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single-pot solid-phase-enhanced sample prepn. (SP3) on a liq. handling robot for automated processing (autoSP3) of tissue lysates in a 96-well format. AutoSP3 performs unbiased protein purifn. and digestion, and delivers peptides that can be directly analyzed by LCMS, thereby significantly reducing hands-on time, reducing variability in protein quantification, and improving longitudinal reproducibility. We demonstrate the distinguishing ability of autoSP3 to process low-input samples, reproducibly quantifying 500-1,000 proteins from 100 to 1,000 cells. Furthermore, we applied this approach to a cohort of clin. FFPE pulmonary adenocarcinoma (ADC) samples and recapitulated their sepn. into known histol. growth patterns. Finally, we integrated autoSP3 with AFA ultrasonication for the automated end-to-end sample prepn. and LCMS anal. of 96 intact tissue samples. Collectively, this constitutes a generic, scalable, and cost-effective workflow with minimal manual intervention, enabling reproducible tissue proteomics in a broad range of clin. and non-clin. applications.
- 31Hughes, C. S.; Moggridge, S.; Müller, T.; Sorensen, P. H.; Morin, G. B.; Krijgsveld, J. Single-Pot, Solid-Phase-Enhanced Sample Preparation for Proteomics Experiments. Nat. Protoc. 2019, 14, 68– 85, DOI: 10.1038/s41596-018-0082-xGoogle Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXit1yltrzK&md5=d80343142c71d0b55bcd35978e5db19dSingle-pot, solid-phase-enhanced sample preparation for proteomics experimentsHughes, Christopher S.; Moggridge, Sophie; Muller, Torsten; Sorensen, Poul H.; Morin, Gregg B.; Krijgsveld, JeroenNature Protocols (2019), 14 (1), 68-85CODEN: NPARDW; ISSN:1750-2799. (Nature Research)A crit. step in proteomics anal. is the optimal extn. and processing of protein material to ensure the highest sensitivity in downstream detection. Achieving this requires a sample-handling technol. that exhibits unbiased protein manipulation, flexibility in reagent use, and virtually lossless processing. Addressing these needs, the single-pot, solid-phase-enhanced sample-prepn. (SP3) technol. is a paramagnetic bead-based approach for rapid, robust, and efficient processing of protein samples for proteomic anal. SP3 uses a hydrophilic interaction mechanism for exchange or removal of components that are commonly used to facilitate cell or tissue lysis, protein solubilization, and enzymic digestion (e.g., detergents, chaotropes, salts, buffers, acids, and solvents) before downstream proteomic anal. The SP3 protocol consists of nonselective protein binding and rinsing steps that are enabled through the use of ethanol-driven solvation capture on the surface of hydrophilic beads, and elution of purified material in aq. conditions. In contrast to alternative approaches, SP3 combines compatibility with a substantial collection of soln. additives with virtually lossless and unbiased recovery of proteins independent of input quantity, all in a simplified single-tube protocol. The SP3 protocol is simple and efficient, and can be easily completed by a std. user in ∼30 min, including reagent prepn. As a result of these properties, SP3 has successfully been used to facilitate examn. of a broad range of sample types spanning simple and complex protein mixts. in large and very small amts., across numerous organisms. This work describes the steps and extensive considerations involved in performing SP3 in bottom-up proteomics, using a simplified protein cleanup scenario for illustration.
- 32Sielaff, M.; Kuharev, J.; Bohn, T.; Hahlbrock, J.; Bopp, T.; Tenzer, S.; Distler, U. Evaluation of FASP, SP3, and IST Protocols for Proteomic Sample Preparation in the Low Microgram Range. J. Proteome Res. 2017, 16, 4060– 4072, DOI: 10.1021/acs.jproteome.7b00433Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFCrtb%252FP&md5=5f3d5a92a9186e5aaa0633e6c8121a34Evaluation of FASP, SP3, and iST Protocols for Proteomic Sample Preparation in the Low Microgram RangeSielaff, Malte; Kuharev, Joerg; Bohn, Toszka; Hahlbrock, Jennifer; Bopp, Tobias; Tenzer, Stefan; Distler, UteJournal of Proteome Research (2017), 16 (11), 4060-4072CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Efficient and reproducible sample prepn. is a prerequisite for any robust and sensitive quant. bottom-up proteomics workflow. Here, the authors performed an independent comparison between single-pot solid-phase-enhanced sample prepn. (SP3), filter-aided sample prepn. (FASP), and a com. kit based on the in-StageTip (iST) method. The authors assessed their performance for the processing of proteomic samples in the low μg range using varying amts. of HeLa cell lysate (1-20 μg of total protein). All three workflows showed similar performances for 20 μg of starting material. When handling sample sizes below 10 μg, the no. of identified proteins and peptides as well as the quant. reproducibility and precision drastically dropped in case of FASP. In contrast, SP3 and iST provided high proteome coverage even in the low μg range. Even when digesting 1 μg of starting material, both methods still enabled the identification of over 3000 proteins and between 25,000 and 30,000 peptides. On av., the quant. reproducibility between exptl. replicates was slightly higher in case of SP3 (R2 = 0.97 (SP3); R2 = 0.93 (iST)). Applying SP3 toward the characterization of the proteome of FACS-sorted tumor-assocd. macrophages in the B16 tumor model enabled the quantification of 2965 proteins and revealed a "mixed" M1/M2 phenotype.
- 33Wright, M. H.; Clough, B.; Rackham, M. D.; Rangachari, K.; Brannigan, J. A.; Grainger, M.; Moss, D. K.; Bottrill, A. R.; Heal, W. P.; Broncel, M.; Serwa, R. A.; Brady, D.; Mann, D. J.; Leatherbarrow, R. J.; Tewari, R.; Wilkinson, A. J.; Holder, A. A.; Tate, E. W. Validation of N-Myristoyltransferase as an Antimalarial Drug Target Using an Integrated Chemical Biology Approach. Nat. Chem. 2014, 6, 112– 121, DOI: 10.1038/nchem.1830Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFOmtL%252FE&md5=d2fa0f4af88198fc26a53c9a5f853ca1Validation of N-myristoyltransferase as an antimalarial drug target using an integrated chemical biology approachWright, Megan H.; Clough, Barbara; Rackham, Mark D.; Rangachari, Kaveri; Brannigan, James A.; Grainger, Munira; Moss, David K.; Bottrill, Andrew R.; Heal, William P.; Broncel, Malgorzata; Serwa, Remigiusz A.; Brady, Declan; Mann, David J.; Leatherbarrow, Robin J.; Tewari, Rita; Wilkinson, Anthony J.; Holder, Anthony A.; Tate, Edward W.Nature Chemistry (2014), 6 (2), 112-121CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Malaria is an infectious disease caused by parasites of the genus Plasmodium, which leads to approx. one million deaths per annum worldwide. Chem. validation of new antimalarial targets is urgently required in view of rising resistance to current drugs. One such putative target is the enzyme N-myristoyltransferase, which catalyzes the attachment of the fatty acid myristate to protein substrates (N-myristoylation). Here, the authors report an integrated chem. biol. approach to explore protein myristoylation in the major human parasite P. falciparum, combining chem. proteomic tools for identification of the myristoylated and glycosylphosphatidylinositol-anchored proteome with selective small-mol. N-mytransferase inhibitors. The authors demonstrate that N-mytransferase is an essential and chem. tractable target in malaria parasites both in vitro and in vivo, and show that selective inhibition of N-myristoylation leads to catastrophic and irreversible failure to assemble the inner membrane complex, a crit. subcellular organelle in the parasite life cycle. The authors' studies provide the basis for the development of new antimalarials targeting N-mytransferase.
- 34Yan, T.; Desai, H. S.; Boatner, L. M.; Yen, S. L.; Cao, J.; Palafox, M. F.; Jami-Alahmadi, Y.; Backus, K. SP3-FAIMS Chemoproteomics for High Coverage Profiling of the Human Cysteinome. ChemBioChem 2021, 22, 1841– 1851, DOI: 10.1002/cbic.202000870Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXkt1aisr4%253D&md5=f45fdb9a576f4541259cb15519dbeb8bSP3-FAIMS Chemoproteomics for High-Coverage Profiling of the Human Cysteinome**Yan, Tianyang; Desai, Heta S.; Boatner, Lisa M.; Yen, Stephanie L.; Cao, Jian; Palafox, Maria F.; Jami-Alahmadi, Yasaman; Backus, Keriann M.ChemBioChem (2021), 22 (10), 1841-1851CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Chemoproteomics has enabled the rapid and proteome-wide discovery of functional, redox-sensitive, and ligandable cysteine residues. Despite widespread adoption and considerable advances in both sample-prepn. workflows and MS instrumentation, chemoproteomics expts. still typically only identify a small fraction of all cysteines encoded by the human genome. Here, we develop an optimized sample-prepn. workflow that combines enhanced peptide labeling with single-pot, solid-phase-enhanced sample-prepn. (SP3) to improve the recovery of biotinylated peptides, even from small sample sizes. By combining this improved workflow with online high-field asym. waveform ion mobility spectrometry (FAIMS) sepn. of labeled peptides, we achieve unprecedented coverage of >14000 unique cysteines in a single-shot 70 min expt. Showcasing the wide utility of the SP3-FAIMS chemoproteomic method, we find that it is also compatible with competitive small-mol. screening by isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP). In aggregate, our anal. of 18 samples from seven cell lines identified 34225 unique cysteines using only ∼28 h of instrument time. The comprehensive spectral library and improved coverage provided by the SP3-FAIMS chemoproteomics method will provide the tech. foundation for future studies aimed at deciphering the functions and druggability of the human cysteineome.
- 35Klont, F.; Kwiatkowski, M.; Faiz, A.; van den Bosch, T.; Pouwels, S. D.; Dekker, F. J.; ten Hacken, N. H. T.; Horvatovich, P.; Bischoff, R. Adsorptive Microtiter Plates As Solid Supports in Affinity Purification Workflows. J. Proteome Res. 2021, 20, 5218– 5221, DOI: 10.1021/acs.jproteome.1c00623Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitlSltrnJ&md5=0152dd555344eac0ff59eea9163a1e40Adsorptive Microtiter Plates As Solid Supports in Affinity Purification WorkflowsKlont, Frank; Kwiatkowski, Marcel; Faiz, Alen; van den Bosch, Thea; Pouwels, Simon D.; Dekker, Frank J.; ten Hacken, Nick H. T.; Horvatovich, Peter; Bischoff, RainerJournal of Proteome Research (2021), 20 (11), 5218-5221CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Affinity ligands such as antibodies are widely used in (bio)medical research for purifying proteins from complex biol. samples. These ligands are generally immobilized onto solid supports which facilitate the sepn. of a captured protein from the sample matrix. Adsorptive microtiter plates are commonly used as solid supports prior to immunochem. detection (e.g., immunoassays) but hardly ever prior to liq. chromatog.-mass spectrometry (LC-MS-)-based detection. Here, we describe the use of adsorptive microtiter plates for protein enrichment prior to LC-MS detection, and we discuss opportunities and challenges of corresponding workflows, based on examples of targeted (i.e., sol. receptor for advanced glycation end-products (sRAGE) in human serum) and discovery-based workflows (i.e., transcription factor p65 (NF-κB) in lysed murine RAW 264.7 macrophages and peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP5) in lysed human A549 alveolar basal epithelial cells). Thereby, we aim to highlight the potential usefulness of adsorptive microtiter plates in affinity purifn. workflows prior to LC-MS detection, which could increase their usage in mass spectrometry-based protein research.
- 36Makowski, M. M.; Gräwe, C.; Foster, B. M.; Nguyen, N. V.; Bartke, T.; Vermeulen, M. Global Profiling of Protein–DNA and Protein–Nucleosome Binding Affinities Using Quantitative Mass Spectrometry. Nat. Commun. 2018, 9, 1653 DOI: 10.1038/s41467-018-04084-0Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MjmslaqsA%253D%253D&md5=4cfa8d72422fe923a20279d154c5a0ceGlobal profiling of protein-DNA and protein-nucleosome binding affinities using quantitative mass spectrometryMakowski Matthew M; Grawe Cathrin; Vermeulen Michiel; Makowski Matthew M; Grawe Cathrin; Vermeulen Michiel; Foster Benjamin M; Bartke Till; Foster Benjamin M; Nguyen Nhuong V; Bartke Till; Foster Benjamin M; Nguyen Nhuong V; Bartke TillNature communications (2018), 9 (1), 1653 ISSN:.Interaction proteomics studies have provided fundamental insights into multimeric biomolecular assemblies and cell-scale molecular networks. Significant recent developments in mass spectrometry-based interaction proteomics have been fueled by rapid advances in label-free, isotopic, and isobaric quantitation workflows. Here, we report a quantitative protein-DNA and protein-nucleosome binding assay that uses affinity purifications from nuclear extracts coupled with isobaric chemical labeling and mass spectrometry to quantify apparent binding affinities proteome-wide. We use this assay with a variety of DNA and nucleosome baits to quantify apparent binding affinities of monomeric and multimeric transcription factors and chromatin remodeling complexes.
- 37Tian, Y.-P.; Zhang, X.-J.; Wu, J.-Y.; Fun, H.-K.; Jiang, M.-H.; Xu, Z.-Q.; Usman, A.; Chantrapromma, S.; Thompson, L. K. Structural Diversity and Properties of a Series of Dinuclear and Mononuclear Copper(Ii) and Copper(i) Carboxylato Complexes. New J. Chem. 2002, 26, 1468– 1473, DOI: 10.10309/b203334hGoogle Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xnt1aiurs%253D&md5=df05eb4d9775770e588acae4427dbefdStructural diversity and properties of a series of dinuclear and mononuclear copper(II) and copper(I) carboxylato complexesTian, Yu-Peng; Zhang, Xuan-Jun; Wu, Jie-Ying; Fun, Hoong-Kun; Jiang, Min-Hua; Xu, Zhi-Qiang; Usman, Anwar; Chantrapromma, Suchada; Thompson, Laurence K.New Journal of Chemistry (2002), 26 (10), 1468-1473CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)The syntheses, crystal structures, magnetic and photoluminescence properties of dinuclear and mononuclear copper(II) and copper(I) N-carbazolylacetate [N-carbazolylacetic acid = Hcabo] with different carboxylato coordination modes are reported. Although the carboxylato group has different coordination modes, the same carboxylate ligand binding to copper ion via four coordinating modes is rare. The crystal structure of [Cu2(Cabo)4(DMF)2]·2DMF (1) consists of a sym. dimeric Cu(II) carboxylato paddle-wheel core and oxygen atoms from DMF at the apical positions. Dinuclear [Cu2(Cabo)3(phen)2]ClO4·H2O·C2H5OH (2) (phen = 1,10-phenanthroline) consists of an unusual dimeric core with two copper atoms bridged by three carboxylates one of which is in the η:η:μ2 bridging mode and the other two are in the rarer monoat. bridging mode. To the authors' knowledge, the present bridging mode was not reported hitherto. The crystal structures of [Cu(Cabo)2phen] and [Cu(Cabo)(PPh3)2] are also reported. Magnetic susceptibilities were measured in the temp. range 2-300 K paddle-wheel copper(II) ions in 1 are strongly coupled antiferromagnetically with 2J = -356.4(6) cm-1, whereas complex 2 shows weak antiferromagnetic interaction with a 2J value of -12.8(4) cm-1. Copper(I) N-carbazolylacetate with strong fluorescence in the solid state as well as high thermal stability was obtained by redn. of the copper(II) N-carbazolylacetate using PPh3 (triphenylphosphine) in DMF soln.
- 38Stojceva Radovanovic, B. C.; Premovic, P. I. Thermal Behaviour of Cu(II)-Urea Complex. J. Therm. Anal. 1992, 38, 715– 719, DOI: 10.1007/bf01979401Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xls1aqs7g%253D&md5=8ea035b99db690cab65d2539510fbf4aThermal behavior of copper(II)-urea complexStojceva Radovanovic, B. C.; Premovic, P. I.Journal of Thermal Analysis (1992), 38 (4), 715-19CODEN: JTHEA9; ISSN:0368-4466.[CuL4]Cl2 (L = urea) was prepd. and its structure was established by FTIR, ESR, at. absorption spectroscopy, and elemental anal. The thermal behavior of [CuL4]Cl2 was studied by TG, DTA, FTIR, and ESR. The decompn. of the complex occurs in 4 stages of wt. loss of different intermediates followed by 3 endothermal effects. The complex is thermally stable at ≤428 K. The ESR and FTIR behavior of [CuL4]Cl2 during thermolysis was studied at 428-633 K. In this temp. range the complex decompn. occurred forming thermodynamically stable regions of Cu(II) which are ferromagnetically coupled.
- 39Hughes, C. S.; Foehr, S.; Garfield, D. A.; Furlong, E. E.; Steinmetz, L. M.; Krijgsveld, J. Ultrasensitive Proteome Analysis Using Paramagnetic Bead Technology. Mol. Syst. Biol. 2014, 10, 757, DOI: 10.15252/msb.20145625Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M3ksF2rsA%253D%253D&md5=0a3dce5610fc3f4b8097c6de64ffb9fdUltrasensitive proteome analysis using paramagnetic bead technologyHughes Christopher S; Foehr Sophia; Garfield David A; Furlong Eileen E; Steinmetz Lars M; Krijgsveld JeroenMolecular systems biology (2014), 10 (), 757 ISSN:.In order to obtain a systems-level understanding of a complex biological system, detailed proteome information is essential. Despite great progress in proteomics technologies, thorough interrogation of the proteome from quantity-limited biological samples is hampered by inefficiencies during processing. To address these challenges, here we introduce a novel protocol using paramagnetic beads, termed Single-Pot Solid-Phase-enhanced Sample Preparation (SP3). SP3 provides a rapid and unbiased means of proteomic sample preparation in a single tube that facilitates ultrasensitive analysis by outperforming existing protocols in terms of efficiency, scalability, speed, throughput, and flexibility. To illustrate these benefits, characterization of 1,000 HeLa cells and single Drosophila embryos is used to establish that SP3 provides an enhanced platform for profiling proteomes derived from sub-microgram amounts of material. These data present a first view of developmental stage-specific proteome dynamics in Drosophila at a single-embryo resolution, permitting characterization of inter-individual expression variation. Together, the findings of this work position SP3 as a superior protocol that facilitates exciting new directions in multiple areas of proteomics ranging from developmental biology to clinical applications.
- 40Truttmann, M. C.; Zheng, X.; Hanke, L.; Damon, J. R.; Grootveld, M.; Krakowiak, J.; Pincus, D.; Ploegh, H. L. Unrestrained AMPylation Targets Cytosolic Chaperones and Activates the Heat Shock Response. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, E152– E160, DOI: 10.1073/pnas.1619234114Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXivFWr&md5=56e77b18492c14dd54d98f0fb23e45c9Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock responseTruttmann, Matthias C.; Zheng, Xu; Hanke, Leo; Damon, Jadyn R.; Grootveld, Monique; Krakowiak, Joanna; Pincus, David; Ploegh, Hidde L.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (2), E152-E160CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Protein AMPylation is a conserved posttranslational modification with emerging roles in endoplasmic reticulum homeostasis. However, the range of substrates and cell biol. consequences of AMPylation remain poorly defined. We expressed human and Caenorhabditis elegans AMPylation enzymes-huntingtin yeast-interacting protein E (HYPE) and filamentation-induced by cAMP (FIC)-1, resp.-in Saccharomyces cerevisiae, a eukaryote that lacks endogenous protein AMPylation. Expression of HYPE and FIC-1 in yeast induced a strong cytoplasmic Hsf1-mediated heat shock response, accompanied by attenuation of protein translation, massive protein aggregation, growth arrest, and lethality. Overexpression of Ssa2, a cytosolic heat shock protein (Hsp)70, was sufficient to partially rescue growth. In human cell lines, overexpression of active HYPE similarly induced protein aggregation and the HSF1-dependent heat shock response. Excessive AMPylation also abolished HSP70-dependent influenza virus replication. Our findings suggest a mode of Hsp70 inactivation by AMPylation and point toward a role for protein AMPylation in the regulation of cellular protein homeostasis beyond the endoplasmic reticulum.
- 41Sanyal, A.; Dutta, S.; Camara, A.; Chandran, A.; Koller, A.; Watson, B. G.; Sengupta, R.; Ysselstein, D.; Montenegro, P.; Cannon, J.; Rochet, J.-C.; Mattoo, S. Alpha-Synuclein Is a Target of Fic-Mediated Adenylylation/AMPylation: Possible Implications for Parkinson’s Disease. J. Mol. Biol. 2019, 431, 2266– 2282, DOI: 10.1016/j.jmb.2019.04.026Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXovVCnsb4%253D&md5=fd934dd43a0e1ef1c177648e6da8f78aAlpha-synuclein is a target of fic-mediated adenylylation/AMPylation: Possible Implications for Parkinson's DiseaseSanyal, Anwesha; Dutta, Sayan; Camara, Ali; Chandran, Aswathy; Koller, Antonius; Watson, Ben G.; Sengupta, Ranjan; Ysselstein, Daniel; Montenegro, Paola; Cannon, Jason; Rochet, Jean-Christophe; Mattoo, SeemaJournal of Molecular Biology (2019), 431 (12), 2266-2282CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)During disease, cells experience various stresses that manifest as an accumulation of misfolded proteins and eventually lead to cell death. To combat this stress, cells activate a pathway called unfolded protein response that functions to maintain endoplasmic reticulum (ER) homeostasis and dets. cell fate. We recently reported a hitherto unknown mechanism of regulating ER stress via a novel post-translational modification called Fic-mediated adenylylation/AMPylation. Specifically, we showed that the human Fic (filamentation induced by cAMP) protein, HYPE/FicD, catalyzes the addn. of an adenosine monophosphate (AMP) to the ER chaperone, BiP, to alter the cell's unfolded protein response-mediated response to misfolded proteins. Here, we report that we have now identified a second target for HYPE-alpha-synuclein (αSyn), a presynaptic protein involved in Parkinson's disease. Aggregated αSyn has been shown to induce ER stress and elicit neurotoxicity in Parkinson's disease models. We show that HYPE adenylylates αSyn and reduces phenotypes assocd. with αSyn aggregation invitro, suggesting a possible mechanism by which cells cope with αSyn toxicity.
- 42Raught, B.; Gingras, A.-C.; Sonenberg, N. The Target of Rapamycin (TOR) Proteins. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7037– 7044, DOI: 10.1073/pnas.121145898Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXkslWmsb4%253D&md5=e6e2182a59a006e21c73ee4dccb941a5The target of rapamycin (TOR) proteinsRaught, Brian; Gingras, Anne-Claude; Sonenberg, NahumProceedings of the National Academy of Sciences of the United States of America (2001), 98 (13), 7037-7044CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A review, with 132 refs. Rapamycin potently inhibits downstream signaling from the target of rapamycin (TOR) proteins. These evolutionarily conserved protein kinases coordinate the balance between protein synthesis and protein degrdn. in response to nutrient quality and quantity. The TOR proteins regulate (i) the initiation and elongation phases of translation, (ii) ribosome biosynthesis, (ii) amino acid import, (iv) the transcription of numerous enzymes involved in multiple metabolic pathways, and (v) autophagy. Intriguingly, recent studies have also suggested that TOR signaling plays a crit. role in brain development, learning, and memory formation.
- 43Leeman, D. S.; Hebestreit, K.; Ruetz, T.; Webb, A. E.; McKay, A.; Pollina, E. A.; Dulken, B. W.; Zhao, X.; Yeo, R. W.; Ho, T. T.; Mahmoudi, S.; Devarajan, K.; Passegué, E.; Rando, T. A.; Frydman, J.; Brunet, A. Lysosome Activation Clears Aggregates and Enhances Quiescent Neural Stem Cell Activation during Aging. Science 2018, 359, 1277– 1283, DOI: 10.1126/science.aag3048Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXkslCjs74%253D&md5=e1b5eb1b30d5eb3a36d37bd8173a250eLysosome activation clears aggregates and enhances quiescent neural stem cell activation during agingLeeman, Dena S.; Hebestreit, Katja; Ruetz, Tyson; Webb, Ashley E.; McKay, Andrew; Pollina, Elizabeth A.; Dulken, Ben W.; Zhao, Xiaoai; Yeo, Robin W.; Ho, Theodore T.; Mahmoudi, Salah; Devarajan, Keerthana; Passegue, Emmanuelle; Rando, Thomas A.; Frydman, Judith; Brunet, AnneScience (Washington, DC, United States) (2018), 359 (6381), 1277-1283CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)In the adult brain, the neural stem cell (NSC) pool comprises quiescent and activated populations with distinct roles. Transcriptomic anal. revealed that quiescent and activated NSCs exhibited differences in their protein homeostasis network. Whereas activated NSCs had active proteasomes, quiescent NSCs contained large lysosomes. Quiescent NSCs from young mice accumulated protein aggregates, and many of these aggregates were stored in large lysosomes. Perturbation of lysosomal activity in quiescent NSCs affected protein-aggregate accumulation and the ability of quiescent NSCs to activate. During aging, quiescent NSCs displayed defects in their lysosomes, increased accumulation of protein aggregates, and reduced ability to activate. Enhancement of the lysosome pathway in old quiescent NSCs cleared protein aggregates and ameliorated the ability of quiescent NSCs to activate, allowing them to regain a more youthful state.
- 44Yoshimori, T.; Yamamoto, A.; Moriyama, Y.; Futai, M.; Tashiro, Y. Bafilomycin A1, a Specific Inhibitor of Vacuolar-Type H(+)-ATPase, Inhibits Acidification and Protein Degradation in Lysosomes of Cultured Cells. J. Biol. Chem. 1991, 266, 17707– 17712, DOI: 10.1016/s0021-9258(19)47429-2Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXltFWhs78%253D&md5=1cee87bb38ff07e9cfc3aa55124f1f5eBafilomycin A1, a specific inhibitor of vacuolar-type hydrogen ion-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cellsYoshimori, Tamotsu; Yamamoto, Akitsugu; Moriyama, Yoshinori; Futai, Masamitsu; Tashiro, YutakaJournal of Biological Chemistry (1991), 266 (26), 17707-12CODEN: JBCHA3; ISSN:0021-9258.Bafilomycin A1 is known as a strong inhibitor of the vacuolar type H+-ATPase in vitro, whereas other type ATPases, e.g. F1,F0-ATPase, are not affected by this antibiotic. The effects of this inhibitor on lysosomes of living cultured cells were tested. The acidification of lysosomes revealed by the incubation with acridine orange was completely inhibited when BNL CL.2 and A431 cells were treated with 0.1-1 μM bafilomycin A1. The effect was reversed by washing the cells. Both studies using less 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and fluorescein isothiocyanate-dextran showed that the intralysosomal pH of A431 cells increased from ∼5.1-5.5 to ∼6.3 in the presence of 1 μM bafilomycin A1. The pH increased gradually in ∼50 min. In the presence of 1 μM bafilomycin A1, 125I-labeled EGF bound to the cell surface at 4° was internalized normally into the cells at 37° but was not degraded at all, in marked contrast to the rapid degrdn. of 125I-EGF in the control cells without the drug. Immunogold electron microscopy showed that EGF was transported into lysosomes irresp. of the addn. of bafilomycin A1. Apparently, the vacuolar type H+-ATPase plays a pivotal role in acidification and protein degrdn. in the lysosomes in vivo.
- 45Zhang, J.-G.; Fariss, M. W. Thenoyltrifluoroacetone, a Potent Inhibitor of Carboxylesterase Activity. Biochem. Pharmacol. 2002, 63, 751– 754, DOI: 10.1016/s0006-2952(01)00871-1Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xjt1Sgt7Y%253D&md5=52a33a6556d3db9f770252f38e8af5d2Thenoyltrifluoroacetone, a potent inhibitor of carboxylesterase activityZhang, Jin-Gang; Fariss, Marc W.Biochemical Pharmacology (2002), 63 (4), 751-754CODEN: BCPCA6; ISSN:0006-2952. (Elsevier Science Inc.)Thenoyltrifluoroacetone (TTFA), a conventional mitochondrial complex II inhibitor, was found to inhibit purified porcine liver carboxylesterase non-competitively with a Ki of 0.61×10-6 M and an IC50 of 0.54×10-6 M. Both rat plasma and liver mitochondrial esterases were inhibited in a concn.-dependent fashion. Results indicate that TTFA is a potent inhibitor of carboxylesterase activity, in addn. to its ability to inhibit mitochondrial complex II activity. Therefore, caution is warranted in using TTFA as a mitochondrial complex inhibitor in combination with esterase substrates, such as fluorescence probes or vitamin E esters.
- 46Mollenhauer, H. H.; Morré, D. J.; Rowe, L. D. Alteration of Intracellular Traffic by Monensin; Mechanism, Specificity and Relationship to Toxicity. Biochim. Biophys. Acta, Rev. Biomembr. 1990, 1031, 225– 246, DOI: 10.1016/0304-4157(90)90008-zGoogle Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXkt1Whsr8%253D&md5=81104fe7af97d6dd7c0ac69026cf1bf6Alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicityMollenhauer, Hilton H.; Morre, D. James; Rowe, Loyd D.Biochimica et Biophysica Acta, Reviews on Biomembranes (1990), 1031 (2), 225-46CODEN: BRBMC5; ISSN:0304-4157.A review with 252 refs. examg. the mechanism of action and specificity of monensin in Na+/H+ exchange and correlating these data with the structural and biochem. information on monensin toxicity derived from animal studies.
- 47Pohlmann, R.; Krüger, S.; Hasilik, A.; von Figura, K. Effect of Monensin on Intracellular Transport and Receptor-Mediated Endocytosis of Lysosomal Enzymes. Biochem. J. 1984, 217, 649– 658, DOI: 10.1042/bj2170649Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXotlCrsQ%253D%253D&md5=6a68d525b77057f059ed574d3db8e139Effect of monensin on intracellular transport and receptor-mediated endocytosis of lysosomal enzymesPohlmann, Regina; Krueger, Susanne; Hasilik, Andrej; Von Figura, KurtBiochemical Journal (1984), 217 (3), 649-58CODEN: BIJOAK; ISSN:0264-6021.In cultured human fibroblasts, monensin, a Na+/H+-exchanging ionophore, (1) inhibits mannose 6-phosphate-sensitive endocytosis of a lysosomal enzyme; (2) enhances secretion of the precursor of cathepsin D, while inhibiting secretion of the precursors of β-hexosaminidase; (3) induces secretion of mature β-hexosaminidase and mature cathepsin D; and (4) inhibits carbohydrate processing in proteolytic maturation of the precursors remaining within the cells; this last effect appears to be secondary to an inhibition of the transport of the precursors. If the treated cells are transferred to a monensin-free medium ∼50% of the accumulated precursors are secreted, and the intracellular enzyme is converted into the mature form. Monensin blocks formation of complex oligosaccharides in lysosomal enzymes. In the presence of monensin, total phosphorylation of glycoproteins is partially inhibited, whereas the secreted glycoproteins are enriched in the phosphorylated species. The suggested inhibition by monensin of the transport within the Golgi app. (Tartakoff, A. M.; 1980) may be the cause of some of the effects obsd. in the present study. Other effects are rather explained by interference by monensin with the acidification in the lysosomal and prelysosomal compartments, which appears to be necessary for the transport of endocytosed and of newly synthesized lysosomal enzymes.
- 48Wang, X.; Wu, X.; Zhang, Z.; Ma, C.; Wu, T.; Tang, S.; Zeng, Z.; Huang, S.; Gong, C.; Yuan, C.; Zhang, L.; Feng, Y.; Huang, B.; Liu, W.; Zhang, B.; Shen, Y.; Luo, W.; Wang, X.; Liu, B.; Lei, Y.; Ye, Z.; Zhao, L.; Cao, D.; Yang, L.; Chen, X.; Haydon, R. C.; Luu, H. H.; Peng, B.; Liu, X.; He, T.-C. Monensin Inhibits Cell Proliferation and Tumor Growth of Chemo-Resistant Pancreatic Cancer Cells by Targeting the EGFR Signaling Pathway. Sci. Rep. 2018, 8, 17914 DOI: 10.1038/s41598-018-36214-5Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslKht7o%253D&md5=c09f916e186df99df103332b48c1380fMonensin inhibits cell proliferation and tumor growth of chemo-resistant pancreatic cancer cells by targeting the EGFR signaling pathwayWang, Xin; Wu, Xingye; Zhang, Zhonglin; Ma, Chao; Wu, Tingting; Tang, Shengli; Zeng, Zongyue; Huang, Shifeng; Gong, Cheng; Yuan, Chengfu; Zhang, Linghuan; Feng, Yixiao; Huang, Bo; Liu, Wei; Zhang, Bo; Shen, Yi; Luo, Wenping; Wang, Xi; Liu, Bo; Lei, Yan; Ye, Zhenyu; Zhao, Ling; Cao, Daigui; Yang, Lijuan; Chen, Xian; Haydon, Rex C.; Luu, Hue H.; Peng, Bing; Liu, Xubao; He, Tong-ChuanScientific Reports (2018), 8 (1), 17914CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly malignancies with <5% five-year survival rate due to late diagnosis, limited treatment options and chemoresistance. There is thus an urgent unmet clin. need to develop effective anticancer drugs to treat pancreatic cancer. Here, we study the potential of repurposing monensin as an anticancer drug for chemo-resistant pancreatic cancer. Using the two commonly-used chemo-resistant pancreatic cancer cell lines PANC-1 and MiaPaCa-2, we show that monensin suppresses cell proliferation and migration, and cell cycle progression, while solicits apoptosis in pancreatic cancer lines at a low micromole range. Moreover, monensin functions synergistically with gemcitabine or EGFR inhibitor erlotinib in suppressing cell growth and inducing cell death of pancreatic cancer cells. Mechanistically, monensin suppresses numerous cancer-assocd. pathways, such as E2F/DP1, STAT1/2, NFkB, AP-1, Elk-1/SRF, and represses EGFR expression in pancreatic cancer lines. Furthermore, the in vivo study shows that monensin blunts PDAC xenograft tumor growth by suppressing cell proliferation via targeting EGFR pathway. Therefore, our findings demonstrate that monensin can be repurposed as an effective anti-pancreatic cancer drug even though more investigations are needed to validate its safety and anticancer efficacy in pre-clin. and clin. models.
- 49Long, J. M.; Holtzman, D. M. Alzheimer Disease: An Update on Pathobiology and Treatment Strategies. Cell 2019, 179, 312– 339, DOI: 10.1016/j.cell.2019.09.001Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVylsbrN&md5=b68edceb2c7ebe4515efded93c9281c3Alzheimer Disease: An Update on Pathobiology and Treatment StrategiesLong, Justin M.; Holtzman, David M.Cell (Cambridge, MA, United States) (2019), 179 (2), 312-339CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A review. Alzheimer disease (AD) is a heterogeneous disease with a complex pathobiol. The presence of extracellular β-amyloid deposition as neuritic plaques and intracellular accumulation of hyperphosphorylated tau as neurofibrillary tangles remains the primary neuropathol. criteria for AD diagnosis. However, a no. of recent fundamental discoveries highlight important pathol. roles for other crit. cellular and mol. processes. Despite this, no disease-modifying treatment currently exists, and numerous phase 3 clin. trials have failed to demonstrate benefits. Here, we review recent advances in our understanding of AD pathobiol. and discuss current treatment strategies, highlighting recent clin. trials and opportunities for developing future disease-modifying therapies.
- 50Yang, X.; Qian, K. Protein O-GlcNAcylation: Emerging Mechanisms and Functions. Nat. Rev. Mol. Cell Biol. 2017, 18, 452– 465, DOI: 10.1038/nrm.2017.22Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnsV2mtbs%253D&md5=291f00ada663647822444a8fa5fca532Protein O-GlcNAcylation: emerging mechanisms and functionsYang, Xiaoyong; Qian, KevinNature Reviews Molecular Cell Biology (2017), 18 (7), 452-465CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. O-GlcNAcylation - the attachment of O-linked N-acetylglucosamine (O-GlcNAc) moieties to cytoplasmic, nuclear and mitochondrial proteins - is a post-translational modification that regulates fundamental cellular processes in metazoans. A single pair of enzymes - O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) - controls the dynamic cycling of this protein modification in a nutrient- and stress-responsive manner. Recent years have seen remarkable advances in our understanding of O-GlcNAcylation at levels that range from structural and mol. biol. to cell signalling and gene regulation to physiol. and disease. New mechanisms and functions of O-GlcNAcylation that are emerging from these recent developments enable us to begin constructing a unified conceptual framework through which the significance of this modification in cellular and organismal physiol. can be understood.
- 51Li, J.; Wang, J.; Wen, L.; Zhu, H.; Li, S.; Huang, K.; Jiang, K.; Li, X.; Ma, C.; Qu, J.; Parameswaran, A.; Song, J.; Zhao, W.; Wang, P. G. An OGA-Resistant Probe Allows Specific Visualization and Accurate Identification of O -GlcNAc-Modified Proteins in Cells. ACS Chem. Biol. 2016, 11, 3002– 3006, DOI: 10.1021/acschembio.6b00678Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFSlsb%252FP&md5=ff09bf8c6987de47cf6d424507c19019An OGA-Resistant Probe Allows Specific Visualization and Accurate Identification of O-GlcNAc-Modified Proteins in CellsLi, Jing; Wang, Jiajia; Wen, Liuqing; Zhu, He; Li, Shanshan; Huang, Kenneth; Jiang, Kuan; Li, Xu; Ma, Cheng; Qu, Jingyao; Parameswaran, Aishwarya; Song, Jing; Zhao, Wei; Wang, Peng GeorgeACS Chemical Biology (2016), 11 (11), 3002-3006CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chem. probes such as GlcNAc or GalNAc analogs bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modifications. However, these chem. probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. The authors report here the development of a novel probe, 1,3,6-tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-D-glucopyranose (Ac34dGlcNAz), that can be processed by the GalNAc salvage pathway, and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of hydroxyl group at C4, this probe is less incorporated into α/β 4-GlcNAc or GalNAc contg. glycoconjugates. Furthermore, the O-4dGlcNAz modification was resistant to the hydrolysis of O-GlcNAcase (OGA), which greatly enhanced the efficiency of incorporation for O-GlcNAcylation. Combined with a click reaction, Ac34dGlcNAz allowed the selective visualization of O-GlcNAc in cells, and accurate identification of O-GlcNAc-modified proteins with LC-MS/MS. This probe represents a more potent and selective tool in tracking, capturing, and identifying O-GlcNAc-modified proteins in cells and cell lysates.
- 52Pedowitz, N. J.; Pratt, M. R. Design and Synthesis of Metabolic Chemical Reporters for the Visualization and Identification of Glycoproteins. RSC Chem. Biol. 2021, 2, 306– 321, DOI: 10.1039/d1cb00010aGoogle Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXktlGlt70%253D&md5=9cca66336cc96323cc8c31e80436a6ddDesign and synthesis of metabolic chemical reporters for the visualization and identification of glycoproteinsPedowitz, Nichole J.; Pratt, Matthew R.RSC Chemical Biology (2021), 2 (2), 306-321CODEN: RCBSBP; ISSN:2633-0679. (Royal Society of Chemistry)A review. Glycosylation events play an invaluable role in regulating cellular processes including enzymic activity, immune recognition, protein stability, and cell-cell interactions. However, researchers have yet to realize the full range of glycan mediated biol. functions due to a lack of appropriate chem. tools. Fortunately, the past 25 years has seen the emergence of modified sugar analogs, termed metabolic chem. reporters (MCRs), which are metabolized by endogenous enzymes to label complex glycan structures. Here, we review the major reporters for each class of glycosylation and highlight recent applications that have made a tremendous impact on the field of glycobiol.
- 53Zhang, C.; Zhang, C.; Dai, P.; Vinogradov, A.; Gates, Z. Site-Selective Cysteine-Cyclooctyne Conjugation. Angew. Chem., Int. Ed. 2018, 57, 6459– 6463, DOI: 10.1002/anie.201800860Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXosFSqtLs%253D&md5=e21a09d9578aaa87167af5ebdda954e1Site-Selective Cysteine-Cyclooctyne ConjugationZhang, Chi; Dai, Peng; Vinogradov, Alexander A.; Gates, Zachary P.; Pentelute, Bradley L.Angewandte Chemie, International Edition (2018), 57 (22), 6459-6463CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors report a site-selective cysteine-cyclooctyne conjugation reaction between a seven-residue peptide tag (DBCO-tag, Leu-Cys-Tyr-Pro-Trp-Val-Tyr) at the N or C terminus of a peptide or protein and various aza-dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO-tag increases the rate of the thiol-yne reaction 220-fold, thereby enabling selective conjugation of DBCO-tag to DBCO-linked fluorescent probes, affinity tags, and cytotoxic drug mols. Fusion of DBCO-tag with the protein of interest enables regioselective cysteine modification on proteins that contain multiple endogenous cysteines; these examples include green fluorescent protein and the antibody trastuzumab. Short peptide tags can aid in accelerating bond-forming reactions that are often slow to non-existent in water.
- 54Wulff-Fuentes, E.; Berendt, R. R.; Massman, L.; Danner, L.; Malard, F.; Vora, J.; Kahsay, R.; Stichelen, S. O.-V. The Human O-GlcNAcome Database and Meta-Analysis. Sci. Data 2021, 8, 25 DOI: 10.1038/s41597-021-00810-4Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXlsVOrsb4%253D&md5=467cd56066d8f2b4a6847a717b7526e4The human O-GlcNAcome database and meta-analysisWulff-Fuentes, Eugenia; Berendt, Rex R.; Massman, Logan; Danner, Laura; Malard, Florian; Vora, Jeet; Kahsay, Robel; Olivier-Van Stichelen, StephanieScientific Data (2021), 8 (1), 25CODEN: SDCABS; ISSN:2052-4463. (Nature Research)Abstr.: Over the past 35 years, ∼1700 articles have characterized protein O-GlcNAcylation. Found in almost all living organisms, this post-translational modification of serine and threonine residues is highly conserved and key to biol. processes. With half of the primary research articles using human models, the O-GlcNAcome recently reached a milestone of 5000 human proteins identified. Herein, we provide an extensive inventory of human O-GlcNAcylated proteins, their O-GlcNAc sites, identification methods, and corresponding refs. (www.oglcnac.mcw.edu). In the absence of a comprehensive online resource for O-GlcNAcylated proteins, this list serves as the only database of O-GlcNAcylated proteins. Based on the thorough anal. of the amino acid sequence surrounding 7002 O-GlcNAc sites, we progress toward a more robust semi-consensus sequence for O-GlcNAcylation. Moreover, we offer a comprehensive meta-anal. of human O-GlcNAcylated proteins for protein domains, cellular and tissue distribution, and pathways in health and diseases, reinforcing that O-GlcNAcylation is a master regulator of cell signaling, equal to the widely studied phosphorylation.
- 55Petelski, A. A.; Emmott, E.; Leduc, A.; Huffman, R. G.; Specht, H.; Perlman, D. H.; Slavov, N. Multiplexed Single-Cell Proteomics Using SCoPE2. Nat. Protoc. 2021, 16, 5398– 5425, DOI: 10.1038/s41596-021-00616-zGoogle Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitlOlurrI&md5=912ffb4f0fd8ad003b1eb5a704300b41Multiplexed single-cell proteomics using SCoPE2Petelski, Aleksandra A.; Emmott, Edward; Leduc, Andrew; Huffman, R. Gray; Specht, Harrison; Perlman, David H.; Slavov, NikolaiNature Protocols (2021), 16 (12), 5398-5425CODEN: NPARDW; ISSN:1750-2799. (Nature Portfolio)Many biol. systems are composed of diverse single cells. This diversity necessitates functional and mol. single-cell anal. Single-cell protein anal. has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Prepn. (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed anal. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ∼200 single cells per 24 h using only std. com. equipment. We emphasize exptl. steps and benchmarks required for achieving quant. protein anal.
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Abstract
Figure 1
Figure 1. Schematic overview of the chemical proteomic workflow. (A) Key steps of the standard chemical proteomic workflow and (B) schematic characterization of the SP2E workflow and basic parameters of the procedure in comparison to the previously used workflow with avidin-coated agarose beads. In the table below the SP2E workflow, the typical times required to proceed with 8 samples (large scale) or 24 samples (small scale) are shown. For comparison, the previously used workflow would typically take about 8 h for eight samples.
Figure 2
Figure 2. Development and optimization of the SP2E workflow using the AMPylation probe. (A) Pro-N6pA probe structure and the workflow used for the optimization of the SP2E method. (B) Optimization of the lysis buffer based on the efficiency of the CuAAC click chemistry. Lysis buffer compositions: line 1 (control cells treated with plain dimethyl sulfoxide (DMSO) and lysed in 1% NP-40, 0.2% SDS in 20 mM HEPES), line 2 (1% NP-40 in PBS), line 3 (1% NP-40, 0.2% SDS in PBS), line 4 (0.5% Triton in PBS), line 5 (0.5% Triton, 0.2% SDS in PBS), line 6 (1% NP-40 in 20 mM HEPES), line 7 (1% NP-40, 0.2% SDS in 20 mM HEPES), line 8 (0.5% Triton in 20 mM Hepes), line 9 (0.5% Triton, 0.2% SDS in 20 mM Hepes), and line 10 (8 M urea in 0.1 M Tris/HCl). (C) In-gel fluorescence showing the click reaction time optimization. In the control C, cells were treated with plain DMSO and the lysate was incubated with the click reaction mixture for 90 min. (D) Heatmap visualizing the SP2E workflow optimization based on fold enrichment of six marker proteins. Condition 1 (without added urea to the click reaction mixture before protein loading onto carboxylate magnetic beads), condition 2 (with added urea to the click reaction before protein loading onto carboxylate beads and one pot clean up and enrichment of modified proteins), and condition 3 (with added urea into the click reaction, but the spatial separation of the protein clean up and enrichment). The numbers in boxes represent fold enrichments. (E) Volcano plot showing significantly enriched proteins (red dots) using the pro-N6pA AMPylation probe with highlighted marker proteins (green dots) using the optimized SP2E workflow (condition 3 from Figure 2D); n = 4, cutoff lines at p-value >0.05 and 2-fold enrichment. (F) Plot displaying total fluorescence intensity from the in-gel analysis of the time optimization of biotin–streptavidin complex formation.
Figure 3
Figure 3. Analysis of protein AMPylation under different stress conditions using the SP2E workflow. (A) Design of the experiment to test the impact of various inhibitors on protein AMPylation. (B) Volcano plot showing the enrichment of AMPylated proteins (pro-N6pA vs DMSO) from SH-SY5Y cells using the SP2E protocol; n = 4, cutoff lines at p-value >0.05 and 2-fold enrichment. (C) PCA of the inhibitor-treated cells and controls displaying separation of the monensin and bafilomycin as well as pro-N6pA-treated cells. Samples that were treated with DMSO and either rapamycin, 2-deoxy-d-glucose, TTFA, or without any inhibitor are depicted in red. Samples that were treated with pro-N6pA and either rapamycin, 2-deoxy-d-glucose, TTFA, or without any inhibitor are depicted in purple. (D) Representative heatmaps visualizing the Pearson correlation coefficients of LFQ intensities of DMSO and pro-N6pA replicates. (E) Profile plot displays the APP LFQ intensities under various conditions. The APP was not found in any other conditions, for example, in cells only treated with DMSO or some other inhibitors. (F) Profile plot displays the PLD3 LFQ intensities under various conditions.
Figure 4
Figure 4. Monensin concentration-dependent increase in APP and PLD3 modification. (A) PCA displays distinct changes in the enriched proteins with increasing monensin concentration. (B) Profile plot of the PLD3 LFQ intensities shows a rapid increase in the PLD3 modification with a 2 nM monensin concentration. (C) Monensin concentration-dependent enrichment of the modified PLD3. For the enrichment, the SP2E protocol was used but the proteins were released from the streptavidin beads by the loading buffer, separated by SDS-PAGE, and analyzed via western blotting with the anti-PLD3 antibody. (D) Enrichment of the modified PLD3 after the treatment with bafilomycin (100 nM) and monensin (2 μM). For the enrichment, the SP2E protocol was used but the proteins were released from the streptavidin beads by loading buffer, separated by SDS-PAGE, and analyzed via western blotting with the anti-PLD3 antibody. (E) Western blotting of the whole proteome from cells treated with bafilomycin (100 nM) and monensin (2 μM) stained with the anti-PLD3 antibody. (F) In contrast to PLD3, the profile plot of the APP LFQ intensity reveals that APP is only enriched with the pro-N6pA probe with 1 and 2 μM monensin in cell culture media.
Figure 5
Figure 5. Analysis of O-GlcNAcylation by the Ac34dGlcNAz probe, SPAAC, and SP2E workflow. (A) Chemical structure of the Ac34dGlcNAz probe for metabolic labeling of O-GlcNAcylated proteins and the DBCO–biotin reagent to functionalize the probe-modified proteins by SPAAC. (B) Volcano plot visualizing the enrichment of the O-GlcNAcylated proteins; n = 4, cutoff lines at p-value >0.05 and 2-fold enrichment. Red dots are significantly enriched proteins. (C) PCA graph points to a clear separation of the control and probe-treated samples. Of note, component 1 possesses a high value of 81.7%. (D) Box plot shows the total number of imputed values across the replicates in DMSO and Ac34dGlcNAz-treated cells. The number of imputed values indicates how many proteins were not identified in the sample but were found in at least one other sample/replicate. The increased number of imputed values in the DMSO controls demonstrates the efficiency of washing steps removing the nonspecific biding proteins. (E) Diagram showing the overlap between all significantly enriched proteins using the Ac34dGlcNAz probe and previously described O-GlcNAcylated proteins. (F) Enrichment of the modified NUP62 with the agarose-based and the SP2E protocol. In addition, the SP2E enrichment was performed with 400 μg of the protein input. Enriched proteins were released from the streptavidin beads by loading buffer, separated by SDS-PAGE, and analyzed via western blotting with the anti-NUP62 antibody.
Figure 6
Figure 6. Scale-down of the SP2E workflow into a 96-well plate format. (A) SP2E protocol with 100 μg of the input protein performed in 1.5 mL tubes visualized in the volcano plot. (B) Optimization of the LC-MS/MS measurement with 100 μg protein input using the 96-well plate format SP2E protocol. (C) Heatmaps representing the Pearson correlation coefficients between the replicates. (D) Volcano plot showing the enrichment of O-GlcNAcylated proteins starting from 100 μg of the input protein in a 96-well plate format. (E) PCA of the small-scale Ac34dGlcNAz enrichment shows very good separation of controls from probe-treated samples, with component 1 value corresponding to 74%. All volcano plots, n = 4, cutoff lines at p-value >0.05 and 2-fold enrichment. Red dots are significantly enriched proteins.
References
This article references 55 other publications.
- 1Becker, T.; Cappel, C.; Matteo, F. D.; Sonsalla, G.; Kaminska, E.; Spada, F.; Cappello, S.; Damme, M.; Kielkowski, P. AMPylation Profiling during Neuronal Differentiation Reveals Extensive Variation on Lysosomal Proteins. iScience 2021, 24, 103521 DOI: 10.1016/j.isci.2021.1035211https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XptFGnsLY%253D&md5=6913a9e390099fa9b4960841ff07a1fdAMPylation profiling during neuronal differentiation reveals extensive variation on lysosomal proteinsBecker, Tobias; Cappel, Cedric; Di Matteo, Francesco; Sonsalla, Giovanna; Kaminska, Ewelina; Spada, Fabio; Cappello, Silvia; Damme, Markus; Kielkowski, PaveliScience (2021), 24 (12), 103521CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)Protein AMPylation is a posttranslational modification with an emerging role in neurodevelopment. In metazoans two highly conserved protein AMP-transferases together with a diverse group of AMPylated proteins have been identified using chem. proteomics and biochem. techniques. However, the function of AMPylation remains largely unknown. Particularly problematic is the localization of thus far identified AMPylated proteins and putative AMP-transferases. We show that protein AMPylation is likely a posttranslational modification of luminal lysosomal proteins characteristic in differentiating neurons. Through a combination of chem. proteomics, gel-based sepn. of modified and unmodified proteins, and an activity assay, we det. that the modified, lysosomal sol. form of exonuclease PLD3 increases dramatically during neuronal maturation and that AMPylation correlates with its catalytic activity. Together, our findings indicate that AMPylation is a so far unknown lysosomal posttranslational modification connected to neuronal differentiation and it may provide a mol. rationale behind lysosomal storage diseases and neurodegeneration.
- 2Mansfield, S. G.; Gordon-Weeks, P. R. Dynamic Post-Translational Modification of Tubulin in Rat Cerebral Cortical Neurons Extending Neurites in Culture: Effects of Taxol. J. Neurocytol. 1991, 20, 654– 666, DOI: 10.1007/bf011870672https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XhslWjurw%253D&md5=bd2a37c3b87c4b50742676042356ca01Dynamic post-translational modification of tubulin in rat cerebral cortical neurons extending neurites in culture: effects of taxolMansfield, S. G.; Gordon-Weeks, P. R.Journal of Neurocytology (1991), 20 (8), 654-66CODEN: JNCYA2; ISSN:0300-4864.Dissocd. embryonic (E18-E20) rat cortical neurons were grown in culture and double-labeled by immunofluorescence with antibodies directed against tyrosinated (YL 1/2), detyrosinated (SUP GLU), and acetylated (6-11B-1) α-tubulin. Within 90 min of plating, neurons extended growth cones that were YL 1/2+ but SUP GLU- and 6-11B-1-. The neurite that forms behind the advancing growth cone is also, initially, YL 1/2+ and SUP GLU-/6-11B-1-. However, when it has attained a length of about half the cell body diam., it becomes SUP GLU+ and 6-11B-1+. The effects of the microtubule polymg. agent taxol (15 μM) on growth cone and neurite α-tubulins was investigated. Taxol, as reported previously, caused the formation of microtubule loops in the central domain of the growth cone, a loss of filopodia, and the collapse of the growth cone onto the loops. The taxol effects peaked at 60 min, when >85% of neurites showed microtubule loops, and declined thereafter, so that at 420 min in taxol, only ∼23% of neurites had microtubule loops. Over this period there was an inverse correlation between the presence of microtubule loops and growth cones. Taxol had striking effects on the intensity of SUP GLU and 6-11B-1 staining in neurons. In 48 h cultures, a 30 min exposure to taxol enhanced the SUP GLU and 6-11B-1 staining of dendrites and axons and produced a loss of YL 1/2 staining in axons. Immunoblotting expts. confirmed that there was an overall redn. in YL 1/2 immunoreactivity and an increase in SUP GLU immunoreactivity. These observations support previous suggestions that the neurite microtubules are assembled in the growth cone and post-translationally modified in the neurite and, in addn., imply that growth cones can overcome the effects of taxol in the continued presence of the compd.
- 3Zheng, P.; Obara, C. J.; Szczesna, E.; Nixon-Abell, J.; Mahalingan, K. K.; Roll-Mecak, A.; Lippincott-Schwartz, J.; Blackstone, C. ER Proteins Decipher the Tubulin Code to Regulate Organelle Distribution. Nature 2022, 132– 138, DOI: 10.1038/s41586-021-04204-93https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXislGntbnI&md5=f7c86f932739a0be1fd09eaa4ef504deER proteins decipher the tubulin code to regulate organelle distributionZheng, Pengli; Obara, Christopher J.; Szczesna, Ewa; Nixon-Abell, Jonathon; Mahalingan, Kishore K.; Roll-Mecak, Antonina; Lippincott-Schwartz, Jennifer; Blackstone, CraigNature (London, United Kingdom) (2022), 601 (7891), 132-138CODEN: NATUAS; ISSN:1476-4687. (Nature Portfolio)Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions1,2. However, how cells interpret these post-translational microtubule modification codes to selectively regulate organelle positioning remains largely unknown. The endoplasmic reticulum (ER) is an interconnected network of diverse morphologies that extends promiscuously throughout the cytoplasm3, forming abundant contacts with other organelles4. Dysregulation of endoplasmic reticulum morphol. is tightly linked to neurol. disorders and cancer5,6. Here we demonstrate that three membrane-bound endoplasmic reticulum proteins preferentially interact with different microtubule populations, with CLIMP63 binding centrosome microtubules, kinectin (KTN1) binding perinuclear polyglutamylated microtubules, and p180 binding glutamylated microtubules. Knockout of these proteins or manipulation of microtubule populations and glutamylation status results in marked changes in endoplasmic reticulum positioning, leading to similar redistributions of other organelles. During nutrient starvation, cells modulate CLIMP63 protein levels and p180-microtubule binding to bidirectionally move endoplasmic reticulum and lysosomes for proper autophagic responses.
- 4Aebersold, R.; Agar, J. N.; Amster, I. J.; Baker, M. S.; Bertozzi, C. R.; Boja, E. S.; Costello, C. E.; Cravatt, B. F.; Fenselau, C.; Garcia, B. A.; Ge, Y.; Gunawardena, J.; Hendrickson, R. C.; Hergenrother, P. J.; Huber, C. G.; Ivanov, A. R.; Jensen, O. N.; Jewett, M. C.; Kelleher, N. L.; Kiessling, L. L.; Krogan, N. J.; Larsen, M. R.; Loo, J. A.; Loo, R. R. O.; Lundberg, E.; MacCoss, M. J.; Mallick, P.; Mootha, V. K.; Mrksich, M.; Muir, T. W.; Patrie, S. M.; Pesavento, J. J.; Pitteri, S. J.; Rodriguez, H.; Saghatelian, A.; Sandoval, W.; Schlüter, H.; Sechi, S.; Slavoff, S. A.; Smith, L. M.; Snyder, M. P.; Thomas, P. M.; Uhlén, M.; Eyk, J. E. V.; Vidal, M.; Walt, D. R.; White, F. M.; Williams, E. R.; Wohlschlager, T.; Wysocki, V. H.; Yates, N. A.; Young, N. L.; Zhang, B. How Many Human Proteoforms Are There?. Nat. Chem. Biol. 2018, 14, 206– 214, DOI: 10.1038/nchembio.25764https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXislSntLs%253D&md5=ba124ec3e824ec106295bc127a289e57How many human proteoforms are there?Aebersold, Ruedi; Agar, Jeffrey N.; Amster, I. Jonathan; Baker, Mark S.; Bertozzi, Carolyn R.; Boja, Emily S.; Costello, Catherine E.; Cravatt, Benjamin F.; Fenselau, Catherine; Garcia, Benjamin A.; Ge, Ying; Gunawardena, Jeremy; Hendrickson, Ronald C.; Hergenrother, Paul J.; Huber, Christian G.; Ivanov, Alexander R.; Jensen, Ole N.; Jewett, Michael C.; Kelleher, Neil L.; Kiessling, Laura L.; Krogan, Nevan J.; Larsen, Martin R.; Loo, Joseph A.; Ogorzalek Loo, Rachel R.; Lundberg, Emma; MacCoss, Michael J.; Mallick, Parag; Mootha, Vamsi K.; Mrksich, Milan; Muir, Tom W.; Patrie, Steven M.; Pesavento, James J.; Pitteri, Sharon J.; Rodriguez, Henry; Saghatelian, Alan; Sandoval, Wendy; Schluter, Hartmut; Sechi, Salvatore; Slavoff, Sarah A.; Smith, Lloyd M.; Snyder, Michael P.; Thomas, Paul M.; Uhlen, Mathias; Van Eyk, Jennifer E.; Vidal, Marc; Walt, David R.; White, Forest M.; Williams, Evan R.; Wohlschlager, Therese; Wysocki, Vicki H.; Yates, Nathan A.; Young, Nicolas L.; Zhang, BingNature Chemical Biology (2018), 14 (3), 206-214CODEN: NCBABT; ISSN:1552-4450. (Nature Research)A review. Despite decades of accumulated knowledge about proteins and their posttranslational modifications (PTMs), numerous questions remain regarding their mol. compn. and biol. function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, the authors outline what the authors know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, the authors examine prevailing notions about the no. of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. The authors frame central issues regarding detn. of protein-level variation and PTMs, including some paradoxes present in the field today. The authors use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes". The authors also explore prospects for improving measurements to better regularize protein-level biol. and efficiently assoc. PTMs to function and phenotype.
- 5Brüning, F.; Noya, S. B.; Bange, T.; Koutsouli, S.; Rudolph, J. D.; Tyagarajan, S. K.; Cox, J.; Mann, M.; Brown, S. A.; Robles, M. S. Sleep-Wake Cycles Drive Daily Dynamics of Synaptic Phosphorylation. Science 2019, 366, eaav3617 DOI: 10.1126/science.aav3617There is no corresponding record for this reference.
- 6Truttmann, M. C.; Pincus, D.; Ploegh, H. L. Chaperone AMPylation Modulates Aggregation and Toxicity of Neurodegenerative Disease-Associated Polypeptides. Proc. Natl. Acad. Sci. U.S.A. 2018, 115, 201801989 DOI: 10.1073/pnas.1801989115There is no corresponding record for this reference.
- 7Rogowski, K.; van Dijk, J.; Magiera, M. M.; Bosc, C.; Deloulme, J.-C.; Bosson, A.; Peris, L.; Gold, N. D.; Lacroix, B.; Grau, M. B.; Bec, N.; Larroque, C.; Desagher, S.; Holzer, M.; Andrieux, A.; Moutin, M.-J.; Janke, C. A Family of Protein-Deglutamylating Enzymes Associated with Neurodegeneration. Cell 2010, 143, 564– 578, DOI: 10.1016/j.cell.2010.10.0147https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVahtbvN&md5=b6bf533a3dbf587ae1097bbac7284b30A Family of Protein-Deglutamylating Enzymes Associated with NeurodegenerationRogowski, Krzysztof; van Dijk, Juliette; Magiera, Maria M.; Bosc, Christophe; Deloulme, Jean-Christophe; Bosson, Anouk; Peris, Leticia; Gold, Nicholas D.; Lacroix, Benjamin; Grau, Montserrat Bosch; Bec, Nicole; Larroque, Christian; Desagher, Solange; Holzer, Max; Andrieux, Annie; Moutin, Marie-Jo; Janke, CarstenCell (Cambridge, MA, United States) (2010), 143 (4), 564-578CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Polyglutamylation is a posttranslational modification that generates glutamate side chains on tubulins and other proteins. Although this modification has been shown to be reversible, little is known about the enzymes catalyzing deglutamylation. Here we describe the enzymic mechanism of protein deglutamylation by members of the cytosolic carboxypeptidase (CCP) family. Three enzymes (CCP1, CCP4, and CCP6) catalyze the shortening of polyglutamate chains and a fourth (CCP5) specifically removes the branching point glutamates. In addn., CCP1, CCP4, and CCP6 also remove gene-encoded glutamates from the carboxyl termini of proteins. Accordingly, we show that these enzymes convert detyrosinated tubulin into Δ2-tubulin and also modify other substrates, including myosin light chain kinase 1. We further analyze Purkinje cell degeneration (pcd) mice that lack functional CCP1 and show that microtubule hyperglutamylation is directly linked to neurodegeneration. Taken together, our results reveal that controlling the length of the polyglutamate side chains on tubulin is crit. for neuronal survival.
- 8Hoch, N. C.; Polo, L. M. ADP-Ribosylation: From Molecular Mechanisms to Human Disease. Genet. Mol. Biol. 2020, 43, e20190075 DOI: 10.1590/1678-4685-gmb-2019-00758https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlamsLvP&md5=5f55dfa4e564a5fb84ee2e8d2613f968ADP-ribosylation: from molecular mechanisms to human diseaseHoch, Nicolas C.; Polo, Luis M.Genetics and Molecular Biology (2020), 43 (1Suppl.1), e20190075CODEN: GMBIFG; ISSN:1415-4757. (Sociedade Brasileira de Genetica)Post-translational modification of proteins by ADP-ribosylation, catalyzed by poly (ADP-ribose) polymerases (PARPs) using NAD+ as a substrate, plays central roles in DNA damage signalling and repair, modulates a range of cellular signalling cascades and initiates programmed cell death by parthanatos. Here, we present mechanistic aspects of ADP-ribose modification, PARP activation and the cellular functions of ADP-ribose signalling, and discuss how this knowledge is uncovering therapeutic avenues for the treatment of increasingly prevalent human diseases such as cancer, ischemic damage and neurodegeneration.
- 9Kam, T.-I.; Mao, X.; Park, H.; Chou, S.-C.; Karuppagounder, S. S.; Umanah, G.; Yun, S.; Brahmachari, S.; Panicker, N.; Chen, R.; Andrabi, S. A.; Qi, C.; Poirier, G. G.; Pletnikova, O.; Troncoso, J. C.; Bekris, L. M.; Leverenz, J. B.; Pantelyat, A.; Ko, H.; Rosenthal, L. S.; Dawson, T. M.; Dawson, V. L. Poly(ADP-Ribose) Drives Pathologic α-Synuclein Neurodegeneration in Parkinson’s Disease. Science 2018, 362, eaat8407 DOI: 10.1126/science.aat8407There is no corresponding record for this reference.
- 10Smith, L. M.; Thomas, P. M.; Shortreed, M. R.; Schaffer, L. V.; Fellers, R. T.; LeDuc, R. D.; Tucholski, T.; Ge, Y.; Agar, J. N.; Anderson, L. C.; Chamot-Rooke, J.; Gault, J.; Loo, J. A.; Paša-Tolić, L.; Robinson, C. V.; Schlüter, H.; Tsybin, Y. O.; Vilaseca, M.; Vizcaíno, J. A.; Danis, P. O.; Kelleher, N. L. A Five-Level Classification System for Proteoform Identifications. Nat. Method 2019, 16, 939– 940, DOI: 10.1038/s41592-019-0573-x10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1Citb7L&md5=1a45637ecf9d919d47da51c1993c392bA five-level classification system for proteoform identificationsSmith, Lloyd M.; Thomas, Paul M.; Shortreed, Michael R.; Schaffer, Leah V.; Fellers, Ryan T.; LeDuc, Richard D.; Tucholski, Trisha; Ge, Ying; Agar, Jeffrey N.; Anderson, Lissa C.; Chamot-Rooke, Julia; Gault, Joseph; Loo, Joseph A.; Pasa-Tolic, Ljiljana; Robinson, Carol V.; Schluter, Hartmut; Tsybin, Yury O.; Vilaseca, Marta; Vizcaino, Juan Antonio; Danis, Paul O.; Kelleher, Neil L.Nature Methods (2019), 16 (10), 939-940CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)There is no expanded citation for this reference.
- 11Smith, L. M.; Agar, J. N.; Chamot-Rooke, J.; Danis, P. O.; Ge, Y.; Loo, J. A.; Paša-Tolić, L.; Tsybin, Y. O.; Kelleher, N. L.; The Consortium for Top-Down Proteomics The Human Proteoform Project: Defining the Human Proteome. Sci. Adv. 2021, 7, eabk0734 DOI: 10.1126/sciadv.abk0734There is no corresponding record for this reference.
- 12Laughlin, S. T.; Bertozzi, C. R. Metabolic Labeling of Glycans with Azido Sugars and Subsequent Glycan-Profiling and Visualization via Staudinger Ligation. Nat. Protoc. 2007, 2, 2930– 2944, DOI: 10.1038/nprot.2007.42212https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlSlsr%252FJ&md5=cc811cc5d3612b077dc95fde283fcc1eMetabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger ligationLaughlin, Scott T.; Bertozzi, Carolyn R.Nature Protocols (2007), 2 (11), 2930-2944CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Metabolic labeling of glycans with a bioorthogonal chem. reporter such as the azide enables their visualization in cells and organisms as well as the enrichment of specific glycoprotein types for proteomic anal. This process involves two steps. Azido sugars are fed to cells or organisms and integrated by the glycan biosynthetic machinery into various glycoconjugates. The azido sugars are then covalently tagged with imaging probes or epitope tags, either ex vivo or in vivo, using an azide-specific reaction. This protocol details the syntheses of the azido sugars N-azidoacetylmannosamine (ManNAz), N-azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine (GlcNAz) and 6-azidofucose (6AzFuc), and the detection reagents phosphine-FLAG and phosphine-FLAG-His6. Applications to the visualization of cellular glycans and enrichment of glycoproteins for proteomic anal. are described. The synthesis of the azido sugars (ManNAz, GalNAz, GlcNAz or 6AzFuc) or detection reagents (phosphine-FLAG or phosphine-FLAG-His6) can be completed in approx. 1 wk. A cell metabolic labeling expt. can be completed in approx. 4 d.
- 13Kallemeijn, W. W.; Lanyon-Hogg, T.; Panyain, N.; Grocin, A. G.; Ciepla, P.; Morales-Sanfrutos, J.; Tate, E. W. Proteome-Wide Analysis of Protein Lipidation Using Chemical Probes: In-Gel Fluorescence Visualization, Identification and Quantification of N-Myristoylation, N- and S-Acylation, O-Cholesterylation, S-Farnesylation and S-Geranylgeranylation. Nat. Protoc. 2021, 16, 5083– 5122, DOI: 10.1038/s41596-021-00601-613https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXisVSht7bI&md5=879b68d06e6accf4fa072a36791b562cProteome-wide analysis of protein lipidation using chemical probes: in-gel fluorescence visualization, identification and quantification of N-myristoylation, N- and S-acylation, O-cholesterylation, S-farnesylation and S-geranylgeranylationKallemeijn, Wouter W.; Lanyon-Hogg, Thomas; Panyain, Nattawadee; Goya Grocin, Andrea; Ciepla, Paulina; Morales-Sanfrutos, Julia; Tate, Edward W.Nature Protocols (2021), 16 (11), 5083-5122CODEN: NPARDW; ISSN:1750-2799. (Nature Portfolio)Protein lipidation is one of the most widespread post-translational modifications (PTMs) found in nature, regulating protein function, structure and subcellular localization. Lipid transferases and their substrate proteins are also attracting increasing interest as drug targets because of their dysregulation in many disease states. However, the inherent hydrophobicity and potential dynamic nature of lipid modifications makes them notoriously challenging to detect by many anal. methods. Chem. proteomics provides a powerful approach to identify and quantify these diverse protein modifications by combining bespoke chem. tools for lipidated protein enrichment with quant. mass spectrometry-based proteomics. Here, we report a robust and proteome-wide approach for the exploration of five major classes of protein lipidation in living cells, through the use of specific chem. probes for each lipid PTM. In-cell labeling of lipidated proteins is achieved by the metabolic incorporation of a lipid probe that mimics the specific natural lipid, concomitantly wielding an alkyne as a bio-orthogonal labeling tag. After incorporation, the chem. tagged proteins can be coupled to multifunctional capture reagents by using click chem., allowing in-gel fluorescence visualization or enrichment via affinity handles for quant. chem. proteomics based on label-free quantification (LFQ) or tandem mass-tag (TMT) approaches. In this protocol, we describe the application of lipid probes for N-myristoylation, N- and S-acylation, O-cholesterylation, S-farnesylation and S-geranylgeranylation in multiple cell lines to illustrate both the workflow and data obtained in these expts. We provide detailed workflows for method optimization, sample prepn. for chem. proteomics and data processing. A properly trained researcher (e.g., technician, graduate student or postdoc) can complete all steps from optimizing metabolic labeling to data processing within 3 wk. This protocol enables sensitive and quant. anal. of lipidated proteins at a proteome-wide scale at native expression levels, which is crit. to understanding the role of lipid PTMs in health and disease.
- 14Martin, B. R.; Wang, C.; Adibekian, A.; Tully, S. E.; Cravatt, B. F. Global Profiling of Dynamic Protein Palmitoylation. Nat. Method 2012, 9, 84– 89, DOI: 10.1038/nmeth.176914https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVagtL3F&md5=d2e067d1896d6cfb00f87a2d2c06c9d8Global profiling of dynamic protein palmitoylationMartin, Brent R.; Wang, Chu; Adibekian, Alexander; Tully, Sarah E.; Cravatt, Benjamin F.Nature Methods (2012), 9 (1), 84-89CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The reversible thioester linkage of palmitic acid on cysteines, known as protein S-palmitoylation, facilitates the membrane assocn. and proper subcellular localization of proteins. Here we report the metabolic incorporation of the palmitic acid analog 17-octadecynoic acid (17-ODYA) in combination with stable-isotope labeling with amino acids in cell culture (SILAC) and pulse-chase methods to generate a global quant. map of dynamic protein palmitoylation events in cells. We distinguished stably palmitoylated proteins from those that turn over rapidly. Treatment with a serine lipase-selective inhibitor identified a pool of dynamically palmitoylated proteins regulated by palmitoyl-protein thioesterases. This subset was enriched in oncoproteins and other proteins linked to aberrant cell growth, migration and cancer. Our method provides a straightforward way to characterize global palmitoylation dynamics in cells and confirms enzyme-mediated depalmitoylation as a crit. regulatory mechanism for a specific subset of rapidly cycling palmitoylated proteins.
- 15Parker, C. G.; Pratt, M. R. Click Chemistry in Proteomic Investigations. Cell 2020, 180, 605– 632, DOI: 10.1016/j.cell.2020.01.02515https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjsVOlsr8%253D&md5=5960886d7bd84bf58863f59f75de62a7Click Chemistry in Proteomic InvestigationsParker, Christopher G.; Pratt, Matthew R.Cell (Cambridge, MA, United States) (2020), 180 (4), 605-632CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A review. Despite advances in genetic and proteomic techniques, a complete portrait of the proteome and its complement of dynamic interactions and modifications remains a lofty, and as of yet, unrealized, objective. Specifically, traditional biol. and anal. approaches have not been able to address key questions relating to the interactions of proteins with small mols., including drugs, drug candidates, metabolites, or protein post-translational modifications (PTMs). Fortunately, chemists have bridged this exptl. gap through the creation of bioorthogonal reactions. These reactions allow for the incorporation of chem. groups with highly selective reactivity into small mols. or protein modifications without perturbing their biol. function, enabling the selective installation of an anal. tag for downstream investigations. The introduction of chem. strategies to parse and enrich subsets of the "functional" proteome has empowered mass spectrometry (MS)-based methods to delve more deeply and precisely into the biochem. state of cells and its perturbations by small mols. In this Primer, we discuss how one of the most versatile bioorthogonal reactions, "click chem.", has been exploited to overcome limitations of biol. approaches to enable the selective marking and functional investigation of crit. protein-small-mol. interactions and PTMs in native biol. environments.
- 16Kielkowski, P.; Buchsbaum, I. Y.; Becker, T.; Bach, K.; Cappello, S.; Sieber, S. A. A Pronucleotide Probe for Live-Cell Imaging of Protein AMPylation. ChemBioChem 2020, 21, 1285– 1287, DOI: 10.1002/cbic.20190071616https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1yis7c%253D&md5=381685eedb002cfdfd5ad0f86a942349A Pronucleotide Probe for Live-Cell Imaging of Protein AMPylationKielkowski, Pavel; Buchsbaum, Isabel Y.; Becker, Tobias; Bach, Kathrin; Cappello, Silvia; Sieber, Stephan A.ChemBioChem (2020), 21 (9), 1285-1287CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Conjugation of proteins to AMP (AMPylation) is a prevalent post-translational modification (PTM) in human cells, involved in the regulation of unfolded protein response and neural development. Here we present a tailored pronucleotide probe suitable for in situ imaging and chem. proteomics profiling of AMPylated proteins. Using straightforward strain-promoted azide-alkyne click chem., the probe provides stable fluorescence labeling in living cells.
- 17Kielkowski, P.; Buchsbaum, I. Y.; Kirsch, V. C.; Bach, N. C.; Drukker, M.; Cappello, S.; Sieber, S. A. FICD Activity and AMPylation Remodelling Modulate Human Neurogenesis. Nat. Commun. 2020, 11, 517 DOI: 10.1038/s41467-019-14235-617https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXksFanurs%253D&md5=a1eddc8f5fa51409da12fe58890cb2cdFICD activity and AMPylation remodelling modulate human neurogenesisKielkowski, Pavel; Buchsbaum, Isabel Y.; Kirsch, Volker C.; Bach, Nina C.; Drukker, Micha; Cappello, Silvia; Sieber, Stephan A.Nature Communications (2020), 11 (1), 517CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Posttranslational modification (PTM) of proteins represents an important cellular mechanism for controlling diverse functions such as signaling, localization or protein-protein interactions. AMPylation (also termed adenylylation) has recently been discovered as a prevalent PTM for regulating protein activity. In human cells AMPylation has been exclusively studied with the FICD protein. Here we investigate the role of AMPylation in human neurogenesis by introducing a cell-permeable propargyl adenosine pronucleotide probe to infiltrate cellular AMPylation pathways and report distinct modifications in intact cancer cell lines, human-derived stem cells, neural progenitor cells (NPCs), neurons and cerebral organoids (COs) via LC-MS/MS as well as imaging methods. A total of 162 AMP modified proteins were identified. FICD-dependent AMPylation remodeling accelerates differentiation of neural progenitor cells into mature neurons in COs, demonstrating a so far unknown trigger of human neurogenesis.
- 18Sinha, A.; Mann, M. A Beginner’s Guide to Mass Spectrometry–Based Proteomics. Biochemist 2020, 42, 64– 69, DOI: 10.1042/bio2020005718https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitlalsrfM&md5=d2b1b3b39f4d66e6a1b90d6e4f360787A beginner's guide to mass spectrometry-based proteomicsSinha, Ankit; Mann, MatthiasBiochemist (2020), 42 (5), 64-69CODEN: BCHMFZ; ISSN:1740-1194. (Portland Press Ltd.)Mass spectrometry (MS)-based proteomics is the most comprehensive approach for the quant. profiling of proteins, their interactions and modifications. It is a challenging topic as a firm grasp requires expertise in biochem. for sample prepn., anal. chem. for instrumentation and computational biol. for data anal. In this short guide, we highlight the various components of a mass spectrometer, the sample prepn. process for conversion of proteins into peptides, and quantification and anal. strategies. The advancing technol. of MS-based proteomics now opens up opportunities in clin. applications and single-cell anal.
- 19Cox, J.; Mann, M. MaxQuant Enables High Peptide Identification Rates, Individualized p.p.b.-Range Mass Accuracies and Proteome-Wide Protein Quantification. Nat. Biotechol. 2008, 26, 1367– 1372, DOI: 10.1038/nbt.151119https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVWjtLzJ&md5=675d31ca84e3a7e4fb9bdd601d8075eaMaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantificationCox, Juergen; Mann, MatthiasNature Biotechnology (2008), 26 (12), 1367-1372CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Efficient anal. of very large amts. of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resoln., quant. MS data. Using correlation anal. and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over std. techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome expt. and allows statistically robust identification and quantification of >4000 proteins in mammalian cell lysates.
- 20Tyanova, S.; Temu, T.; Sinitcyn, P.; Carlson, A.; Hein, M. Y.; Geiger, T.; Mann, M.; Cox, J. The Perseus Computational Platform for Comprehensive Analysis of (Prote)Omics Data. Nat. Method 2016, 13, 731– 740, DOI: 10.1038/nmeth.390120https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVKntbnN&md5=f8c3e2876e4d724518054bb1a2d1e6eeThe Perseus computational platform for comprehensive analysis of (prote)omics dataTyanova, Stefka; Temu, Tikira; Sinitcyn, Pavel; Carlson, Arthur; Hein, Marco Y.; Geiger, Tamar; Mann, Matthias; Cox, JuergenNature Methods (2016), 13 (9), 731-740CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A main bottleneck in proteomics is the downstream biol. anal. of highly multivariate quant. protein abundance data generated using mass-spectrometry-based anal. We developed the Perseus software platform (http://www.perseus-framework.org) to support biol. and biomedical researchers in interpreting protein quantification, interaction and post-translational modification data. Perseus contains a comprehensive portfolio of statistical tools for high-dimensional omics data anal. covering normalization, pattern recognition, time-series anal., cross-omics comparisons and multiple-hypothesis testing. A machine learning module supports the classification and validation of patient groups for diagnosis and prognosis, and it also detects predictive protein signatures. Central to Perseus is a user-friendly, interactive workflow environment that provides complete documentation of computational methods used in a publication. All activities in Perseus are realized as plugins, and users can extend the software by programming their own, which can be shared through a plugin store. We anticipate that Perseus's arsenal of algorithms and its intuitive usability will empower interdisciplinary anal. of complex large data sets.
- 21Yu, F.; Teo, G. C.; Kong, A. T.; Haynes, S. E.; Avtonomov, D. M.; Geiszler, D. J.; Nesvizhskii, A. I. Identification of Modified Peptides Using Localization-Aware Open Search. Nat. Commun. 2020, 11, 4065 DOI: 10.1038/s41467-020-17921-y21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1Wrtr7J&md5=f15cad0240097b55bd077ad641788807Identification of modified peptides using localization-aware open searchYu, Fengchao; Teo, Guo Ci; Kong, Andy T.; Haynes, Sarah E.; Avtonomov, Dmitry M.; Geiszler, Daniel J.; Nesvizhskii, Alexey I.Nature Communications (2020), 11 (1), 4065CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Abstr.: Identification of post-translationally or chem. modified peptides in mass spectrometry-based proteomics expts. is a crucial yet challenging task. We have recently introduced a fragment ion indexing method and the MSFragger search engine to empower an open search strategy for comprehensive anal. of modified peptides. However, this strategy does not consider fragment ions shifted by unknown modifications, preventing modification localization and limiting the sensitivity of the search. Here we present a localization-aware open search method, in which both modification-contg. (shifted) and regular fragment ions are indexed and used in scoring. We also implement a fast mass calibration and optimization method, allowing optimization of the mass tolerances and other key search parameters. We demonstrate that MSFragger with mass calibration and localization-aware open search identifies modified peptides with significantly higher sensitivity and accuracy. Comparing MSFragger to other modification-focused tools (pFind3, MetaMorpheus, and TagGraph) shows that MSFragger remains an excellent option for fast, comprehensive, and sensitive searches for modified peptides in shotgun proteomics data.
- 22Grammel, M.; Luong, P.; Orth, K.; Hang, H. C. A Chemical Reporter for Protein AMPylation. J. Am. Chem. Soc. 2011, 133, 17103– 17105, DOI: 10.1021/ja205137d22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1ymsr7F&md5=9e83c16371afb9fba2a17d5ea4a763ebA Chemical Reporter for Protein AMPylationGrammel, Markus; Luong, Phi; Orth, Kim; Hang, Howard C.Journal of the American Chemical Society (2011), 133 (43), 17103-17105CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein AMPylation is an emerging post-translational modification, which plays key roles in bacterial pathogenesis and cell biol. Enzymes with AMPylation activity, referred to as AMPylators, have been identified in several bacterial pathogens and eukaryotes. To facilitate the study of this unique modification, the authors developed an alkynyl chem. reporter for detection and identification of protein AMPylation substrates. Covalent functionalization of AMPylation substrates with the alkynyl reporter in lieu of adenylyl 5'-monophosphate (AMP) allows their subsequent bioorthogonal ligation with azide-fluorescent dyes or affinity enrichment tags. This chem. reporter is transferred by a range of AMPylators onto their cognate protein substrates and allows rapid detection and identification of AMPylated substrates.
- 23Kliza, K. W.; Liu, Q.; Roosenboom, L. W. M.; Jansen, P. W. T. C.; Filippov, D. V.; Vermeulen, M. Reading ADP-Ribosylation Signaling Using Chemical Biology and Interaction Proteomics. Mol. Cell 2021, 81, 4552.e8– 4567.e8, DOI: 10.1016/j.molcel.2021.08.037There is no corresponding record for this reference.
- 24Yang, Y.-Y.; Ascano, J. M.; Hang, H. C. Bioorthogonal Chemical Reporters for Monitoring Protein Acetylation. J. Am. Chem. Soc. 2010, 132, 3640– 3641, DOI: 10.1021/ja908871t24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXisFGltrw%253D&md5=e7584db37fc5210158c281f7f85e23cbBioorthogonal Chemical Reporters for Monitoring Protein AcetylationYang, Yu-Ying; Ascano, Janice M.; Hang, Howard C.Journal of the American Chemical Society (2010), 132 (11), 3640-3641CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Protein acetylation is a key post-translational modification that regulates diverse biol. activities in eukaryotes. Bioorthogonal chem. reporters that enable direct in-gel fluorescent visualization and proteome-wide identification of acetylated proteins via copper-catalyzed azide-alkyne cycloaddn. are prepd. 3-Butynoyl, 4-pentynoyl, and 5-hexynoyl-CoA thioesters are prepd. as alkyne-contg. acetyl-CoA analogs; the sodium salts of 3-butynoic, 4-pentynoic, and 5-hexynoic acids are prepd. as alkyne-contg. acetate analogs. 4-Pentynoyl-CoA and 5-hexynoyl-CoA function as efficient substrates of the lysine acetyltransferase p300 and serve as sensitive reagents for monitoring p300-catalyzed protein acetylation in vitro. Sodium 3-butynoate, sodium 4-pentynoate, and sodium 5-hexynoate are metabolically incorporated onto cellular proteins through biosynthetic mechanisms for profiling of acetylated proteins in diverse cell types. Mass spectrometric anal. of the enriched 4-pentynoate-labeled proteins revealed many reported acetylated proteins as well as new candidate acetylated proteins from Jurkat T cells and also specific sites of lysine acetylation.
- 25Sieber, S. A.; Cappello, S.; Kielkowski, P. From Young to Old: AMPylation Hits the Brain. Cell. Chem. Biol. 2020, 27, 773– 779, DOI: 10.1016/j.chembiol.2020.05.00925https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtFGhs7fJ&md5=eeaffb75cfd1779ad287f0971bb51e95From Young to Old: AMPylation Hits the BrainSieber, Stephan A.; Cappello, Silvia; Kielkowski, PavelCell Chemical Biology (2020), 27 (7), 773-779CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)Protein post-translational modifications (PTMs) are implicated in numerous physiol. processes and significantly contribute to complex regulatory networks of protein functions. Recently, a protein PTM called AMPylation was found to play a role in modulation of neurodevelopment and neurodegeneration. Combination of biochem. and chem. proteomic studies has uncovered the prevalence of this PTM in regulation of diverse metabolic pathways. In metazoans, thus far two protein AMP transferases have been identified to introduce AMPylation: FICD and SELO. These two proteins were found to be involved in unfolded protein response and redox homeostasis on the cellular level and in the case of FICD to adjust the development of glial cells and neurons in Drosophila and cerebral organoids, resp. Together with findings on AMPylation and its assocn. with toxic protein aggregation, we summarize in this Perspective the knowledge and putative future directions of protein AMPylation research.
- 26Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. A Strain-Promoted [3 + 2] Azide–Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems. J. Am. Chem. Soc. 2004, 126, 15046– 15047, DOI: 10.1021/ja044996f26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXpt1Sks7s%253D&md5=37af3dbaa89ae4cffaba2dee30e50ec0A strain-promoted [3+2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systemsAgard, Nicholas J.; Prescher, Jennifer A.; Bertozzi, Carolyn R.Journal of the American Chemical Society (2004), 126 (46), 15046-15047CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Selective chem. reactions that are orthogonal to the diverse functionality of biol. systems have become important tools in the field of chem. biol. Two notable examples are the Staudinger ligation of azides and phosphines and the Cu(I)-catalyzed [3+2] cycloaddn. of azides and alkynes ("click chem."). The Staudinger ligation has sufficient biocompatibility for performance in living animals but suffers from phosphine oxidn. and synthetic challenges. Click chem. obviates the requirement of phosphines, but the Cu(I) catalyst is toxic to cells, thereby precluding in vivo applications. Here we present a strain-promoted [3+2] cycloaddn. between cyclooctynes and azides that proceeds under physiol. conditions without the need for a catalyst. The utility of the reaction was demonstrated by selective modification of biomols. in vitro and on living cells, with no apparent toxicity.
- 27Zecha, J.; Satpathy, S.; Kanashova, T.; Avanessian, S. C.; Kane, M. H.; Clauser, K. R.; Mertins, P.; Carr, S. A.; Kuster, B. TMT Labeling for the Masses: A Robust and Cost-Efficient, In-Solution Labeling Approach* [S]. Mol. Cell. Proteomics 2019, 18, 1468– 1478, DOI: 10.1074/mcp.tir119.00138527https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvFWms7fP&md5=230b43b5f8d2584131c63edc7ea87fe7TMT labeling for the masses: a robust and cost-efficient, in-solution labeling approachZecha, Jana; Satpathy, Shankha; Kanashova, Tamara; Avanessian, Shayan C.; Kane, M. Harry; Clauser, Karl R.; Mertins, Philipp; Carr, Steven A.; Kuster, BernhardMolecular & Cellular Proteomics (2019), 18 (7), 1468-1478CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)Isobaric stable isotope labeling using, for example, tandem mass tags (TMTs) is increasingly being applied for large-scale proteomic studies. Expts. focusing on proteoform anal. in drug time course or perturbation studies or in large patient cohorts greatly benefit from the reproducible quantification of single peptides across samples. However, such studies often require labeling of hundreds of micrograms of peptides such that the cost for labeling reagents represents a major contribution to the overall cost of an expt. Here, we describe and evaluate a robust and cost-effective protocol for TMT labeling that reduces the quantity of required labeling reagent by a factor of eight and achieves complete labeling. Under- and overlabeling of peptides derived from complex digests of tissues and cell lines were systematically evaluated using peptide quantities of between 12.5 and 800μg and TMT-to-peptide ratios (wt/wt) ranging from 8:1 to 1:2 at different TMT and peptide concns. When reaction vols. were reduced to maintain TMT and peptide concns. of at least 10 mM and 2 g/l, resp., TMT-to-peptide ratios as low as 1:1 (wt/wt) resulted in labeling efficiencies of > 99% and excellent intra- and interlab. reproducibility. The utility of the optimized protocol was further demonstrated in a deepscale proteome and phosphoproteome anal. of patientderived xenograft tumor tissue benchmarked against the labeling procedure recommended by the TMT vendor. Finally, we discuss the impact of labeling reaction parameters for N-hydroxysuccinimide ester-based chem. and provide guidance on adopting efficient labeling protocols for different peptide quantities.
- 28Cox, J.; Hein, M. Y.; Luber, C. A.; Paron, I.; Nagaraj, N.; Mann, M. Accurate Proteome-Wide Label-Free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQ. Mol. Cell. Proteomics 2014, 13, 2513– 2526, DOI: 10.1074/mcp.M113.03159128https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsVynurrI&md5=f3f1c7dc8fbf729c568446968b89f37cAccurate Proteome-wide Label-free Quantification by Delayed Normalization and Maximal Peptide Ratio Extraction, Termed MaxLFQCox, Juergen; Hein, Marco Y.; Luber, Christian A.; Paron, Igor; Nagaraj, Nagarjuna; Mann, MatthiasMolecular & Cellular Proteomics (2014), 13 (9), 2513-2526CODEN: MCPOBS; ISSN:1535-9484. (American Society for Biochemistry and Molecular Biology)Protein quantification without isotopic labels has been a long-standing interest in the proteomics field. However, accurate and robust proteome-wide quantification with label-free approaches remains a challenge. We developed a new intensity detn. and normalization procedure called MaxLFQ that is fully compatible with any peptide or protein sepn. prior to LC-MS anal. Protein abundance profiles are assembled using the max. possible information from MS signals, given that the presence of quantifiable peptides varies from sample to sample. For a benchmark dataset with two proteomes mixed at known ratios, we accurately detected the mixing ratio over the entire protein expression range, with greater precision for abundant proteins. The significance of individual label-free quantifications was obtained via a t test approach. For a second benchmark dataset, we accurately quantify fold changes over several orders of magnitude, a task that is challenging with label-based methods. MaxLFQ is a generic label-free quantification technol. that is readily applicable to many biol. questions; it is compatible with std. statistical anal. workflows, and it has been validated in many and diverse biol. projects. Our algorithms can handle very large expts. of 500+ samples in a manageable computing time. It is implemented in the freely available MaxQuant computational proteomics platform and works completely seamlessly at the click of a button.
- 29Wiśniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M. Universal Sample Preparation Method for Proteome Analysis. Nat. Method 2009, 6, 359– 362, DOI: 10.1038/nmeth.132229https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXks12ksb0%253D&md5=f34cb14143462984852497dc1f9ee5c2Universal sample preparation method for proteome analysisWisniewski, Jacek R.; Zougman, Alexandre; Nagaraj, Nagarjuna; Mann, MatthiasNature Methods (2009), 6 (5), 359-362CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)The authors describe a method, filter-aided sample prepn. (FASP), which combines the advantages of in-gel and in-soln. digestion for mass spectrometry-based proteomics. The authors completely solubilized the proteome in SDS, which the authors then exchanged by urea on a std. filtration device. Peptides eluted after digestion on the filter were pure, allowing single-run analyses of organelles and an unprecedented depth of proteome coverage.
- 30Müller, T.; Kalxdorf, M.; Longuespée, R.; Kazdal, D. N.; Stenzinger, A.; Krijgsveld, J. Automated Sample Preparation with SP3 for Low-input Clinical Proteomics. Mol. Syst. Biol. 2020, 16, e9111 DOI: 10.15252/msb.2019911130https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhvFOis7o%253D&md5=5bf4201cc5b52478697c68c60cae6ee0Automated sample preparation with SP3 for low-input clinical proteomicsMueller, Torsten; Kalxdorf, Mathias; Longuespee, Remi; Kazdal, Daniel N.; Stenzinger, Albrecht; Krijgsveld, JeroenMolecular Systems Biology (2020), 16 (1), e9111CODEN: MSBOC3; ISSN:1744-4292. (Wiley-VCH Verlag GmbH & Co. KGaA)High-throughput and streamlined workflows are essential in clin. proteomics for standardized processing of samples from a variety of sources, including fresh-frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single-pot solid-phase-enhanced sample prepn. (SP3) on a liq. handling robot for automated processing (autoSP3) of tissue lysates in a 96-well format. AutoSP3 performs unbiased protein purifn. and digestion, and delivers peptides that can be directly analyzed by LCMS, thereby significantly reducing hands-on time, reducing variability in protein quantification, and improving longitudinal reproducibility. We demonstrate the distinguishing ability of autoSP3 to process low-input samples, reproducibly quantifying 500-1,000 proteins from 100 to 1,000 cells. Furthermore, we applied this approach to a cohort of clin. FFPE pulmonary adenocarcinoma (ADC) samples and recapitulated their sepn. into known histol. growth patterns. Finally, we integrated autoSP3 with AFA ultrasonication for the automated end-to-end sample prepn. and LCMS anal. of 96 intact tissue samples. Collectively, this constitutes a generic, scalable, and cost-effective workflow with minimal manual intervention, enabling reproducible tissue proteomics in a broad range of clin. and non-clin. applications.
- 31Hughes, C. S.; Moggridge, S.; Müller, T.; Sorensen, P. H.; Morin, G. B.; Krijgsveld, J. Single-Pot, Solid-Phase-Enhanced Sample Preparation for Proteomics Experiments. Nat. Protoc. 2019, 14, 68– 85, DOI: 10.1038/s41596-018-0082-x31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXit1yltrzK&md5=d80343142c71d0b55bcd35978e5db19dSingle-pot, solid-phase-enhanced sample preparation for proteomics experimentsHughes, Christopher S.; Moggridge, Sophie; Muller, Torsten; Sorensen, Poul H.; Morin, Gregg B.; Krijgsveld, JeroenNature Protocols (2019), 14 (1), 68-85CODEN: NPARDW; ISSN:1750-2799. (Nature Research)A crit. step in proteomics anal. is the optimal extn. and processing of protein material to ensure the highest sensitivity in downstream detection. Achieving this requires a sample-handling technol. that exhibits unbiased protein manipulation, flexibility in reagent use, and virtually lossless processing. Addressing these needs, the single-pot, solid-phase-enhanced sample-prepn. (SP3) technol. is a paramagnetic bead-based approach for rapid, robust, and efficient processing of protein samples for proteomic anal. SP3 uses a hydrophilic interaction mechanism for exchange or removal of components that are commonly used to facilitate cell or tissue lysis, protein solubilization, and enzymic digestion (e.g., detergents, chaotropes, salts, buffers, acids, and solvents) before downstream proteomic anal. The SP3 protocol consists of nonselective protein binding and rinsing steps that are enabled through the use of ethanol-driven solvation capture on the surface of hydrophilic beads, and elution of purified material in aq. conditions. In contrast to alternative approaches, SP3 combines compatibility with a substantial collection of soln. additives with virtually lossless and unbiased recovery of proteins independent of input quantity, all in a simplified single-tube protocol. The SP3 protocol is simple and efficient, and can be easily completed by a std. user in ∼30 min, including reagent prepn. As a result of these properties, SP3 has successfully been used to facilitate examn. of a broad range of sample types spanning simple and complex protein mixts. in large and very small amts., across numerous organisms. This work describes the steps and extensive considerations involved in performing SP3 in bottom-up proteomics, using a simplified protein cleanup scenario for illustration.
- 32Sielaff, M.; Kuharev, J.; Bohn, T.; Hahlbrock, J.; Bopp, T.; Tenzer, S.; Distler, U. Evaluation of FASP, SP3, and IST Protocols for Proteomic Sample Preparation in the Low Microgram Range. J. Proteome Res. 2017, 16, 4060– 4072, DOI: 10.1021/acs.jproteome.7b0043332https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFCrtb%252FP&md5=5f3d5a92a9186e5aaa0633e6c8121a34Evaluation of FASP, SP3, and iST Protocols for Proteomic Sample Preparation in the Low Microgram RangeSielaff, Malte; Kuharev, Joerg; Bohn, Toszka; Hahlbrock, Jennifer; Bopp, Tobias; Tenzer, Stefan; Distler, UteJournal of Proteome Research (2017), 16 (11), 4060-4072CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Efficient and reproducible sample prepn. is a prerequisite for any robust and sensitive quant. bottom-up proteomics workflow. Here, the authors performed an independent comparison between single-pot solid-phase-enhanced sample prepn. (SP3), filter-aided sample prepn. (FASP), and a com. kit based on the in-StageTip (iST) method. The authors assessed their performance for the processing of proteomic samples in the low μg range using varying amts. of HeLa cell lysate (1-20 μg of total protein). All three workflows showed similar performances for 20 μg of starting material. When handling sample sizes below 10 μg, the no. of identified proteins and peptides as well as the quant. reproducibility and precision drastically dropped in case of FASP. In contrast, SP3 and iST provided high proteome coverage even in the low μg range. Even when digesting 1 μg of starting material, both methods still enabled the identification of over 3000 proteins and between 25,000 and 30,000 peptides. On av., the quant. reproducibility between exptl. replicates was slightly higher in case of SP3 (R2 = 0.97 (SP3); R2 = 0.93 (iST)). Applying SP3 toward the characterization of the proteome of FACS-sorted tumor-assocd. macrophages in the B16 tumor model enabled the quantification of 2965 proteins and revealed a "mixed" M1/M2 phenotype.
- 33Wright, M. H.; Clough, B.; Rackham, M. D.; Rangachari, K.; Brannigan, J. A.; Grainger, M.; Moss, D. K.; Bottrill, A. R.; Heal, W. P.; Broncel, M.; Serwa, R. A.; Brady, D.; Mann, D. J.; Leatherbarrow, R. J.; Tewari, R.; Wilkinson, A. J.; Holder, A. A.; Tate, E. W. Validation of N-Myristoyltransferase as an Antimalarial Drug Target Using an Integrated Chemical Biology Approach. Nat. Chem. 2014, 6, 112– 121, DOI: 10.1038/nchem.183033https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFOmtL%252FE&md5=d2fa0f4af88198fc26a53c9a5f853ca1Validation of N-myristoyltransferase as an antimalarial drug target using an integrated chemical biology approachWright, Megan H.; Clough, Barbara; Rackham, Mark D.; Rangachari, Kaveri; Brannigan, James A.; Grainger, Munira; Moss, David K.; Bottrill, Andrew R.; Heal, William P.; Broncel, Malgorzata; Serwa, Remigiusz A.; Brady, Declan; Mann, David J.; Leatherbarrow, Robin J.; Tewari, Rita; Wilkinson, Anthony J.; Holder, Anthony A.; Tate, Edward W.Nature Chemistry (2014), 6 (2), 112-121CODEN: NCAHBB; ISSN:1755-4330. (Nature Publishing Group)Malaria is an infectious disease caused by parasites of the genus Plasmodium, which leads to approx. one million deaths per annum worldwide. Chem. validation of new antimalarial targets is urgently required in view of rising resistance to current drugs. One such putative target is the enzyme N-myristoyltransferase, which catalyzes the attachment of the fatty acid myristate to protein substrates (N-myristoylation). Here, the authors report an integrated chem. biol. approach to explore protein myristoylation in the major human parasite P. falciparum, combining chem. proteomic tools for identification of the myristoylated and glycosylphosphatidylinositol-anchored proteome with selective small-mol. N-mytransferase inhibitors. The authors demonstrate that N-mytransferase is an essential and chem. tractable target in malaria parasites both in vitro and in vivo, and show that selective inhibition of N-myristoylation leads to catastrophic and irreversible failure to assemble the inner membrane complex, a crit. subcellular organelle in the parasite life cycle. The authors' studies provide the basis for the development of new antimalarials targeting N-mytransferase.
- 34Yan, T.; Desai, H. S.; Boatner, L. M.; Yen, S. L.; Cao, J.; Palafox, M. F.; Jami-Alahmadi, Y.; Backus, K. SP3-FAIMS Chemoproteomics for High Coverage Profiling of the Human Cysteinome. ChemBioChem 2021, 22, 1841– 1851, DOI: 10.1002/cbic.20200087034https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXkt1aisr4%253D&md5=f45fdb9a576f4541259cb15519dbeb8bSP3-FAIMS Chemoproteomics for High-Coverage Profiling of the Human Cysteinome**Yan, Tianyang; Desai, Heta S.; Boatner, Lisa M.; Yen, Stephanie L.; Cao, Jian; Palafox, Maria F.; Jami-Alahmadi, Yasaman; Backus, Keriann M.ChemBioChem (2021), 22 (10), 1841-1851CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)Chemoproteomics has enabled the rapid and proteome-wide discovery of functional, redox-sensitive, and ligandable cysteine residues. Despite widespread adoption and considerable advances in both sample-prepn. workflows and MS instrumentation, chemoproteomics expts. still typically only identify a small fraction of all cysteines encoded by the human genome. Here, we develop an optimized sample-prepn. workflow that combines enhanced peptide labeling with single-pot, solid-phase-enhanced sample-prepn. (SP3) to improve the recovery of biotinylated peptides, even from small sample sizes. By combining this improved workflow with online high-field asym. waveform ion mobility spectrometry (FAIMS) sepn. of labeled peptides, we achieve unprecedented coverage of >14000 unique cysteines in a single-shot 70 min expt. Showcasing the wide utility of the SP3-FAIMS chemoproteomic method, we find that it is also compatible with competitive small-mol. screening by isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP). In aggregate, our anal. of 18 samples from seven cell lines identified 34225 unique cysteines using only ∼28 h of instrument time. The comprehensive spectral library and improved coverage provided by the SP3-FAIMS chemoproteomics method will provide the tech. foundation for future studies aimed at deciphering the functions and druggability of the human cysteineome.
- 35Klont, F.; Kwiatkowski, M.; Faiz, A.; van den Bosch, T.; Pouwels, S. D.; Dekker, F. J.; ten Hacken, N. H. T.; Horvatovich, P.; Bischoff, R. Adsorptive Microtiter Plates As Solid Supports in Affinity Purification Workflows. J. Proteome Res. 2021, 20, 5218– 5221, DOI: 10.1021/acs.jproteome.1c0062335https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitlSltrnJ&md5=0152dd555344eac0ff59eea9163a1e40Adsorptive Microtiter Plates As Solid Supports in Affinity Purification WorkflowsKlont, Frank; Kwiatkowski, Marcel; Faiz, Alen; van den Bosch, Thea; Pouwels, Simon D.; Dekker, Frank J.; ten Hacken, Nick H. T.; Horvatovich, Peter; Bischoff, RainerJournal of Proteome Research (2021), 20 (11), 5218-5221CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)Affinity ligands such as antibodies are widely used in (bio)medical research for purifying proteins from complex biol. samples. These ligands are generally immobilized onto solid supports which facilitate the sepn. of a captured protein from the sample matrix. Adsorptive microtiter plates are commonly used as solid supports prior to immunochem. detection (e.g., immunoassays) but hardly ever prior to liq. chromatog.-mass spectrometry (LC-MS-)-based detection. Here, we describe the use of adsorptive microtiter plates for protein enrichment prior to LC-MS detection, and we discuss opportunities and challenges of corresponding workflows, based on examples of targeted (i.e., sol. receptor for advanced glycation end-products (sRAGE) in human serum) and discovery-based workflows (i.e., transcription factor p65 (NF-κB) in lysed murine RAW 264.7 macrophages and peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP5) in lysed human A549 alveolar basal epithelial cells). Thereby, we aim to highlight the potential usefulness of adsorptive microtiter plates in affinity purifn. workflows prior to LC-MS detection, which could increase their usage in mass spectrometry-based protein research.
- 36Makowski, M. M.; Gräwe, C.; Foster, B. M.; Nguyen, N. V.; Bartke, T.; Vermeulen, M. Global Profiling of Protein–DNA and Protein–Nucleosome Binding Affinities Using Quantitative Mass Spectrometry. Nat. Commun. 2018, 9, 1653 DOI: 10.1038/s41467-018-04084-036https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MjmslaqsA%253D%253D&md5=4cfa8d72422fe923a20279d154c5a0ceGlobal profiling of protein-DNA and protein-nucleosome binding affinities using quantitative mass spectrometryMakowski Matthew M; Grawe Cathrin; Vermeulen Michiel; Makowski Matthew M; Grawe Cathrin; Vermeulen Michiel; Foster Benjamin M; Bartke Till; Foster Benjamin M; Nguyen Nhuong V; Bartke Till; Foster Benjamin M; Nguyen Nhuong V; Bartke TillNature communications (2018), 9 (1), 1653 ISSN:.Interaction proteomics studies have provided fundamental insights into multimeric biomolecular assemblies and cell-scale molecular networks. Significant recent developments in mass spectrometry-based interaction proteomics have been fueled by rapid advances in label-free, isotopic, and isobaric quantitation workflows. Here, we report a quantitative protein-DNA and protein-nucleosome binding assay that uses affinity purifications from nuclear extracts coupled with isobaric chemical labeling and mass spectrometry to quantify apparent binding affinities proteome-wide. We use this assay with a variety of DNA and nucleosome baits to quantify apparent binding affinities of monomeric and multimeric transcription factors and chromatin remodeling complexes.
- 37Tian, Y.-P.; Zhang, X.-J.; Wu, J.-Y.; Fun, H.-K.; Jiang, M.-H.; Xu, Z.-Q.; Usman, A.; Chantrapromma, S.; Thompson, L. K. Structural Diversity and Properties of a Series of Dinuclear and Mononuclear Copper(Ii) and Copper(i) Carboxylato Complexes. New J. Chem. 2002, 26, 1468– 1473, DOI: 10.10309/b203334h37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xnt1aiurs%253D&md5=df05eb4d9775770e588acae4427dbefdStructural diversity and properties of a series of dinuclear and mononuclear copper(II) and copper(I) carboxylato complexesTian, Yu-Peng; Zhang, Xuan-Jun; Wu, Jie-Ying; Fun, Hoong-Kun; Jiang, Min-Hua; Xu, Zhi-Qiang; Usman, Anwar; Chantrapromma, Suchada; Thompson, Laurence K.New Journal of Chemistry (2002), 26 (10), 1468-1473CODEN: NJCHE5; ISSN:1144-0546. (Royal Society of Chemistry)The syntheses, crystal structures, magnetic and photoluminescence properties of dinuclear and mononuclear copper(II) and copper(I) N-carbazolylacetate [N-carbazolylacetic acid = Hcabo] with different carboxylato coordination modes are reported. Although the carboxylato group has different coordination modes, the same carboxylate ligand binding to copper ion via four coordinating modes is rare. The crystal structure of [Cu2(Cabo)4(DMF)2]·2DMF (1) consists of a sym. dimeric Cu(II) carboxylato paddle-wheel core and oxygen atoms from DMF at the apical positions. Dinuclear [Cu2(Cabo)3(phen)2]ClO4·H2O·C2H5OH (2) (phen = 1,10-phenanthroline) consists of an unusual dimeric core with two copper atoms bridged by three carboxylates one of which is in the η:η:μ2 bridging mode and the other two are in the rarer monoat. bridging mode. To the authors' knowledge, the present bridging mode was not reported hitherto. The crystal structures of [Cu(Cabo)2phen] and [Cu(Cabo)(PPh3)2] are also reported. Magnetic susceptibilities were measured in the temp. range 2-300 K paddle-wheel copper(II) ions in 1 are strongly coupled antiferromagnetically with 2J = -356.4(6) cm-1, whereas complex 2 shows weak antiferromagnetic interaction with a 2J value of -12.8(4) cm-1. Copper(I) N-carbazolylacetate with strong fluorescence in the solid state as well as high thermal stability was obtained by redn. of the copper(II) N-carbazolylacetate using PPh3 (triphenylphosphine) in DMF soln.
- 38Stojceva Radovanovic, B. C.; Premovic, P. I. Thermal Behaviour of Cu(II)-Urea Complex. J. Therm. Anal. 1992, 38, 715– 719, DOI: 10.1007/bf0197940138https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38Xls1aqs7g%253D&md5=8ea035b99db690cab65d2539510fbf4aThermal behavior of copper(II)-urea complexStojceva Radovanovic, B. C.; Premovic, P. I.Journal of Thermal Analysis (1992), 38 (4), 715-19CODEN: JTHEA9; ISSN:0368-4466.[CuL4]Cl2 (L = urea) was prepd. and its structure was established by FTIR, ESR, at. absorption spectroscopy, and elemental anal. The thermal behavior of [CuL4]Cl2 was studied by TG, DTA, FTIR, and ESR. The decompn. of the complex occurs in 4 stages of wt. loss of different intermediates followed by 3 endothermal effects. The complex is thermally stable at ≤428 K. The ESR and FTIR behavior of [CuL4]Cl2 during thermolysis was studied at 428-633 K. In this temp. range the complex decompn. occurred forming thermodynamically stable regions of Cu(II) which are ferromagnetically coupled.
- 39Hughes, C. S.; Foehr, S.; Garfield, D. A.; Furlong, E. E.; Steinmetz, L. M.; Krijgsveld, J. Ultrasensitive Proteome Analysis Using Paramagnetic Bead Technology. Mol. Syst. Biol. 2014, 10, 757, DOI: 10.15252/msb.2014562539https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M3ksF2rsA%253D%253D&md5=0a3dce5610fc3f4b8097c6de64ffb9fdUltrasensitive proteome analysis using paramagnetic bead technologyHughes Christopher S; Foehr Sophia; Garfield David A; Furlong Eileen E; Steinmetz Lars M; Krijgsveld JeroenMolecular systems biology (2014), 10 (), 757 ISSN:.In order to obtain a systems-level understanding of a complex biological system, detailed proteome information is essential. Despite great progress in proteomics technologies, thorough interrogation of the proteome from quantity-limited biological samples is hampered by inefficiencies during processing. To address these challenges, here we introduce a novel protocol using paramagnetic beads, termed Single-Pot Solid-Phase-enhanced Sample Preparation (SP3). SP3 provides a rapid and unbiased means of proteomic sample preparation in a single tube that facilitates ultrasensitive analysis by outperforming existing protocols in terms of efficiency, scalability, speed, throughput, and flexibility. To illustrate these benefits, characterization of 1,000 HeLa cells and single Drosophila embryos is used to establish that SP3 provides an enhanced platform for profiling proteomes derived from sub-microgram amounts of material. These data present a first view of developmental stage-specific proteome dynamics in Drosophila at a single-embryo resolution, permitting characterization of inter-individual expression variation. Together, the findings of this work position SP3 as a superior protocol that facilitates exciting new directions in multiple areas of proteomics ranging from developmental biology to clinical applications.
- 40Truttmann, M. C.; Zheng, X.; Hanke, L.; Damon, J. R.; Grootveld, M.; Krakowiak, J.; Pincus, D.; Ploegh, H. L. Unrestrained AMPylation Targets Cytosolic Chaperones and Activates the Heat Shock Response. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, E152– E160, DOI: 10.1073/pnas.161923411440https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXivFWr&md5=56e77b18492c14dd54d98f0fb23e45c9Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock responseTruttmann, Matthias C.; Zheng, Xu; Hanke, Leo; Damon, Jadyn R.; Grootveld, Monique; Krakowiak, Joanna; Pincus, David; Ploegh, Hidde L.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (2), E152-E160CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Protein AMPylation is a conserved posttranslational modification with emerging roles in endoplasmic reticulum homeostasis. However, the range of substrates and cell biol. consequences of AMPylation remain poorly defined. We expressed human and Caenorhabditis elegans AMPylation enzymes-huntingtin yeast-interacting protein E (HYPE) and filamentation-induced by cAMP (FIC)-1, resp.-in Saccharomyces cerevisiae, a eukaryote that lacks endogenous protein AMPylation. Expression of HYPE and FIC-1 in yeast induced a strong cytoplasmic Hsf1-mediated heat shock response, accompanied by attenuation of protein translation, massive protein aggregation, growth arrest, and lethality. Overexpression of Ssa2, a cytosolic heat shock protein (Hsp)70, was sufficient to partially rescue growth. In human cell lines, overexpression of active HYPE similarly induced protein aggregation and the HSF1-dependent heat shock response. Excessive AMPylation also abolished HSP70-dependent influenza virus replication. Our findings suggest a mode of Hsp70 inactivation by AMPylation and point toward a role for protein AMPylation in the regulation of cellular protein homeostasis beyond the endoplasmic reticulum.
- 41Sanyal, A.; Dutta, S.; Camara, A.; Chandran, A.; Koller, A.; Watson, B. G.; Sengupta, R.; Ysselstein, D.; Montenegro, P.; Cannon, J.; Rochet, J.-C.; Mattoo, S. Alpha-Synuclein Is a Target of Fic-Mediated Adenylylation/AMPylation: Possible Implications for Parkinson’s Disease. J. Mol. Biol. 2019, 431, 2266– 2282, DOI: 10.1016/j.jmb.2019.04.02641https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXovVCnsb4%253D&md5=fd934dd43a0e1ef1c177648e6da8f78aAlpha-synuclein is a target of fic-mediated adenylylation/AMPylation: Possible Implications for Parkinson's DiseaseSanyal, Anwesha; Dutta, Sayan; Camara, Ali; Chandran, Aswathy; Koller, Antonius; Watson, Ben G.; Sengupta, Ranjan; Ysselstein, Daniel; Montenegro, Paola; Cannon, Jason; Rochet, Jean-Christophe; Mattoo, SeemaJournal of Molecular Biology (2019), 431 (12), 2266-2282CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)During disease, cells experience various stresses that manifest as an accumulation of misfolded proteins and eventually lead to cell death. To combat this stress, cells activate a pathway called unfolded protein response that functions to maintain endoplasmic reticulum (ER) homeostasis and dets. cell fate. We recently reported a hitherto unknown mechanism of regulating ER stress via a novel post-translational modification called Fic-mediated adenylylation/AMPylation. Specifically, we showed that the human Fic (filamentation induced by cAMP) protein, HYPE/FicD, catalyzes the addn. of an adenosine monophosphate (AMP) to the ER chaperone, BiP, to alter the cell's unfolded protein response-mediated response to misfolded proteins. Here, we report that we have now identified a second target for HYPE-alpha-synuclein (αSyn), a presynaptic protein involved in Parkinson's disease. Aggregated αSyn has been shown to induce ER stress and elicit neurotoxicity in Parkinson's disease models. We show that HYPE adenylylates αSyn and reduces phenotypes assocd. with αSyn aggregation invitro, suggesting a possible mechanism by which cells cope with αSyn toxicity.
- 42Raught, B.; Gingras, A.-C.; Sonenberg, N. The Target of Rapamycin (TOR) Proteins. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 7037– 7044, DOI: 10.1073/pnas.12114589842https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXkslWmsb4%253D&md5=e6e2182a59a006e21c73ee4dccb941a5The target of rapamycin (TOR) proteinsRaught, Brian; Gingras, Anne-Claude; Sonenberg, NahumProceedings of the National Academy of Sciences of the United States of America (2001), 98 (13), 7037-7044CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A review, with 132 refs. Rapamycin potently inhibits downstream signaling from the target of rapamycin (TOR) proteins. These evolutionarily conserved protein kinases coordinate the balance between protein synthesis and protein degrdn. in response to nutrient quality and quantity. The TOR proteins regulate (i) the initiation and elongation phases of translation, (ii) ribosome biosynthesis, (ii) amino acid import, (iv) the transcription of numerous enzymes involved in multiple metabolic pathways, and (v) autophagy. Intriguingly, recent studies have also suggested that TOR signaling plays a crit. role in brain development, learning, and memory formation.
- 43Leeman, D. S.; Hebestreit, K.; Ruetz, T.; Webb, A. E.; McKay, A.; Pollina, E. A.; Dulken, B. W.; Zhao, X.; Yeo, R. W.; Ho, T. T.; Mahmoudi, S.; Devarajan, K.; Passegué, E.; Rando, T. A.; Frydman, J.; Brunet, A. Lysosome Activation Clears Aggregates and Enhances Quiescent Neural Stem Cell Activation during Aging. Science 2018, 359, 1277– 1283, DOI: 10.1126/science.aag304843https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXkslCjs74%253D&md5=e1b5eb1b30d5eb3a36d37bd8173a250eLysosome activation clears aggregates and enhances quiescent neural stem cell activation during agingLeeman, Dena S.; Hebestreit, Katja; Ruetz, Tyson; Webb, Ashley E.; McKay, Andrew; Pollina, Elizabeth A.; Dulken, Ben W.; Zhao, Xiaoai; Yeo, Robin W.; Ho, Theodore T.; Mahmoudi, Salah; Devarajan, Keerthana; Passegue, Emmanuelle; Rando, Thomas A.; Frydman, Judith; Brunet, AnneScience (Washington, DC, United States) (2018), 359 (6381), 1277-1283CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)In the adult brain, the neural stem cell (NSC) pool comprises quiescent and activated populations with distinct roles. Transcriptomic anal. revealed that quiescent and activated NSCs exhibited differences in their protein homeostasis network. Whereas activated NSCs had active proteasomes, quiescent NSCs contained large lysosomes. Quiescent NSCs from young mice accumulated protein aggregates, and many of these aggregates were stored in large lysosomes. Perturbation of lysosomal activity in quiescent NSCs affected protein-aggregate accumulation and the ability of quiescent NSCs to activate. During aging, quiescent NSCs displayed defects in their lysosomes, increased accumulation of protein aggregates, and reduced ability to activate. Enhancement of the lysosome pathway in old quiescent NSCs cleared protein aggregates and ameliorated the ability of quiescent NSCs to activate, allowing them to regain a more youthful state.
- 44Yoshimori, T.; Yamamoto, A.; Moriyama, Y.; Futai, M.; Tashiro, Y. Bafilomycin A1, a Specific Inhibitor of Vacuolar-Type H(+)-ATPase, Inhibits Acidification and Protein Degradation in Lysosomes of Cultured Cells. J. Biol. Chem. 1991, 266, 17707– 17712, DOI: 10.1016/s0021-9258(19)47429-244https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3MXltFWhs78%253D&md5=1cee87bb38ff07e9cfc3aa55124f1f5eBafilomycin A1, a specific inhibitor of vacuolar-type hydrogen ion-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cellsYoshimori, Tamotsu; Yamamoto, Akitsugu; Moriyama, Yoshinori; Futai, Masamitsu; Tashiro, YutakaJournal of Biological Chemistry (1991), 266 (26), 17707-12CODEN: JBCHA3; ISSN:0021-9258.Bafilomycin A1 is known as a strong inhibitor of the vacuolar type H+-ATPase in vitro, whereas other type ATPases, e.g. F1,F0-ATPase, are not affected by this antibiotic. The effects of this inhibitor on lysosomes of living cultured cells were tested. The acidification of lysosomes revealed by the incubation with acridine orange was completely inhibited when BNL CL.2 and A431 cells were treated with 0.1-1 μM bafilomycin A1. The effect was reversed by washing the cells. Both studies using less 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and fluorescein isothiocyanate-dextran showed that the intralysosomal pH of A431 cells increased from ∼5.1-5.5 to ∼6.3 in the presence of 1 μM bafilomycin A1. The pH increased gradually in ∼50 min. In the presence of 1 μM bafilomycin A1, 125I-labeled EGF bound to the cell surface at 4° was internalized normally into the cells at 37° but was not degraded at all, in marked contrast to the rapid degrdn. of 125I-EGF in the control cells without the drug. Immunogold electron microscopy showed that EGF was transported into lysosomes irresp. of the addn. of bafilomycin A1. Apparently, the vacuolar type H+-ATPase plays a pivotal role in acidification and protein degrdn. in the lysosomes in vivo.
- 45Zhang, J.-G.; Fariss, M. W. Thenoyltrifluoroacetone, a Potent Inhibitor of Carboxylesterase Activity. Biochem. Pharmacol. 2002, 63, 751– 754, DOI: 10.1016/s0006-2952(01)00871-145https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xjt1Sgt7Y%253D&md5=52a33a6556d3db9f770252f38e8af5d2Thenoyltrifluoroacetone, a potent inhibitor of carboxylesterase activityZhang, Jin-Gang; Fariss, Marc W.Biochemical Pharmacology (2002), 63 (4), 751-754CODEN: BCPCA6; ISSN:0006-2952. (Elsevier Science Inc.)Thenoyltrifluoroacetone (TTFA), a conventional mitochondrial complex II inhibitor, was found to inhibit purified porcine liver carboxylesterase non-competitively with a Ki of 0.61×10-6 M and an IC50 of 0.54×10-6 M. Both rat plasma and liver mitochondrial esterases were inhibited in a concn.-dependent fashion. Results indicate that TTFA is a potent inhibitor of carboxylesterase activity, in addn. to its ability to inhibit mitochondrial complex II activity. Therefore, caution is warranted in using TTFA as a mitochondrial complex inhibitor in combination with esterase substrates, such as fluorescence probes or vitamin E esters.
- 46Mollenhauer, H. H.; Morré, D. J.; Rowe, L. D. Alteration of Intracellular Traffic by Monensin; Mechanism, Specificity and Relationship to Toxicity. Biochim. Biophys. Acta, Rev. Biomembr. 1990, 1031, 225– 246, DOI: 10.1016/0304-4157(90)90008-z46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXkt1Whsr8%253D&md5=81104fe7af97d6dd7c0ac69026cf1bf6Alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicityMollenhauer, Hilton H.; Morre, D. James; Rowe, Loyd D.Biochimica et Biophysica Acta, Reviews on Biomembranes (1990), 1031 (2), 225-46CODEN: BRBMC5; ISSN:0304-4157.A review with 252 refs. examg. the mechanism of action and specificity of monensin in Na+/H+ exchange and correlating these data with the structural and biochem. information on monensin toxicity derived from animal studies.
- 47Pohlmann, R.; Krüger, S.; Hasilik, A.; von Figura, K. Effect of Monensin on Intracellular Transport and Receptor-Mediated Endocytosis of Lysosomal Enzymes. Biochem. J. 1984, 217, 649– 658, DOI: 10.1042/bj217064947https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXotlCrsQ%253D%253D&md5=6a68d525b77057f059ed574d3db8e139Effect of monensin on intracellular transport and receptor-mediated endocytosis of lysosomal enzymesPohlmann, Regina; Krueger, Susanne; Hasilik, Andrej; Von Figura, KurtBiochemical Journal (1984), 217 (3), 649-58CODEN: BIJOAK; ISSN:0264-6021.In cultured human fibroblasts, monensin, a Na+/H+-exchanging ionophore, (1) inhibits mannose 6-phosphate-sensitive endocytosis of a lysosomal enzyme; (2) enhances secretion of the precursor of cathepsin D, while inhibiting secretion of the precursors of β-hexosaminidase; (3) induces secretion of mature β-hexosaminidase and mature cathepsin D; and (4) inhibits carbohydrate processing in proteolytic maturation of the precursors remaining within the cells; this last effect appears to be secondary to an inhibition of the transport of the precursors. If the treated cells are transferred to a monensin-free medium ∼50% of the accumulated precursors are secreted, and the intracellular enzyme is converted into the mature form. Monensin blocks formation of complex oligosaccharides in lysosomal enzymes. In the presence of monensin, total phosphorylation of glycoproteins is partially inhibited, whereas the secreted glycoproteins are enriched in the phosphorylated species. The suggested inhibition by monensin of the transport within the Golgi app. (Tartakoff, A. M.; 1980) may be the cause of some of the effects obsd. in the present study. Other effects are rather explained by interference by monensin with the acidification in the lysosomal and prelysosomal compartments, which appears to be necessary for the transport of endocytosed and of newly synthesized lysosomal enzymes.
- 48Wang, X.; Wu, X.; Zhang, Z.; Ma, C.; Wu, T.; Tang, S.; Zeng, Z.; Huang, S.; Gong, C.; Yuan, C.; Zhang, L.; Feng, Y.; Huang, B.; Liu, W.; Zhang, B.; Shen, Y.; Luo, W.; Wang, X.; Liu, B.; Lei, Y.; Ye, Z.; Zhao, L.; Cao, D.; Yang, L.; Chen, X.; Haydon, R. C.; Luu, H. H.; Peng, B.; Liu, X.; He, T.-C. Monensin Inhibits Cell Proliferation and Tumor Growth of Chemo-Resistant Pancreatic Cancer Cells by Targeting the EGFR Signaling Pathway. Sci. Rep. 2018, 8, 17914 DOI: 10.1038/s41598-018-36214-548https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslKht7o%253D&md5=c09f916e186df99df103332b48c1380fMonensin inhibits cell proliferation and tumor growth of chemo-resistant pancreatic cancer cells by targeting the EGFR signaling pathwayWang, Xin; Wu, Xingye; Zhang, Zhonglin; Ma, Chao; Wu, Tingting; Tang, Shengli; Zeng, Zongyue; Huang, Shifeng; Gong, Cheng; Yuan, Chengfu; Zhang, Linghuan; Feng, Yixiao; Huang, Bo; Liu, Wei; Zhang, Bo; Shen, Yi; Luo, Wenping; Wang, Xi; Liu, Bo; Lei, Yan; Ye, Zhenyu; Zhao, Ling; Cao, Daigui; Yang, Lijuan; Chen, Xian; Haydon, Rex C.; Luu, Hue H.; Peng, Bing; Liu, Xubao; He, Tong-ChuanScientific Reports (2018), 8 (1), 17914CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly malignancies with <5% five-year survival rate due to late diagnosis, limited treatment options and chemoresistance. There is thus an urgent unmet clin. need to develop effective anticancer drugs to treat pancreatic cancer. Here, we study the potential of repurposing monensin as an anticancer drug for chemo-resistant pancreatic cancer. Using the two commonly-used chemo-resistant pancreatic cancer cell lines PANC-1 and MiaPaCa-2, we show that monensin suppresses cell proliferation and migration, and cell cycle progression, while solicits apoptosis in pancreatic cancer lines at a low micromole range. Moreover, monensin functions synergistically with gemcitabine or EGFR inhibitor erlotinib in suppressing cell growth and inducing cell death of pancreatic cancer cells. Mechanistically, monensin suppresses numerous cancer-assocd. pathways, such as E2F/DP1, STAT1/2, NFkB, AP-1, Elk-1/SRF, and represses EGFR expression in pancreatic cancer lines. Furthermore, the in vivo study shows that monensin blunts PDAC xenograft tumor growth by suppressing cell proliferation via targeting EGFR pathway. Therefore, our findings demonstrate that monensin can be repurposed as an effective anti-pancreatic cancer drug even though more investigations are needed to validate its safety and anticancer efficacy in pre-clin. and clin. models.
- 49Long, J. M.; Holtzman, D. M. Alzheimer Disease: An Update on Pathobiology and Treatment Strategies. Cell 2019, 179, 312– 339, DOI: 10.1016/j.cell.2019.09.00149https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVylsbrN&md5=b68edceb2c7ebe4515efded93c9281c3Alzheimer Disease: An Update on Pathobiology and Treatment StrategiesLong, Justin M.; Holtzman, David M.Cell (Cambridge, MA, United States) (2019), 179 (2), 312-339CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A review. Alzheimer disease (AD) is a heterogeneous disease with a complex pathobiol. The presence of extracellular β-amyloid deposition as neuritic plaques and intracellular accumulation of hyperphosphorylated tau as neurofibrillary tangles remains the primary neuropathol. criteria for AD diagnosis. However, a no. of recent fundamental discoveries highlight important pathol. roles for other crit. cellular and mol. processes. Despite this, no disease-modifying treatment currently exists, and numerous phase 3 clin. trials have failed to demonstrate benefits. Here, we review recent advances in our understanding of AD pathobiol. and discuss current treatment strategies, highlighting recent clin. trials and opportunities for developing future disease-modifying therapies.
- 50Yang, X.; Qian, K. Protein O-GlcNAcylation: Emerging Mechanisms and Functions. Nat. Rev. Mol. Cell Biol. 2017, 18, 452– 465, DOI: 10.1038/nrm.2017.2250https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnsV2mtbs%253D&md5=291f00ada663647822444a8fa5fca532Protein O-GlcNAcylation: emerging mechanisms and functionsYang, Xiaoyong; Qian, KevinNature Reviews Molecular Cell Biology (2017), 18 (7), 452-465CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)A review. O-GlcNAcylation - the attachment of O-linked N-acetylglucosamine (O-GlcNAc) moieties to cytoplasmic, nuclear and mitochondrial proteins - is a post-translational modification that regulates fundamental cellular processes in metazoans. A single pair of enzymes - O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) - controls the dynamic cycling of this protein modification in a nutrient- and stress-responsive manner. Recent years have seen remarkable advances in our understanding of O-GlcNAcylation at levels that range from structural and mol. biol. to cell signalling and gene regulation to physiol. and disease. New mechanisms and functions of O-GlcNAcylation that are emerging from these recent developments enable us to begin constructing a unified conceptual framework through which the significance of this modification in cellular and organismal physiol. can be understood.
- 51Li, J.; Wang, J.; Wen, L.; Zhu, H.; Li, S.; Huang, K.; Jiang, K.; Li, X.; Ma, C.; Qu, J.; Parameswaran, A.; Song, J.; Zhao, W.; Wang, P. G. An OGA-Resistant Probe Allows Specific Visualization and Accurate Identification of O -GlcNAc-Modified Proteins in Cells. ACS Chem. Biol. 2016, 11, 3002– 3006, DOI: 10.1021/acschembio.6b0067851https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFSlsb%252FP&md5=ff09bf8c6987de47cf6d424507c19019An OGA-Resistant Probe Allows Specific Visualization and Accurate Identification of O-GlcNAc-Modified Proteins in CellsLi, Jing; Wang, Jiajia; Wen, Liuqing; Zhu, He; Li, Shanshan; Huang, Kenneth; Jiang, Kuan; Li, Xu; Ma, Cheng; Qu, Jingyao; Parameswaran, Aishwarya; Song, Jing; Zhao, Wei; Wang, Peng GeorgeACS Chemical Biology (2016), 11 (11), 3002-3006CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chem. probes such as GlcNAc or GalNAc analogs bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modifications. However, these chem. probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. The authors report here the development of a novel probe, 1,3,6-tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-D-glucopyranose (Ac34dGlcNAz), that can be processed by the GalNAc salvage pathway, and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of hydroxyl group at C4, this probe is less incorporated into α/β 4-GlcNAc or GalNAc contg. glycoconjugates. Furthermore, the O-4dGlcNAz modification was resistant to the hydrolysis of O-GlcNAcase (OGA), which greatly enhanced the efficiency of incorporation for O-GlcNAcylation. Combined with a click reaction, Ac34dGlcNAz allowed the selective visualization of O-GlcNAc in cells, and accurate identification of O-GlcNAc-modified proteins with LC-MS/MS. This probe represents a more potent and selective tool in tracking, capturing, and identifying O-GlcNAc-modified proteins in cells and cell lysates.
- 52Pedowitz, N. J.; Pratt, M. R. Design and Synthesis of Metabolic Chemical Reporters for the Visualization and Identification of Glycoproteins. RSC Chem. Biol. 2021, 2, 306– 321, DOI: 10.1039/d1cb00010a52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXktlGlt70%253D&md5=9cca66336cc96323cc8c31e80436a6ddDesign and synthesis of metabolic chemical reporters for the visualization and identification of glycoproteinsPedowitz, Nichole J.; Pratt, Matthew R.RSC Chemical Biology (2021), 2 (2), 306-321CODEN: RCBSBP; ISSN:2633-0679. (Royal Society of Chemistry)A review. Glycosylation events play an invaluable role in regulating cellular processes including enzymic activity, immune recognition, protein stability, and cell-cell interactions. However, researchers have yet to realize the full range of glycan mediated biol. functions due to a lack of appropriate chem. tools. Fortunately, the past 25 years has seen the emergence of modified sugar analogs, termed metabolic chem. reporters (MCRs), which are metabolized by endogenous enzymes to label complex glycan structures. Here, we review the major reporters for each class of glycosylation and highlight recent applications that have made a tremendous impact on the field of glycobiol.
- 53Zhang, C.; Zhang, C.; Dai, P.; Vinogradov, A.; Gates, Z. Site-Selective Cysteine-Cyclooctyne Conjugation. Angew. Chem., Int. Ed. 2018, 57, 6459– 6463, DOI: 10.1002/anie.20180086053https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXosFSqtLs%253D&md5=e21a09d9578aaa87167af5ebdda954e1Site-Selective Cysteine-Cyclooctyne ConjugationZhang, Chi; Dai, Peng; Vinogradov, Alexander A.; Gates, Zachary P.; Pentelute, Bradley L.Angewandte Chemie, International Edition (2018), 57 (22), 6459-6463CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)The authors report a site-selective cysteine-cyclooctyne conjugation reaction between a seven-residue peptide tag (DBCO-tag, Leu-Cys-Tyr-Pro-Trp-Val-Tyr) at the N or C terminus of a peptide or protein and various aza-dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO-tag increases the rate of the thiol-yne reaction 220-fold, thereby enabling selective conjugation of DBCO-tag to DBCO-linked fluorescent probes, affinity tags, and cytotoxic drug mols. Fusion of DBCO-tag with the protein of interest enables regioselective cysteine modification on proteins that contain multiple endogenous cysteines; these examples include green fluorescent protein and the antibody trastuzumab. Short peptide tags can aid in accelerating bond-forming reactions that are often slow to non-existent in water.
- 54Wulff-Fuentes, E.; Berendt, R. R.; Massman, L.; Danner, L.; Malard, F.; Vora, J.; Kahsay, R.; Stichelen, S. O.-V. The Human O-GlcNAcome Database and Meta-Analysis. Sci. Data 2021, 8, 25 DOI: 10.1038/s41597-021-00810-454https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXlsVOrsb4%253D&md5=467cd56066d8f2b4a6847a717b7526e4The human O-GlcNAcome database and meta-analysisWulff-Fuentes, Eugenia; Berendt, Rex R.; Massman, Logan; Danner, Laura; Malard, Florian; Vora, Jeet; Kahsay, Robel; Olivier-Van Stichelen, StephanieScientific Data (2021), 8 (1), 25CODEN: SDCABS; ISSN:2052-4463. (Nature Research)Abstr.: Over the past 35 years, ∼1700 articles have characterized protein O-GlcNAcylation. Found in almost all living organisms, this post-translational modification of serine and threonine residues is highly conserved and key to biol. processes. With half of the primary research articles using human models, the O-GlcNAcome recently reached a milestone of 5000 human proteins identified. Herein, we provide an extensive inventory of human O-GlcNAcylated proteins, their O-GlcNAc sites, identification methods, and corresponding refs. (www.oglcnac.mcw.edu). In the absence of a comprehensive online resource for O-GlcNAcylated proteins, this list serves as the only database of O-GlcNAcylated proteins. Based on the thorough anal. of the amino acid sequence surrounding 7002 O-GlcNAc sites, we progress toward a more robust semi-consensus sequence for O-GlcNAcylation. Moreover, we offer a comprehensive meta-anal. of human O-GlcNAcylated proteins for protein domains, cellular and tissue distribution, and pathways in health and diseases, reinforcing that O-GlcNAcylation is a master regulator of cell signaling, equal to the widely studied phosphorylation.
- 55Petelski, A. A.; Emmott, E.; Leduc, A.; Huffman, R. G.; Specht, H.; Perlman, D. H.; Slavov, N. Multiplexed Single-Cell Proteomics Using SCoPE2. Nat. Protoc. 2021, 16, 5398– 5425, DOI: 10.1038/s41596-021-00616-z55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitlOlurrI&md5=912ffb4f0fd8ad003b1eb5a704300b41Multiplexed single-cell proteomics using SCoPE2Petelski, Aleksandra A.; Emmott, Edward; Leduc, Andrew; Huffman, R. Gray; Specht, Harrison; Perlman, David H.; Slavov, NikolaiNature Protocols (2021), 16 (12), 5398-5425CODEN: NPARDW; ISSN:1750-2799. (Nature Portfolio)Many biol. systems are composed of diverse single cells. This diversity necessitates functional and mol. single-cell anal. Single-cell protein anal. has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Prepn. (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed anal. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ∼200 single cells per 24 h using only std. com. equipment. We emphasize exptl. steps and benchmarks required for achieving quant. protein anal.
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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.2c00284.
Supporting figures, cell culture conditions, SP2E workflow, LC-MS/MS acquisition conditions, data analysis, and list of reagents (PDF)
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