Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

You’ve supercharged your research process with ACS and Mendeley!

STEP 1:
Click to create an ACS ID

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

MENDELEY PAIRING EXPIRED
Your Mendeley pairing has expired. Please reconnect
ACS Publications. Most Trusted. Most Cited. Most Read
Native Capillary Isoelectric Focusing for the Separation of Protein Complex Isoforms and Subcomplexes

OR SEARCH CITATIONS

My Activity
Publications

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:Anal. Chem. 2010, 82, 15, 6643-6651
    Article

    Native Capillary Isoelectric Focusing for the Separation of Protein Complex Isoforms and Subcomplexes
    Click to copy article linkArticle link copied!

    View Author Information
    Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Rd. La Jolla, California 92037, Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 02142, Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, Department of Biochemistry, University of Washington, Seattle, Washington 98195
    * To whom correspondence should be addressed. Phone: 858-784-8862; Fax: 858-784-8883; E-mail: [email protected]
    †The Scripps Research Institute.
    ‡Whitehead Institute for Biomedical Research.
    ∥University of Washington.
    §Howard Hughes Medical Institute.
    Other Access Options

    Analytical Chemistry

    Cite this: Anal. Chem. 2010, 82, 15, 6643–6651
    Click to copy citationCitation copied!
    https://doi.org/10.1021/ac101235k
    Published July 8, 2010
    Copyright © 2010 American Chemical Society
    Request reuse permissions

    Abstract

    Click to copy section linkSection link copied!

    Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically relevant HIS-tag and FLAG-tag purified protein complexes, we demonstrate the separations of protein complex isoforms of the mammalian target of rapamycin complex (mTORC1 and 2) and the subcomplexes and different phosphorylation states of the Dam1 complex. The high efficiency capillary isoelectric focusing separation allowed for resolution of protein complexes and subcomplexes similar in size and biochemical composition. By performing separations with native buffers and reduced temperature (15 °C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection (<45 min). Increasing the separation temperature allowed us to monitor dissociation of the Dam1 complex into its subcomplexes (25 °C) and eventually its individual protein components (30 °C). The separation of two different phosphorylation states of the Dam1 complex, generated from an in vitro kinase assay with Mps1 kinase, was straightforward due to the large pI shift upon multiple phosphorylation events. The separation of the protein complex isoforms of mTORC, on the other hand, required the addition of a small pI range (4−6.5) of ampholytes to improve resolution and stability of the complexes. We show that native capillary isoelectric focusing is a powerful method for the difficult separations of large, similar, unstable protein complexes. This method shows potential for differentiation of protein complex isoform and subcomplex compositions, post-translational modifications, architectures, stabilities, equilibria, and relative abundances under biologically relevant conditions.

    Copyright © 2010 American Chemical Society

    Subjects

    what are subjects

    Read this article

    To access this article, please review the available access options below.

    Get instant access

    Purchase Access

    Read this article for 48 hours. Check out below using your ACS ID or as a guest.

    Recommended

    Access through Your Institution

    You may have access to this article through your institution.

    Your institution does not have access to this content. Add or change your institution or let them know you’d like them to include access.

    Cited By

    Click to copy section linkSection link copied!

    This article is cited by 32 publications.

    1. Ziting Gao, Zachary Hutchins, Zongbo Li, Wenwan Zhong. Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for Separation of Extracellular Vesicles. Analytical Chemistry 2022, 94 (41) , 14083-14091. https://doi.org/10.1021/acs.analchem.2c03550
    2. Tian Xu, Linjie Han, Liangliang Sun. Automated Capillary Isoelectric Focusing-Mass Spectrometry with Ultrahigh Resolution for Characterizing Microheterogeneity and Isoelectric Points of Intact Protein Complexes. Analytical Chemistry 2022, 94 (27) , 9674-9682. https://doi.org/10.1021/acs.analchem.2c00975
    3. Kevin Jooß, Luis F. Schachner, Rachel Watson, Zachary B. Gillespie, Sarah A. Howard, Marcus A. Cheek, Matthew J. Meiners, Amin Sobh, Jonathan D. Licht, Michael-Christopher Keogh, Neil L. Kelleher. Separation and Characterization of Endogenous Nucleosomes by Native Capillary Zone Electrophoresis–Top-Down Mass Spectrometry. Analytical Chemistry 2021, 93 (12) , 5151-5160. https://doi.org/10.1021/acs.analchem.0c04975
    4. M. Rachel Mehaffey, Qiangwei Xia, Jennifer S. Brodbelt. Uniting Native Capillary Electrophoresis and Multistage Ultraviolet Photodissociation Mass Spectrometry for Online Separation and Characterization of Escherichia coli Ribosomal Proteins and Protein Complexes. Analytical Chemistry 2020, 92 (22) , 15202-15211. https://doi.org/10.1021/acs.analchem.0c03784
    5. Arseniy M. Belov, Rosa Viner, Marcia R. Santos, David M. Horn, Marshall Bern, Barry L. Karger, Alexander R. Ivanov. Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry. Journal of the American Society for Mass Spectrometry 2017, 28 (12) , 2614-2634. https://doi.org/10.1007/s13361-017-1781-1
    6. Xuemei Han, Yueju Wang, Aaron Aslanian, Bryan Fonslow, Beth Graczyk, Trisha N. Davis, and John R. Yates, III . In-Line Separation by Capillary Electrophoresis Prior to Analysis by Top-Down Mass Spectrometry Enables Sensitive Characterization of Protein Complexes. Journal of Proteome Research 2014, 13 (12) , 6078-6086. https://doi.org/10.1021/pr500971h
    7. Mohammad Pirmoradian, Bo Zhang, Konstantin Chingin, Juan Astorga-Wells, and Roman A. Zubarev . Membrane-Assisted Isoelectric Focusing Device As a Micropreparative Fractionator for Two-Dimensional Shotgun Proteomics. Analytical Chemistry 2014, 86 (12) , 5728-5732. https://doi.org/10.1021/ac404180e
    8. Yaoyang Zhang, Bryan R. Fonslow, Bing Shan, Moon-Chang Baek, and John R. Yates, III . Protein Analysis by Shotgun/Bottom-up Proteomics. Chemical Reviews 2013, 113 (4) , 2343-2394. https://doi.org/10.1021/cr3003533
    9. Yana V. Miteva, Hanna G. Budayeva, and Ileana M. Cristea . Proteomics-Based Methods for Discovery, Quantification, and Validation of Protein–Protein Interactions. Analytical Chemistry 2013, 85 (2) , 749-768. https://doi.org/10.1021/ac3033257
    10. Hu Zhou, Zhibing Ning, Amanda E. Starr, Mohamed Abu-Farha, and Daniel Figeys . Advancements in Top-Down Proteomics. Analytical Chemistry 2012, 84 (2) , 720-734. https://doi.org/10.1021/ac202882y
    11. Stefani N. Thomas, Yunhu Wan, Zhongping Liao, Phyllis I. Hanson, and Austin J. Yang . Stable Isotope Labeling with Amino Acids in Cell Culture Based Mass Spectrometry Approach to Detect Transient Protein Interactions Using Substrate Trapping. Analytical Chemistry 2011, 83 (14) , 5511-5518. https://doi.org/10.1021/ac200950k
    12. Ann‐Katrin Schwenzer, Lena Kruse, Kevin Jooß, Christian Neusüß. Capillary electrophoresis‐mass spectrometry for protein analyses under native conditions: Current progress and perspectives. PROTEOMICS 2024, 24 (3-4) https://doi.org/10.1002/pmic.202300135
    13. Kevin Jooß, John P. McGee, Rafael D. Melani, Neil L. Kelleher. Standard procedures for native CZE‐MS of proteins and protein complexes up to 800 kDa. ELECTROPHORESIS 2021, 42 (9-10) , 1050-1059. https://doi.org/10.1002/elps.202000317
    14. Patricia M. Morgan, Mara G. Freire, Ana Paula M. Tavares, Antonysamy Michael, Xiaoying Zhang. Extraction and Purification of IgY. 2021, 135-160. https://doi.org/10.1007/978-3-030-72688-1_11
    15. Yu Sun, Shuai Zhang. Application of Capillary Electrophoresis for Ephedrine and Pseudoephedrine Detection: a Review. International Journal of Electrochemical Science 2020, 15 (3) , 2614-2623. https://doi.org/10.20964/2020.03.38
    16. Cassandra M. Modahl, Rajeev Kungur Brahma, Cho Yeow Koh, Narumi Shioi, R. Manjunatha Kini. Omics Technologies for Profiling Toxin Diversity and Evolution in Snake Venom: Impacts on the Discovery of Therapeutic and Diagnostic Agents. Annual Review of Animal Biosciences 2020, 8 (1) , 91-116. https://doi.org/10.1146/annurev-animal-021419-083626
    17. Claire M. Delahunty, John R. Yates. Protein–Protein Interactions. 2019, 125-144. https://doi.org/10.1002/9781119081661.ch5
    18. Parviz Ghezellou, Vannuruswamy Garikapati, Seyed Mahdi Kazemi, Kerstin Strupat, Alireza Ghassempour, Bernhard Spengler. A perspective view of top‐down proteomics in snake venom research. Rapid Communications in Mass Spectrometry 2019, 33 (S1) , 20-27. https://doi.org/10.1002/rcm.8255
    19. Julien Camperi, Bart De Cock, Valérie Pichon, Audrey Combes, Jean Guibourdenche, Thierry Fournier, Yvan Vander Heyden, Debby Mangelings, Nathalie Delaunay. First characterizations by capillary electrophoresis of human Chorionic Gonadotropin at the intact level. Talanta 2019, 193 , 77-86. https://doi.org/10.1016/j.talanta.2018.09.095
    20. Petra Gronemeyer, Holger Thiess, Steffen Zobel‐Roos, Reinhard Ditz, Jochen Strube. Integration of Upstream and Downstream in Continuous Biomanufacturing. 2017, 481-510. https://doi.org/10.1002/9783527699902.ch17
    21. Nazar Zaki, Elfadil A. Mohamed, Antonio Mora. Characterization of Protein Complexes and Subcomplexes in Protein-Protein Interaction Databases. Biochemistry Research International 2015, 2015 , 1-12. https://doi.org/10.1155/2015/245075
    22. Zhongxiu Jiang, Yun Qin, Zhen Peng, Shenghua Chen, Shu Chen, Chunyan Deng, Juan Xiang. The simultaneous detection of free and total prostate antigen in serum samples with high sensitivity and specificity by using the dual-channel surface plasmon resonance. Biosensors and Bioelectronics 2014, 62 , 268-273. https://doi.org/10.1016/j.bios.2014.06.060
    23. Ni Hou, Yu Chen, Shiyong Yu, Zongliang Quan, Chenhua Pan, Yulin Deng, Lina Geng. Native Protein Separation by Isoelectric Focusing and Blue Gel Electrophoresis-Coupled Two-Dimensional Microfluidic Chip Electrophoresis. Chromatographia 2014, 77 (19-20) , 1339-1346. https://doi.org/10.1007/s10337-014-2728-3
    24. Spyridon Konstantinidis, Simyee Kong, Nigel Titchener‐Hooker. Identifying analytics for high throughput bioprocess development studies. Biotechnology and Bioengineering 2013, 110 (7) , 1924-1935. https://doi.org/10.1002/bit.24850
    25. Ryan Bonn, Sushma Rampal, Tracey Rae, Jeffrey Fishpaugh. CIEF method optimization: Development of robust and reproducible protein reagent characterization in the clinical immunodiagnostic industry. ELECTROPHORESIS 2013, 34 (6) , 825-832. https://doi.org/10.1002/elps.201200529
    26. Elena L Rudashevskaya, Roberto Sacco, Klaus Kratochwill, Marie L Huber, Matthias Gstaiger, Giulio Superti-Furga, Keiryn L Bennett. A method to resolve the composition of heterogeneous affinity-purified protein complexes assembled around a common protein by chemical cross-linking, gel electrophoresis and mass spectrometry. Nature Protocols 2013, 8 (1) , 75-97. https://doi.org/10.1038/nprot.2012.133
    27. Hou-yu Wang, Si Li, Yun-yun Tang, Jing-yu Dong, Liu-yin Fan, Cheng-xi Cao. Determination of free acidic and alkaline residues of protein via moving reaction boundary titration in microdevice electrophoresis. The Analyst 2013, 138 (12) , 3544. https://doi.org/10.1039/c3an36494a
    28. Mahmoud F. Bahnasy, Charles A. Lucy. A versatile semi-permanent sequential bilayer/diblock polymer coating for capillary isoelectric focusing. Journal of Chromatography A 2012, 1267 , 89-95. https://doi.org/10.1016/j.chroma.2012.07.016
    29. Eunmi Ban, Soo Hyun Park, Min‐Jung Kang, Hyun‐Jung Lee, Eun Joo Song, Young Sook Yoo. Growing trend of CE at the omics level: The frontier of systems biology – An update. ELECTROPHORESIS 2012, 33 (1) , 2-13. https://doi.org/10.1002/elps.201100344
    30. Noor Remmerie, Thomas De Vijlder, Kris Laukens, Thanh Hai Dang, Filip Lemière, Inge Mertens, Dirk Valkenborg, Ronny Blust, Erwin Witters. Next generation functional proteomics in non-model plants: A survey on techniques and applications for the analysis of protein complexes and post-translational modifications. Phytochemistry 2011, 72 (10) , 1192-1218. https://doi.org/10.1016/j.phytochem.2011.01.003
    31. Aline Staub, Davy Guillarme, Julie Schappler, Jean-Luc Veuthey, Serge Rudaz. Intact protein analysis in the biopharmaceutical field. Journal of Pharmaceutical and Biomedical Analysis 2011, 55 (4) , 810-822. https://doi.org/10.1016/j.jpba.2011.01.031
    32. Bin Hu, Shuiping Yang, Ming Li, Haiwei Gu, Huanwen Chen. Direct detection of native proteins in biological matrices using extractive electrospray ionization mass spectrometry. The Analyst 2011, 136 (18) , 3599. https://doi.org/10.1039/c1an15410a
    Download PDF
    Go to Analytical Chemistry
    Get e-Alerts
    Get e-Alerts

    Analytical Chemistry

    Cite this: Anal. Chem. 2010, 82, 15, 6643–6651
    Click to copy citationCitation copied!
    https://doi.org/10.1021/ac101235k
    Published July 8, 2010
    Copyright © 2010 American Chemical Society
    Request reuse permissions

    Article Views

    1847

    Altmetric

    -

    Citations

    32
    Learn about these metrics

    Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

    Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

    The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.