In-Line Separation by Capillary Electrophoresis Prior to Analysis by Top-Down Mass Spectrometry Enables Sensitive Characterization of Protein Complexes
Open accessAbstract
Intact protein analysis via top-down mass spectrometry (MS) provides a bird’s eye view over the protein complexes and complex protein mixtures with the unique capability of characterizing protein variants, splice isoforms, and combinatorial post-translational modifications (PTMs). Here we applied capillary electrophoresis (CE) through a sheathless CE–electrospray ionization interface coupled to an LTQ Velos Orbitrap Elite mass spectrometer to analyze the Dam1 complex from Saccharomyces cerevisiae. We achieved a 100-fold increase in sensitivity compared to a reversed-phase liquid chromatography coupled MS analysis of recombinant Dam1 complex with a total loading of 2.5 ng (12 amol). N-terminal processing forms of individual subunits of the Dam1 complex were observed as well as their phosphorylation stoichiometry upon Mps1p kinase treatment.
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Cites
Large-scale analysis of the yeast proteome by multidimensional protein identification technology
Washburn, M. P., Wolters, D. A., & Yates, J. R. (2001) · Nature Biotechnology · doi:10.1038/85686 · Cited by 4,759 · see citing works
Self-citationPeng, J., Elias, J. E., Thoreen, C. C., Licklider, L., & Gygi, S. P. (2002) · Journal of Proteome Research · doi:10.1021/pr025556v · Cited by 1,652 · see citing works
Beausoleil, S. A., Villén, J., Gerber, S. A., Rush, J., & Gygi, S. P. (2006) · Nature Biotechnology · doi:10.1038/nbt1240 · Cited by 1,577 · see citing works
Tabb, D. L., McDonald, W. H., & Yates, J. R. (2002) · Journal of Proteome Research · doi:10.1021/pr015504q · Cited by 1,369 · see citing works
Self-citationThe Orbitrap: a new mass spectrometer
Hu, Q., Noll, R. J., Li, H., Makarov, A., Hardman, M., & Cooks, R. G. (2005) · Journal of Mass Spectrometry · doi:10.1002/jms.856 · Cited by 1,338 · see citing works
Mapping intact protein isoforms in discovery mode using top-down proteomics
Tran, J. C., Zamdborg, L., Ahlf, D. R., Lee, J. E., Catherman, A. D., Durbin, K. R., Tipton, J. D., Vellaichamy, A., Kellie, J. F., Li, M., et al. (2011) · Nature · doi:10.1038/nature10575 · Cited by 665 · see citing works
Phospho-Regulation of Kinetochore-Microtubule Attachments by the Aurora Kinase Ipl1p
Cheeseman, I. M., Anderson, S., Jwa, M., et al. (2002) · Cell · doi:10.1016/s0092-8674(02)00973-x · Cited by 616 · see citing works
Top Down versus Bottom Up Protein Characterization by Tandem High-Resolution Mass Spectrometry
Kelleher, N. L., Lin, H., Valaskovic, G. A., Aaserud, D. J., Fridriksson, E. K., & McLafferty, F. W. (1999) · Journal of the American Chemical Society · doi:10.1021/ja973655h · Cited by 606 · see citing works
Improved electrospray ionization interface for capillary zone electrophoresis-mass spectrometry
Smith, R. D., Barinaga, C. J., & Udseth, H. R. (1988) · Analytical Chemistry · doi:10.1021/ac00169a022 · Cited by 534 · see citing works
Mass Spectrometry: Bottom-Up or Top-Down?
Chait, B. T. (2006) · Science · doi:10.1126/science.1133987 · Cited by 442 · see citing works
Peer Reviewed: Top-Down Proteomics
Kelleher, N. (2004) · Analytical Chemistry · doi:10.1021/ac0415657 · Cited by 386 · see citing works
Ge, Y., Lawhorn, B. G., ElNaggar, M., Strauss, E., Park, J.-H., Begley, T. P., & McLafferty, F. W. (2002) · Journal of the American Chemical Society · doi:10.1021/ja011335z · Cited by 356 · see citing works
McDonald, W. H., Tabb, D. L., Sadygov, R. G., MacCoss, M. J., Venable, J. D., Graumann, J., Johnson, J. R., Cociorva, D., & Yates, J. R. (2004) · Rapid Communications in Mass Spectrometry · doi:10.1002/rcm.1603 · Cited by 345 · see citing works
Self-citationExtending Top-Down Mass Spectrometry to Proteins with Masses Greater Than 200 Kilodaltons
Han, X., Jin, M., Breuker, K., & McLafferty, F. W. (2006) · Science · doi:10.1126/science.1128868 · Cited by 328 · see citing works
Self-citationYates, J. R., & Kelleher, N. L. (2013) · Analytical Chemistry · doi:10.1021/ac401484r · Cited by 324 · see citing works
Self-citationMoini, M. (2007) · Analytical Chemistry · doi:10.1021/ac0704560 · Cited by 322 · see citing works
Wojcik, R., Dada, O. O., Sadilek, M., & Dovichi, N. J. (2010) · Rapid Communications in Mass Spectrometry · doi:10.1002/rcm.4672 · Cited by 234 · see citing works
LeDuc, R. D., Taylor, G. K., Kim, Y.-B., Januszyk, T. E., Bynum, L., Solà, J., Garavelli, J., & Kelleher, N. (2004) · Nucleic Acids Res. · doi:10.1093/nar/gkh447 · Cited by 192 · see citing works
The Dam1 kinetochore complex harnesses microtubule dynamics to produce force and movement
Asbury, C. L., Gestaut, D. R., Powers, A. F., Franck, A. D., & Davis, T. N. (2006) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0602249103 · Cited by 186 · see citing works
Self-citationProcessing Complex Mixtures of Intact Proteins for Direct Analysis by Mass Spectrometry
Meng, F., Cargile, B. J., Patrie, S. M., Johnson, J. R., McLoughlin, S. M., & Kelleher, N. L. (2002) · Analytical Chemistry · doi:10.1021/ac020049i · Cited by 183 · see citing works
Busnel, J., Schoenmaker, B., Ramautar, R., Carrasco‐Pancorbo, A., Ratnayake, C. K., Feitelson, J. S., Chapman, J. D., Deelder, A. M., & Mayboroda, O. A. (2010) · Analytical Chemistry · doi:10.1021/ac102159d · Cited by 165 · see citing works
Ansong, C., Wu, S., Meng, D., Liu, X., Brewer, H. M., Deatherage Kaiser, B. L., Nakayasu, E. S., Cort, J. R., Pevzner, P., Smith, R. D., et al. (2013) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1221210110 · Cited by 157 · see citing works
Faserl, K., Sarg, B., Kremser, L., & Lindner, H. H. (2011) · Analytical Chemistry · doi:10.1021/ac2010372 · Cited by 153 · see citing works
Mitotic Spindle Integrity and Kinetochore Function Linked by the Duo1p/Dam1p Complex
Cheeseman, I. M., Enquist-Newman, M., Müller‐Reichert, T., et al. (2001) · The Journal of Cell Biology · doi:10.1083/jcb.152.1.197 · Cited by 148 · see citing works
Wang, Y., Fonslow, B. R., Wong, C. C. L., Nakorchevsky, A., & Yates, J. R. (2012) · Analytical Chemistry · doi:10.1021/ac301091m · Cited by 144 · see citing works
Self-citationGestaut, D. R., Graczyk, B., Cooper, J. R., et al. (2008) · Nature Cell Biology · doi:10.1038/ncb1702 · Cited by 142 · see citing works
Self-citationCantin, G. T., Shock, T. R., Park, S. K., et al. (2007) · Analytical Chemistry · doi:10.1021/ac0618730 · Cited by 135 · see citing works
Ramautar, R., Busnel, J., Deelder, A. M., & Mayboroda, O. A. (2011) · Analytical Chemistry · doi:10.1021/ac202407v · Cited by 129 · see citing works
A protease for 'middle-down' proteomics
Wu, C., Tran, J. C., Zamdborg, L., Durbin, K. R., Li, M., Ahlf, D. R., Early, B. P., Thomas, P. M., Sweedler, J. V., & Kelleher, N. L. (2012) · Nature Methods · doi:10.1038/nmeth.2074 · Cited by 128 · see citing works
Mps1 Phosphorylation of Dam1 Couples Kinetochores to Microtubule Plus Ends at Metaphase
Shimogawa, M. M., Graczyk, B., Gardner, M. K., et al. (2006) · Current Biology · doi:10.1016/j.cub.2006.06.063 · Cited by 107 · see citing works
Self-citationSystematic Phosphorylation Analysis of Human Mitotic Protein Complexes
Hegemann, B., Hutchins, J. R. A., Hudecz, O., Novatchkova, M., Rameseder, J., Sykora, M. M., Liu, S., Mazanek, M., Lénárt, P., Hèriché, J.-K., et al. (2011) · Science Signaling · doi:10.1126/scisignal.2001993 · Cited by 105 · see citing works
A Robust Two-Dimensional Separation for Top-Down Tandem Mass Spectrometry of the Low-Mass Proteome
Lee, J. E., Kellie, J. F., Tran, J. C., Tipton, J., Catherman, A. D., Thomas, H. M., Ahlf, D. R., Durbin, K. R., Vellaichamy, A., Ntai, I., et al. (2009) · Journal of the American Society for Mass Spectrometry · doi:10.1016/j.jasms.2009.08.001 · Cited by 104 · see citing works
Roth, M. J., Forbes, A. J., Boyne, M., Kim, Y.-B., Robinson, D., & Kelleher, N. (2005) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m500064-mcp200 · Cited by 96 · see citing works
Optimizing capillary electrophoresis for top‐down proteomics of 30–80 kDa proteins
Li, Y., Compton, P. D., Tran, J. C., Ntai, I., & Kelleher, N. L. (2014) · PROTEOMICS · doi:10.1002/pmic.201300381 · Cited by 95 · see citing works
Vellaichamy, A., Tran, J. C., Catherman, A. D., Lee, J. E., Kellie, J. F., Sweet, S., Zamdborg, L., Thomas, P. M., Ahlf, D. R., Durbin, K. R., et al. (2010) · Analytical Chemistry · doi:10.1021/ac9021083 · Cited by 94 · see citing works
Evaluation of the Compact High-Field Orbitrap for Top-Down Proteomics of Human Cells
Ahlf, D. R., Compton, P. D., Tran, J. C., Early, B. P., Thomas, P. M., & Kelleher, N. L. (2012) · Journal of Proteome Research · doi:10.1021/pr3004216 · Cited by 89 · see citing works
Automatic Validation of Phosphopeptide Identifications from Tandem Mass Spectra
Lu, B., Ruse, C. I., Xu, T., Park, S. K., & Yates, J. R. (2007) · Analytical Chemistry · doi:10.1021/ac061334v · Cited by 89 · see citing works
Self-citationInterpreting Top-Down Mass Spectra Using Spectral Alignment
Frank, A. M., Pesavento, J. J., Mizzen, C. A., Kelleher, N. L., & Pevzner, P. A. (2008) · Analytical Chemistry · doi:10.1021/ac702324u · Cited by 77 · see citing works
Zhong, X., Maxwell, E. J., & Chen, D. D. Y. (2011) · Analytical Chemistry · doi:10.1021/ac200636y · Cited by 70 · see citing works
Automated Proteomics of E. coli via Top-Down Electron-Transfer Dissociation Mass Spectrometry
Bunger, M. K., Cargile, B. J., Ngunjiri, A., Bundy, J. L., & Stephenson, J. L. (2008) · Analytical Chemistry · doi:10.1021/ac7018409 · Cited by 67 · see citing works
Sun, L., Knierman, M. D., Zhu, G., & Dovic̀hi, N. J. (2013) · Analytical Chemistry · doi:10.1021/ac4008122 · Cited by 65 · see citing works
“Proteotyping”: Population Proteomics of Human Leukocytes Using Top Down Mass Spectrometry
Roth, M. J., Parks, B. A., Ferguson, J. T., Boyne, M. T., & Kelleher, N. L. (2008) · Analytical Chemistry · doi:10.1021/ac800141g · Cited by 65 · see citing works
Haselberg, R., de Jong, G. J., & Somsen, G. W. (2010) · Analytica Chimica Acta · doi:10.1016/j.aca.2010.08.032 · Cited by 64 · see citing works
Kellie, J. F., Catherman, A. D., Durbin, K. R., Tran, J. C., Tipton, J., Norris, J. L., Witkowski, C. E., Thomas, P. M., & Kelleher, N. (2011) · Analytical Chemistry · doi:10.1021/ac202384v · Cited by 62 · see citing works
Precursor ion independent algorithm for top-down shotgun proteomics
Tsai, Y. S., Scherl, A., Shaw, J. L., Mackay, C. L., Shaffer, S. A., Langridge‐Smith, P. R. R., & Goodlett, D. R. (2009) · Journal of the American Society for Mass Spectrometry · doi:10.1016/j.jasms.2009.07.024 · Cited by 59 · see citing works
Yeast Dam1p has a role at the kinetochore in assembly of the mitotic spindle
Jones, M. H., He, X., Giddings, T. H., et al. (2001) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.241417098 · Cited by 58 · see citing works
Zhao, Y., Sun, L., Champion, M. M., Knierman, M. D., & Dovichi, N. J. (2014) · Analytical Chemistry · doi:10.1021/ac500092q · Cited by 56 · see citing works
Asymmetrical Emitter Geometries for Increased Range of Stable Electrospray Flow Rates
Maxwell, E. J., Zhong, X., & Chen, D. D. Y. (2010) · Analytical Chemistry · doi:10.1021/ac1017953 · Cited by 50 · see citing works
Heemskerk, A. A. M., Wuhrer, M., Busnel, J., Koeleman, C. A. M., Selman, M. H. J., Vidarsson, G., Kapur, R., Schoenmaker, B., Derks, R. J. E., Deelder, A. M., et al. (2013) · ELECTROPHORESIS · doi:10.1002/elps.201200357 · Cited by 43 · see citing works
Fonslow, B. R., Kang, S. A. W., Gestaut, D. R., Graczyk, B., Davis, T. N., Sabatini, D. M., & Yates, J. R. (2010) · Analytical Chemistry · doi:10.1021/ac101235k · Cited by 42 · see citing works
Self-citationMoini, M., & Huang, H.-Y. (2004) · Electrophoresis · doi:10.1002/elps.200305906 · Cited by 42 · see citing works
Plo1 phosphorylates Dam1 to promote chromosome bi-orientation in fission yeast
Buttrick, G. J., Lancaster, T. C., Meadows, J. C., et al. (2012) · Journal of Cell Science · doi:10.1242/jcs.096826 · Cited by 7 · see citing works
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Chen, B., Brown, K. A., Lin, Z., & Ge, Y. (2018) · Analytical Chemistry · doi:10.1021/acs.analchem.7b04747 · Cited by 210 · see citing works
McCool, E. N., Lubeckyj, R. A., Shen, X., Chen, D., Kou, Q., Liu, X., & Sun, L. (2018) · Analytical Chemistry · doi:10.1021/acs.analchem.8b00693 · Cited by 117 · see citing works
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- Authors
- Han, X., Wang, Y., Aslanian, A., Fonslow, B., Graczyk, B., Davis, T. N., & Yates, J. R.
- Affiliations
- Scripps Research Institute (US)
- University of Washington (US)
- Corresponding author
- Yates, John R.
- Venue
- Journal of Proteome Research
- Publisher
- American Chemical Society (ACS)
- Published
- 2014-11-09
- Volume
- 13
- Issue
- 12
- Pages
- 6078-6086
- ISSN
- 1535-3893 (ISSN-L)
- 1535-3907
- PMC
- PMC4262260
- OpenAlex
- W2323293250
- Cited by
- 72
- References
- 52
- Citations by year
- 2026: 3 · 2025: 5 · 2024: 5 · 2023: 3 · 2022: 10 · 2021: 9 · 2020: 2 · 2019: 12 · 2018: 5 · 2017: 8 · 2016: 8 · 2015: 2
- FWCI
- 2.57
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- Journal of Proteome Research (publishedVersion, other-oa)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health (8P41GM103533-17, R01 MH067880)
- National Heart, Lung, and Blood Institute (HHSN268201000035C)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Capillary Electrophoresis Applications
- Keywords
- top-down mass spectrometry, capillary electrophoresis, Dam1 complex, Saccharomyces cerevisiae, protein complexes, top-down proteomics, phosphorylation stoichiometry, Mps1 kinase, N-terminal processing, post-translational modification, protein isoforms, sensitivity enhancement
- MeSH terms
- Binding Sites, Microtubule-Associated Proteins, Molecular Weight, Phosphorylation, Reproducibility of Results, Protein Serine-Threonine Kinases, Cell Cycle Proteins, Electrophoresis, Capillary, Protein Subunits, Spectrometry, Mass, Electrospray Ionization, Saccharomyces cerevisiae Proteins, Proteomics
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