Quantitation and Identification of Thousands of Human Proteoforms below 30 kDa
Open accessAbstract
Top-down proteomics is capable of identifying and quantitating unique proteoforms through the analysis of intact proteins. We extended the coverage of the label-free technique, achieving differential analysis of whole proteins <30 kDa from the proteomes of growing and senescent human fibroblasts. By integrating improved control software with more instrument time allocated for quantitation of intact ions, we were able to collect protein data between the two cell states, confidently comparing 1577 proteoform levels. To then identify and characterize proteoforms, our advanced acquisition software, named Autopilot, employed enhanced identification efficiency in identifying 1180 unique Swiss-Prot accession numbers at 1% false-discovery rate. This coverage of the low mass proteome is equivalent to the largest previously reported but was accomplished in 23% of the total acquisition time. By maximizing both the number of quantified proteoforms and their identification rate in an integrated software environment, this work significantly advances proteoform-resolved analyses of complex systems.
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Global top-down proteomics using the Orbitrap has enabled identification and quantification of thousands of proteoforms.
“Moreover, global top-down proteomics using the Orbitrap has enabled the identification and quantification of thousands of proteoforms [84,100].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [100]
CAD and HCD are the most prevalent and robust techniques for protein fragmentation, especially for global top-down proteomics studies.
“Collisionally activated dissociation (CAD) and higher-energy collisional dissociation (HCD) are the most prevalent and robust techniques for protein fragmentation especially for global top-down proteomics studies [100,113].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [100]
Top-down proteomics is currently applied mainly to proteoforms smaller than 30 kDa, because high-resolution FTMS performs poorly for larger proteoforms and intact proteomes in the 30-60 kDa range are highly complex.
“top-down proteomics (TDP) is currently mostly applied to the characterization of proteoforms <30 kDa due to the poor performance of high-resolution FTMS for the analysis of larger proteoforms and the high complexity of intact proteomes in the 30–60 kDa mass range.”
From Proton Transfer Charge Reduction Enables High-Throughput Top-Down Analysis of Large Proteoforms (2019) · cites this paper as [17]
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Cites
Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.
Shevchenko, A., Wilm, M., Vorm, O., & Mann, M. F. (1996) · Analytical Chemistry · doi:10.1021/ac950914h · Cited by 9,047 · see citing works
Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a
Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D., & Lowe, S. W. (1997) · Cell · doi:10.1016/s0092-8674(00)81902-9 · Cited by 5,178 · see citing works
Wessel, D., & Flügge, U. (1984) · Analytical Biochemistry · doi:10.1016/0003-2697(84)90782-6 · Cited by 3,997 · see citing works
Gillet, L. C., Navarro, P., Tate, S., Röst, H., Selevsek, N., Reiter, L., Bonner, R., & Aebersold, R. (2012) · Molecular & Cellular Proteomics · doi:10.1074/mcp.o111.016717 · Cited by 2,877 · see citing works
Debacq-Chainiaux, F., Erusalimsky, J. D., Campisi, J., & Toussaint, O. (2009) · Nature Protocols · doi:10.1038/nprot.2009.191 · Cited by 1,668 · see citing works
Proteoform: a single term describing protein complexity
The Consortium for Top Down Proteomics, Smith, L. M., & Kelleher, N. L. (2013) · Nature Methods · doi:10.1038/nmeth.2369 · Cited by 1,528 · see citing works
Self-citationMapping 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
Self-citationA key role for mitochondrial gatekeeper pyruvate dehydrogenase in oncogene-induced senescence
Kaplon, J., Zheng, L., Meissl, K., Chaneton, B., Selivanov, V. A., Mackay, G., van der Burg, S. H., Verdegaal, E. M. E., Cascante, M., Shlomi, T., et al. (2013) · Nature · doi:10.1038/nature12154 · Cited by 633 · see citing works
A novel role for high-mobility group A proteins in cellular senescence and heterochromatin formation
Narita, M., Narita, M., Krizhanovsky, V., Nuñez, S., Chicas, A., Hearn, S. A., Myers, M. P., & Lowe, S. W. (2006) · Cell · doi:10.1016/j.cell.2006.05.052 · Cited by 629 · see citing works
Self-citationThe biology of replicative senescence
Campisi, J. (1997) · European Journal of Cancer · doi:10.1016/s0959-8049(96)00058-5 · Cited by 538 · see citing works
Hebert, A. S., Richards, A. L., Bailey, D. J., Ulbrich, A., Coughlin, E. E., Westphall, M. S., & Coon, J. J. (2014) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m113.034769 · Cited by 528 · see citing works
Many roads lead to oncogene-induced senescence
Courtois-Cox, S., Jones, S. L., & Cichowski, K. (2008) · Oncogene · doi:10.1038/sj.onc.1210950 · Cited by 397 · see citing works
Decision tree–driven tandem mass spectrometry for shotgun proteomics
Swaney, D. L., McAlister, G. C., & Coon, J. J. (2008) · Nature Methods · doi:10.1038/nmeth.1260 · Cited by 312 · see citing works
Chandra, T., Kirschner, K., Thuret, J.-Y., Pope, B. D., Ryba, T., Newman, S., Ahmed, K., Samarajiwa, S. A., Salama, R., Carroll, T., et al. (2012) · Molecular Cell · doi:10.1016/j.molcel.2012.06.010 · Cited by 308 · see citing works
Self-citationTran, J. C., & Doucette, A. A. (2008) · Analytical Chemistry · doi:10.1021/ac702197w · Cited by 206 · see citing works
Bennecke, M., Kriegl, L., Bajbouj, M., Retzlaff, K., Robine, S., Jung, A., Arkan, M. C., Kirchner, T., & Greten, F. R. (2010) · Cancer Cell · doi:10.1016/j.ccr.2010.06.013 · Cited by 186 · see citing works
Approaching complete peroxisome characterization by gas-phase fractionation
Yi, E. C., Marelli, M., Lee, H., Purvine, S. O., Aebersold, R. H., Aitchison, J. D., & Goodlett, D. R. (2002) · Electrophoresis · doi:10.1002/1522-2683(200209)23:18 · Cited by 183 · see citing works
Catherman, A. D., Durbin, K. R., Ahlf, D. R., Early, B. P., Fellers, R. T., Tran, J. C., Thomas, P. M., & Kelleher, N. L. (2013) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m113.030114 · Cited by 157 · see citing works
Self-citationAnsong, 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
Comprehensive Analysis of Protein Modifications by Top-Down Mass Spectrometry
Zhang, H., & Ge, Y. (2011) · Circulation Cardiovascular Genetics · doi:10.1161/circgenetics.110.957829 · Cited by 148 · see citing works
Parks, B. A., Jiang, L., Thomas, P. M., Wenger, C. D., Roth, M. J., Boyne, M. T., Burke, P. V., Kwast, K. E., & Kelleher, N. L. (2007) · Analytical Chemistry · doi:10.1021/ac070553t · Cited by 123 · see citing works
Self-citationApplying Label-Free Quantitation to Top Down Proteomics
Ntai, I., Kim, K., Fellers, R. T., Skinner, O. S., Smith, A. D., Early, B. P., Savaryn, J. P., LeDuc, R. D., Thomas, P. M., & Kelleher, N. L. (2014) · Analytical Chemistry · doi:10.1021/ac500395k · Cited by 102 · see citing works
Self-citationTranscriptional regulation of cellular senescence
Lanigan, F., Geraghty, J. G., & Bracken, A. P. (2011) · Oncogene · doi:10.1038/onc.2011.34 · Cited by 95 · 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
Self-citationMazur, M. T., Cardasis, H. L., Spellman, D. S., Liaw, A., Yates, N. A., & Hendrickson, R. C. (2010) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0910776107 · Cited by 87 · see citing works
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Ntai, I., LeDuc, R. D., Fellers, R. T., Erdmann-Gilmore, P., Davies, S. R., Rumsey, J. M., Early, B. P., Thomas, P. M., Li, S., Compton, P. D., et al. (2015) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m114.047480 · Cited by 85 · see citing works
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Catherman, A. D., Li, M., Tran, J. C., Durbin, K. R., Compton, P. D., Early, B. P., Thomas, P. M., & Kelleher, N. L. (2013) · Analytical Chemistry · doi:10.1021/ac3031527 · Cited by 76 · see citing works
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Meng, F., Wiener, M. C., Sachs, J. R., Burns, C., Verma, P., Paweletz, C. P., Mazur, M. T., Deyanova, E. G., Yates, N. A., & Hendrickson, R. C. (2006) · Journal of the American Society for Mass Spectrometry · doi:10.1016/j.jasms.2006.09.014 · Cited by 74 · see citing works
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Zhang, Q., & Wang, Y. (2010) · Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms · doi:10.1016/j.bbagrm.2009.11.009 · Cited by 73 · see citing works
Improving Qualitative and Quantitative Performance for MSE-based Label-free Proteomics
Bond, N. J., Shliaha, P. V., Lilley, K. S., & Gatto, L. (2013) · Journal of Proteome Research · doi:10.1021/pr300776t · Cited by 69 · see citing works
Weng, M., Lee, J., Wei, S., Li, Q., Shahamatdar, S., Hsu, D. J., Schetter, A. J., Swatkoski, S., Mannan, P., Garfield, S. H., et al. (2012) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1207673110 · Cited by 66 · see citing works
Wu, S., Brown, J. N., Tolić, N., Meng, D., Liu, X., Zhang, H., Zhao, R., Moore, R. J., Pevzner, P. A., Smith, R. D., et al. (2014) · PROTEOMICS · doi:10.1002/pmic.201300378 · Cited by 60 · see citing works
Durbin, K. R., Skinner, O. S., Fellers, R. T., & Kelleher, N. L. (2015) · Journal of the American Society for Mass Spectrometry · doi:10.1007/s13361-015-1078-1 · Cited by 52 · see citing works
Self-citationOncogene‐induced cellular senescence elicits an anti‐ <scp>W</scp> arburg effect
Li, M., Durbin, K. R., Sweet, S. M. M., Tipton, J. D., Zheng, Y., & Kelleher, N. L. (2013) · PROTEOMICS · doi:10.1002/pmic.201200298 · Cited by 43 · see citing works
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Dean, J. P., & Nelson, P. S. (2008) · British Journal of Cancer · doi:10.1038/sj.bjc.6604087 · Cited by 42 · see citing works
Durbin, K. R., Fellers, R. T., Ntai, I., Kelleher, N. L., & Compton, P. D. (2014) · Analytical Chemistry · doi:10.1021/ac402904h · Cited by 35 · see citing works
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Savaryn, J. P., Skinner, O. S., Fornelli, L., Fellers, R. T., Compton, P. D., Terhune, S. S., Abecassis, M. M., & Kelleher, N. L. (2015) · Journal of Proteomics · doi:10.1016/j.jprot.2015.04.025 · Cited by 12 · see citing works
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de Graaf, E. L., Kaplon, J., Zhou, H., Heck, A. J. R., Peeper, D. S., & Altelaar, M. A. F. (2014) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m113.035436 · Cited by 11 · see citing works
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- Authors
- Durbin, K. R., Fornelli, L., Fellers, R. T., Doubleday, P. F., Narita, M., & Kelleher, N. L.
- Affiliations
- Northwestern University (US)
- University of Cambridge (GB)
- Corresponding author
- Kelleher, Neil L.
- ORCID
- Venue
- Journal of Proteome Research
- Publisher
- American Chemical Society (ACS)
- Published
- 2016-02-11
- Volume
- 15
- Issue
- 3
- Pages
- 976-982
- ISSN
- 1535-3893 (ISSN-L)
- 1535-3907
- PMC
- PMC4794255
- OpenAlex
- W2269451983
- Cited by
- 111
- References
- 39
- Citations by year
- 2026: 5 · 2025: 4 · 2024: 4 · 2023: 10 · 2022: 7 · 2021: 12 · 2020: 9 · 2019: 21 · 2018: 10 · 2017: 16 · 2016: 12 · 2015: 1
- FWCI
- 9.80
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Journal of Proteome Research (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Cancer Institute (HHSN261200800001E)
- University of Cambridge
- National Institute on Drug Abuse (P30 DA018310)
- National Institute of General Medical Sciences (GM067193, GM108569)
- Cancer Research UK
- Office for Research, Northwestern University
- Swiss National Science Foundation
- Hutchison Whampoa Limited
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced Biosensing Techniques and Applications
- Keywords
- top-down proteomics, proteoforms, label-free quantification, human fibroblasts, cellular senescence, protein identification
- MeSH terms
- Cells, Cultured, Humans, Software, Cellular Senescence, Protein Isoforms, Proteome, Tandem Mass Spectrometry, Chromatography, Reverse-Phase
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