Top-Down Analysis of Branched Proteins Using Mass Spectrometry
Open accessAbstract
Post-translational modifications by the covalent attachment of Rub1 (NEDD8), ubiquitin, SUMO, and other small signaling proteins have profound impacts on the functions and fates of cellular proteins. Investigations of the relationship of these bioactive structures and their functions are limited by analytical methods that are scarce and tedious. A novel strategy is reported here for the analysis of branched proteins by top-down mass spectrometry and illustrated by application to four recombinant proteins and one synthetic peptide modified by covalent bonds with ubiquitin or Rub1. The approach allows an analyte to be recognized as a branched protein; the participating proteins to be identified; the site of conjugation to be defined; and other chemical, native, and recombinant modifications to be characterized. In addition to the high resolution and high accuracy provided by the mass spectrometer, success is based on sample fragmentation by electron-transfer dissociation assisted by collisional activation and on software designed for graphic interpretation and adapted for branched proteins. The strategy allows for structures of unknown, two-component branched proteins to be elucidated directly the first time and can potentially be extended to more complex systems.
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TDMS is well suited for identifying and characterizing branched systems consisting of two proteins.
“In addition, as demonstrated by Gomes and co-workers33,34, TDMS is well-suited for identification and characterization of branched systems consisting of two proteins.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [34]
TDMS is now used in a wide range of applications, including production of therapeutic proteins such as antibody drug conjugates.
“TDMS is now being used in a wide range of applications, including the production of therapeutic proteins, such as antibody drug conjugates34.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [34]
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Cites
Systematic and Quantitative Assessment of the Ubiquitin-Modified Proteome
Kim, W., Bennett, E. J., Huttlin, E. L., Guo, A., Li, J., Possemato, A. P., Sowa, M. E., Rad, R., Rush, A. J., Comb, M. J., et al. (2011) · Molecular Cell · doi:10.1016/j.molcel.2011.08.025 · Cited by 1,582 · see citing works
Proteasome-Independent Functions of Ubiquitin in Endocytosis and Signaling
Mukhopadhyay, D., & Riezman, H. (2007) · Science · doi:10.1126/science.1127085 · Cited by 1,221 · see citing works
Ubiquitin-like Protein Conjugation: Structures, Chemistry, and Mechanism
Cappadocia, L., & Lima, C. D. (2017) · Chemical Reviews · doi:10.1021/acs.chemrev.6b00737 · Cited by 548 · see citing works
The Ubiquitin System, an Immense Realm
Varshavsky, A. J. (2012) · Annual Review of Biochemistry · doi:10.1146/annurev-biochem-051910-094049 · Cited by 278 · see citing works
Controlled Synthesis of Polyubiquitin Chains
Pickart, C. M., & Raasi, S. (2005) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/s0076-6879(05)99002-2 · Cited by 266 · see citing works
ProSight Lite: Graphical software to analyze top‐down mass spectrometry data
Fellers, R. T., Greer, J. B., Early, B. P., Yu, X., LeDuc, R. D., Kelleher, N. L., & Thomas, P. M. (2015) · PROTEOMICS · doi:10.1002/pmic.201400313 · Cited by 247 · see citing works
A conserved catalytic residue in the ubiquitin‐conjugating enzyme family
Wu, P., Hanlon, M., Eddins, M., Tsui, C., Rogers, R. S., Jensen, J. P., Matunis, M. J., Weissman, A. M., Wolberger, C., & Pickart, C. M. (2003) · The EMBO Journal · doi:10.1093/emboj/cdg501 · Cited by 193 · see citing works
Regulation of p53 by the Ubiquitin-conjugating Enzymes UbcH5B/C in Vivo
Saville, M. K., Sparks, A., Xirodimas, D. P., Wardrop, J., Stevenson, L. F., Bourdon, J.-C., Woods, Y. L., & Lane, D. P. (2004) · Journal of Biological Chemistry · doi:10.1074/jbc.m403362200 · Cited by 158 · see citing works
SUMO: of branched proteins and nuclear bodies
Seeler, J.-S., & Dejean, A. (2001) · Oncogene · doi:10.1038/sj.onc.1204758 · Cited by 153 · see citing works
Bondarenko, P. V., Second, T. P., Zabrouskov, V., Makarov, A., & Zhang, Z. (2009) · Journal of the American Society for Mass Spectrometry · doi:10.1016/j.jasms.2009.03.020 · Cited by 151 · see citing works
MASH Suite Pro: A Comprehensive Software Tool for Top-Down Proteomics
Cai, W., Guner, H., Gregorich, Z. R., Chen, A. J., Ayaz-Guner, S., Peng, Y., Valeja, S. G., Liu, X., & Ge, Y. (2016) · Molecular & Cellular Proteomics · doi:10.1074/mcp.o115.054387 · Cited by 132 · see citing works
Cover Picture: Proteomics 7'15
Fellers, R. T., Greer, J. B., Early, B. P., Yu, X., LeDuc, R. D., Kelleher, N. L., & Thomas, P. M. (2015) · PROTEOMICS · doi:10.1002/pmic.201570050 · Cited by 122 · see citing works
Brunner, A. M., Lössl, P., Liu, F., Huguet, R., Mullen, C., Yamashita, M., Zabrouskov, V., Makarov, A., Altelaar, M. A. F., & Heck, A. J. R. (2015) · Analytical Chemistry · doi:10.1021/acs.analchem.5b00162 · Cited by 121 · see citing works
Li, H., Wolff, J. J., Orden, S. L. V., & Loo, J. A. (2013) · Analytical Chemistry · doi:10.1021/ac4033214 · Cited by 119 · see citing works
Mixed-Linkage Ubiquitin Chains Send Mixed Messages
Nakasone, M. A., Livnat‐Levanon, N., Glickman, M. H., et al. (2013) · Structure · doi:10.1016/j.str.2013.02.019 · Cited by 101 · see citing works
Castañeda, C. A., Liu, J., Chaturvedi, A., Nowicka, U., Cropp, T. A., & Fushman, D. (2011) · Journal of the American Chemical Society · doi:10.1021/ja207220g · Cited by 96 · see citing works
Self-citationBush, D. R., Zang, L., Belov, A. M., Ivanov, A. R., & Karger, B. L. (2016) · Analytical Chemistry · doi:10.1021/acs.analchem.5b03218 · Cited by 92 · see citing works
Activated Ion-Electron Transfer Dissociation Enables Comprehensive Top-Down Protein Fragmentation
Riley, N. M., Westphall, M. S., & Coon, J. J. (2017) · Journal of Proteome Research · doi:10.1021/acs.jproteome.7b00249 · Cited by 79 · see citing works
Fornelli, L., Ayoub, D., Aizikov, K., Liu, X., Damoc, E., Pevzner, P. A., Makarov, A., Beck, A., & Tsybin, Y. O. (2017) · Journal of Proteomics · doi:10.1016/j.jprot.2017.02.013 · Cited by 70 · see citing works
Li, H., Sheng, Y., McGee, W. M., Cammarata, M. B., Holden, D. D., & Loo, J. A. (2017) · Analytical Chemistry · doi:10.1021/acs.analchem.6b02377 · Cited by 62 · see citing works
Ubiquitin-Modifying Enzymes and Regulation of the Inflammasome
Kattah, M. G., Malynn, B. A., & Ma, A. I. (2017) · Journal of Molecular Biology · doi:10.1016/j.jmb.2017.10.001 · Cited by 55 · see citing works
Sequencing Larger Intact Proteins (30–70 kDa) with Activated Ion Electron Transfer Dissociation
Riley, N., Westphall, M., & Coon, J. (2017) · Journal of the American Society for Mass Spectrometry · doi:10.1007/s13361-017-1808-7 · Cited by 55 · see citing works
Vogel, R. I., Coughlin, K. A., Scotti, A., Iizuka, Y., Anchoori, R. K., Roden, R. B. S., Marastoni, M., & Bazzaro, M. (2015) · Oncotarget · doi:10.18632/oncotarget.2904 · Cited by 51 · see citing works
Singh, R. K., Zerath, S., Kleifeld, O., Scheffner, M., Glickman, M. H., & Fushman, D. (2012) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m112.022467 · Cited by 51 · see citing works
Self-citationIlluminating the dark matter of shotgun proteomics
Skinner, O. S., & Kelleher, N. L. (2015) · Nature Biotechnology · doi:10.1038/nbt.3287 · Cited by 49 · see citing works
Cannon, J. R., Martinez‐Fonts, K., Robotham, S. A., Matouschek, A. T., & Brodbelt, J. S. (2015) · Analytical Chemistry · doi:10.1021/ac5038363 · Cited by 46 · see citing works
Holden, D. D., McGee, W. M., & Brodbelt, J. S. (2015) · Analytical Chemistry · doi:10.1021/acs.analchem.5b03911 · Cited by 43 · see citing works
Varadan, R., Assfalg, M., & Fushman, D. (2005) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/s0076-6879(05)99012-5 · Cited by 37 · see citing works
Self-citationInsights into the Molecular Composition of Endogenous Unanchored Polyubiquitin Chains
Strachan, J., Roach, L. V., Sokratous, K., Tooth, D., Long, J., Garner, T. P., Searle, M. S., Oldham, N. J., & Layfield, R. (2012) · Journal of Proteome Research · doi:10.1021/pr201167n · Cited by 35 · see citing works
Morris, J. R., & Garvin, A. J. (2017) · Journal of Molecular Biology · doi:10.1016/j.jmb.2017.05.012 · Cited by 32 · see citing works
Proteomic Identification and Analysis of K63-Linked Ubiquitin Conjugates
Cannon, J. R., Nakasone, M. A., Fushman, D., et al. (2012) · Analytical Chemistry · doi:10.1021/ac302675y · Cited by 17 · see citing works
Self-citationLee, A. E., Geis‐Asteggiante, L., Dixon, E. K., Kim, Y., Kashyap, T. R., Wang, Y., Fushman, D., & Fenselau, C. (2016) · Journal of Mass Spectrometry · doi:10.1002/jms.3759 · Cited by 11 · see citing works
Self-citationPreparing to read the ubiquitin code: top‐down analysis of unanchored ubiquitin tetramers
Lee, A. E., Geis‐Asteggiante, L., Dixon, E. K., Miller, M. B., Wang, Y., Fushman, D., & Fenselau, C. (2016) · Journal of Mass Spectrometry · doi:10.1002/jms.3787 · Cited by 11 · see citing works
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- [9]Bazzaro M Oncotarget, 2015, 6, 4159– 4165. 25784654 PMC4414179
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Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research
Gomes, F. P., & Yates, J. R. (2019) · Mass Spectrometry Reviews · doi:10.1002/mas.21599 · Cited by 112 · see citing works
Self-citationTop‐down analysis of novel synthetic branched proteins
Gomes, F., Lemma, B., Abeykoon, D., Chen, D., Wang, Y., Fushman, D., & Fenselau, C. (2019) · Journal of Mass Spectrometry · doi:10.1002/jms.4303 · Cited by 7 · see citing works
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- Authors
- Chen, D., Gomes, F., Abeykoon, D., Lemma, B., Wang, Y., Fushman, D., & Fenselau, C.
- Affiliations
- University of Maryland, College Park (US)
- University of Maryland, College Park, Maryland 20742, United States
- Corresponding author
- Fenselau, Catherine
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2018-03-07
- Volume
- 90
- Issue
- 6
- Pages
- 4032-4038
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC6146919
- OpenAlex
- W2790832597
- Cited by
- 14
- References
- 33
- Citations by year
- 2025: 1 · 2024: 1 · 2023: 1 · 2022: 1 · 2021: 2 · 2020: 2 · 2019: 4 · 2018: 2
- FWCI
- 1.08
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- NIH Office of the Director (OD019938)
- National Institute of General Medical Sciences (GM021248, GM065334)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
- Keywords
- top-down mass spectrometry, post-translational modification, neddylation, ubiquitin conjugation, SUMO, electron transfer dissociation, collisional activation, protein structure elucidation
- MeSH terms
- NEDD8 Protein, Amino Acid Sequence, Humans, Models, Molecular, Protein Processing, Post-Translational, Proteins, Recombinant Proteins, Saccharomyces cerevisiae, Ubiquitins, Ubiquitin, Saccharomyces cerevisiae Proteins, PTEN Phosphohydrolase, Tandem Mass Spectrometry, Ubiquitination
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