On the Scalability and Requirements of Whole Protein Mass Spectrometry
Open accessAbstract
Top-down proteomics has improved over the past decade despite the significant challenges presented by the analysis of large protein ions. Here, the detection of these high mass species by electrospray-based mass spectrometry (MS) is examined from a theoretical perspective to understand the mass-dependent increases in the number of charge states, isotopic peaks, and interfering species present in typical protein mass spectra. Integrating these effects into a quantitative model captures the reduced ability to detect species over 25 kDa with the speed and sensitivity characteristic of proteomics based on <3 kDa peptide ions. The model quantifies the challenge that top-down proteomics faces with respect to current MS instrumentation and projects that depletion of (13)C and (15)N isotopes can improve detection at high mass by only <2-fold at 100 kDa whereas the effect is up to 5-fold at 10 kDa. Further, we find that supercharging electrosprayed proteins to the point of producing <5 charge states at high mass would improve detection by more than 20-fold.
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
The claims and statistics of this paper haven’t been checked yet.
Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Large proteins have inherently lower signal-to-noise and are underrepresented in top-down studies.
“Often large proteins, which have an inherently lower signal-to-noise, are underrepresented in top-down studies [83].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [83]
ESI protein detection suffers from signal competition with small-molecule contaminants that outcompete large molecules, resulting in the need for highly purified samples.
“Although ESI enables the highly sensitive detection of analytes, protein detection by ESI suffers from signal competition with small-molecule contaminates that outcompete large molecules and results in the need for highly purified samples [23,83].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [83]
It remains challenging to detect and quantify large heart proteins such as cMyBP-C (141 kDa) and MHC (223 kDa) because of the exponential decay in MS signal-to-noise ratio observed with increasing molecular weight, combined with coelution of low-molecular-weight or high-abundance proteins.
“It remains challenging to detect and quantify large heart proteins, such as cMyBP-C (141 kDa) (37, 44, 45) and MHC (223 kDa), because of the exponential decay in MS signal-to-noise ratio observed with the increasing molecular weight (46), combined with coelution of low-molecular-weight or high-abundance proteins.”
From Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics (2020) · cites this paper as [46]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
The self-check needs the paper’s full text.
Cites
Electrospray Ionization for Mass Spectrometry of Large Biomolecules
Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., & Whitehouse, C. M. (1989) · Science · doi:10.1126/science.2675315 · Cited by 7,634 · see citing works
Practical implications of some recent studies in electrospray ionization fundamentals
Cech, N. B., & Enke, C. G. (2001) · Mass Spectrometry Reviews · doi:10.1002/mas.10008 · Cited by 1,436 · see citing works
Current Protocols in Bioinformatics
(2018) · Current Protocols in Bioinformatics · doi:10.1002/(issn)1934-340x · Cited by 641 · see citing works
Covey, T. R., Bonner, R. F., Shushan, B. I., Henion, J. D., & Boyd, R. K. (1988) · Rapid Communications in Mass Spectrometry · doi:10.1002/rcm.1290021111 · Cited by 593 · see citing works
Zhang, Z., & Marshall, A. G. (1998) · Journal of the American Society for Mass Spectrometry · doi:10.1016/s1044-0305(97)00284-5 · Cited by 511 · see citing works
Automated reduction and interpretation of
Horn, D. M., Zubarev, R. A., & McLafferty, F. W. (2000) · Journal of the American Society for Mass Spectrometry · doi:10.1016/s1044-0305(99)00157-9 · Cited by 507 · see citing works
Senko, M. W., Beu, S. C., & McLaffertycor, F. W. (1995) · Journal of the American Society for Mass Spectrometry · doi:10.1016/1044-0305(95)00017-8 · Cited by 451 · see citing works
Effect of reducing disulfide-containing proteins on electrospray ionization mass spectra
Loo, J. A., Edmonds, C. G., Udseth, H. R., & Smith, R. D. (1990) · Analytical Chemistry · doi:10.1021/ac00206a009 · Cited by 274 · see citing works
Light‐Wahl, K. J., Schwartz, B. L., & Smith, R. (1994) · Journal of the American Chemical Society · doi:10.1021/ja00091a035 · Cited by 262 · see citing works
Loo, J. A., Udseth, H. R., Smith, R. D., & Futrell, J. H. (1988) · Rapid Communications in Mass Spectrometry · doi:10.1002/rcm.1290021006 · Cited by 241 · see citing works
Dobó, A., & Kaltashov, I. A. (2001) · Analytical Chemistry · doi:10.1021/ac010713f · Cited by 237 · see citing works
Supercharged Protein and Peptide Ions Formed by Electrospray Ionization
Iavarone, A. T., Jurchen, J. C., & Williams, E. R. (2001) · Analytical Chemistry · doi:10.1021/ac001251t · Cited by 226 · see citing works
Wall, D. B., Kachman, M. T., Gong, S., Hinderer, R., Parus, S., Misek, D. E., Hanash, S., & Lubman, D. M. (2000) · Analytical Chemistry · doi:10.1021/ac991332t · Cited by 211 · see citing works
Tran, J. C., & Doucette, A. A. (2008) · Analytical Chemistry · doi:10.1021/ac702197w · Cited by 206 · see citing works
Isotopic distributions in mass spectra of large molecules
Yergey, J. A., Heller, D. N., Hansen, G., Cotter, R. J., & Fenselau, C. (1983) · Analytical Chemistry · doi:10.1021/ac00253a037 · Cited by 202 · see citing works
Iavarone, A. T., Jurchen, J. C., & Williams, E. R. (2000) · Journal of the American Society for Mass Spectrometry · doi:10.1016/s1044-0305(00)00169-0 · Cited by 199 · see citing works
Rapid Calculation of Isotope Distributions
Rockwood, A. L., Orden, S. L. V., & Smith, R. (1995) · Analytical Chemistry · doi:10.1021/ac00111a031 · Cited by 130 · 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-citationProtein Molecular Mass to 1 Da by 13C, 15N Double-Depletion and FT-ICR Mass Spectrometry
Marshall, A. G., Senko, M. W., Li, W., Li, M., Dillon, S. R., Guan, S., & Logan, T. M. (1997) · Journal of the American Chemical Society · doi:10.1021/ja9630046 · Cited by 110 · 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
Self-citationLimbach, P. A., Grosshans, P. B., & Marshall, A. G. (1993) · Analytical Chemistry · doi:10.1021/ac00050a008 · Cited by 95 · see citing works
Improved fourier-transform ion-cyclotron-resonance mass spectrometry of large biomolecules
Beu, S. C., Senko, M. W., Quinn, J. P., & McLafferty, F. W. (1993) · Journal of the American Society for Mass Spectrometry · doi:10.1016/1044-0305(93)85077-b · Cited by 84 · see citing works
Proteomic profiling of intact proteins using WAX-RPLC 2-D separations and FTICR mass spectrometry.
Sharma, S., Simpson, D., Toliċ, N., Jaitly, N., Mayampurath, A., Smith, R. D., & Paša-Tolić, L. (2007) · Journal of Proteome Research · doi:10.1021/pr060354a · Cited by 77 · see citing works
Valeja, S. G., Tipton, J. D., Emmett, M. R., & Marshall, A. G. (2010) · Analytical Chemistry · doi:10.1021/ac1016858 · Cited by 76 · 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
Meng, F., Du, Y., Miller, L. M., Patrie, S. M., Robinson, D. E., & Kelleher, N. L. (2004) · Analytical Chemistry · doi:10.1021/ac0354903 · Cited by 67 · see citing works
Self-citationLoo, J. A., Udseth, H. R., & Smith, R. (1988) · Journal of Mass Spectrometry · doi:10.1002/bms.1200170511 · Cited by 63 · see citing works
Formation of peptide aggregates during ESI: Size, charge, composition, and contributions to noise
Counterman, A. E., Hilderbrand, A. E., Barnes, C. A. S., et al. (2001) · Journal of the American Society for Mass Spectrometry · doi:10.1016/s1044-0305(01)00283-5 · Cited by 62 · see citing works
Durbin, K. R., Tran, J. C., Zamdborg, L., Sweet, S. M. M., Catherman, A. D., Lee, J. E., Li, M., Kellie, J. F., & Kelleher, N. L. (2010) · PROTEOMICS · doi:10.1002/pmic.201000177 · Cited by 57 · see citing works
Self-citationHofstadler, S. A., Bruce, J. E., Rockwood, A. L., Anderson, G., Winger, B. E., & Smith, R. D. (1994) · International Journal of Mass Spectrometry and Ion Processes · doi:10.1016/0168-1176(94)85020-8 · Cited by 55 · see citing works
The role of conformational flexibility on protein supercharging in native electrospray ionization
Sterling, H. J., Cassou, C. A., Trnka, M. J., Burlingame, A. L., Krantz, B. A., & Williams, E. R. (2011) · Physical Chemistry Chemical Physics · doi:10.1039/c1cp20277d · Cited by 55 · see citing works
High Pressure (>1 atm) Electrospray Ionization Mass Spectrometry
Chen, L. C., Mandal, M. K., & Hiraoka, K. (2011) · Journal of the American Society for Mass Spectrometry · doi:10.1007/s13361-010-0058-8 · Cited by 52 · see citing works
LeDuc, R. D., & Kelleher, N. L. (2007) · Current Protocols in Bioinformatics · doi:10.1002/0471250953.bi1306s19 · Cited by 42 · see citing works
Self-citationIslas, C. A., Suelves, I., Carter, J. F., Herod, A. A., & Kandiyoti, R. (2000) · Rapid Communications in Mass Spectrometry · doi:10.1002/1097-0231(20001015)14:19 · Cited by 35 · see citing works
Gardner, M. W., & Brodbelt, J. S. (2009) · Journal of the American Society for Mass Spectrometry · doi:10.1016/j.jasms.2009.08.008 · Cited by 10 · see citing works
Xiong, Y., Schroeder, K. T., Greenbaum, N. L., et al. (2004) · Analytical Chemistry · doi:10.1021/ac030299e · Cited by 8 · see citing works
Flagged first, then by citations
Research works printed but not linked: 4
- [16]Hofstadler SA Bruce JE Rockwood AL Anderson GA Winger BE Smith RD International Journal of Mass Spectrometry 1994 132 109 127
- [21]Marshall AG Senko MW Li WQ Li M Dillon S Guan SH Logan TM Journal of the American Chemical Society 1997 119 433 434
- [33]Counterman AE Hilderbrand AE Barnes CAS Clemmer DE Journal of the American Society for Mass Spectrometry 2001 12 1020 1035
- [34]Lightwahl KJ Schwartz BL Smith RD Journal of the American Chemical Society 1994 116 5271 5278
Cited by
Sorted by citations
Progress in Top-Down Proteomics and the Analysis of Proteoforms
Toby, T. K., Fornelli, L., & Kelleher, N. L. (2016) · Annual Review of Analytical Chemistry · doi:10.1146/annurev-anchem-071015-041550 · Cited by 576 · see citing works
Self-citationTop Down proteomics: Facts and perspectives
Catherman, A. D., Skinner, O. S., & Kelleher, N. L. (2014) · Biochemical and Biophysical Research Communications · doi:10.1016/j.bbrc.2014.02.041 · Cited by 507 · see citing works
“Sensitivity is also vital, as high molecular weight species such as proteins will have broad isotopic distributions, distributing the signal from a single protein across many peaks [71].”
BackgroundSelf-citationBest practices and benchmarks for intact protein analysis for top-down mass spectrometry
Donnelly, D. P., Rawlins, C. M., DeHart, C. J., Fornelli, L., Schachner, L. F., Lin, Z., Lippens, J. L., Aluri, K. C., Sarin, R., Chen, B., et al. (2019) · Nature Methods · doi:10.1038/s41592-019-0457-0 · Cited by 377 · see citing works
Self-citationTop‐down proteomics in health and disease: Challenges and opportunities
Gregorich, Z. R., & Ge, Y. (2014) · PROTEOMICS · doi:10.1002/pmic.201300432 · Cited by 217 · see citing works
Identification and Quantification of Proteoforms by Mass Spectrometry
Schaffer, L. V., Millikin, R. J., Miller, R. M., Anderson, L. C., Fellers, R. T., Ge, Y., Kelleher, N. L., LeDuc, R. D., Liu, X., Payne, S. H., et al. (2019) · PROTEOMICS · doi:10.1002/pmic.201800361 · Cited by 209 · see citing works
Self-citationTop-Down Proteomics: Ready for Prime Time?
Chen, B., Brown, K. A., Lin, Z., & Ge, Y. (2018) · Analytical Chemistry · doi:10.1021/acs.analchem.7b04747 · Cited by 210 · see citing works
Top-down proteomics: challenges, innovations, and applications in basic and clinical research
Brown, K. A., Melby, J. A., Roberts, D. S., & Ge, Y. (2020) · Expert Review of Proteomics · doi:10.1080/14789450.2020.1855982 · Cited by 152 · see citing works
Cai, W., Tucholski, T., Chen, B., Alpert, A. J., McIlwain, S., Kohmoto, T., Jin, S., & Ge, Y. (2017) · Analytical Chemistry · doi:10.1021/acs.analchem.7b00380 · Cited by 135 · see citing works
Tucholski, T., Cai, W., Gregorich, Z. R., Bayne, E. F., Mitchell, S. D., McIlwain, S. J., de Lange, W. J., Wrobbel, M., Karp, H., Hite, Z., et al. (2020) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2006764117 · Cited by 107 · see citing works
Peng, Y., Gregorich, Z. R., Valeja, S. G., Zhang, H., Cai, W., Chen, Y.-C., Guner, H., Chen, A. J., Schwahn, D. J., Hacker, T. A., et al. (2014) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m114.040675 · Cited by 107 · see citing works
Top-down Proteomics: Technology Advancements and Applications to Heart Diseases
Cai, W., Tucholski, T. M., Gregorich, Z. R., & Ge, Y. (2016) · Expert Review of Proteomics · doi:10.1080/14789450.2016.1209414 · Cited by 99 · 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
Self-citationAnderson, L. C., DeHart, C. J., Kaiser, N. K., Fellers, R. T., Smith, D. F., Greer, J. B., LeDuc, R. D., Blakney, G. T., Thomas, P. M., Kelleher, N. L., et al. (2017) · Journal of Proteome Research · doi:10.1021/acs.jproteome.6b00696 · Cited by 94 · see citing works
Self-citationProton Transfer Charge Reduction Enables High-Throughput Top-Down Analysis of Large Proteoforms
Huguet, R., Mullen, C., Srzentić, K., Greer, J. B., Fellers, R. T., Zabrouskov, V., Syka, J. E. P., Kelleher, N. L., & Fornelli, L. (2019) · Analytical Chemistry · doi:10.1021/acs.analchem.9b03925 · Cited by 94 · see citing works
Self-citationFornelli, L., Durbin, K. R., Fellers, R. T., Early, B. P., Greer, J. B., LeDuc, R. D., Compton, P. D., & Kelleher, N. L. (2017) · Journal of Proteome Research · doi:10.1021/acs.jproteome.6b00698 · Cited by 88 · see citing works
Self-citationFLASHDeconv: Ultrafast, High-Quality Feature Deconvolution for Top-Down Proteomics
Jeong, K., Kim, J., Gaikwad, M., Hidayah, S. N., Heikaus, L., Schlüter, H., & Kohlbacher, O. (2020) · Cell Systems · doi:10.1016/j.cels.2020.01.003 · Cited by 82 · see citing works
Takemori, A., Butcher, D. S., Harman, V. M., Brownridge, P., Shima, K., Higo, D., Ishizaki, J., Hasegawa, H., Suzuki, J., Yamashita, M., et al. (2020) · Journal of Proteome Research · doi:10.1021/acs.jproteome.0c00303 · Cited by 90 · 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-citationA comprehensive pipeline for translational top-down proteomics from a single blood draw
Toby, T. K., Fornelli, L., Srzentić, K., DeHart, C. J., Levitsky, J., Friedewald, J., & Kelleher, N. L. (2019) · Nature Protocols · doi:10.1038/s41596-018-0085-7 · Cited by 86 · see citing works
Self-citationMelani, R. D., Skinner, O. S., Fornelli, L., Domont, G. B., Compton, P. D., & Kelleher, N. L. (2016) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m115.056523 · Cited by 85 · see citing works
Self-citationCai, W., Zhang, J., de Lange, W. J., Gregorich, Z. R., Karp, H., Farrell, E. T., Mitchell, S. D., Tucholski, T., Lin, Z., Biermann, M., et al. (2019) · Circulation Research · doi:10.1161/circresaha.119.315305 · Cited by 78 · see citing works
Lubeckyj, R. A., Basharat, A. R., Shen, X., Liu, X., & Sun, L. (2019) · Journal of the American Society for Mass Spectrometry · doi:10.1007/s13361-019-02167-w · Cited by 72 · see citing works
Specific Enrichment of Phosphoproteins Using Functionalized Multivalent Nanoparticles
Hwang, L., Ayaz-Guner, S., Gregorich, Z. R., Cai, W., Valeja, S. G., Jin, S., & Ge, Y. (2015) · Journal of the American Chemical Society · doi:10.1021/ja511833y · Cited by 70 · see citing works
Native vs Denatured: An in Depth Investigation of Charge State and Isotope Distributions
Kafader, J. O., Melani, R. D., Schachner, L. F., Ives, A. N., Patrie, S. M., Kelleher, N. L., & Compton, P. D. (2020) · Journal of the American Society for Mass Spectrometry · doi:10.1021/jasms.9b00040 · Cited by 51 · see citing works
Self-citationTucholski, T., Knott, S. J., Chen, B., Pistono, P., Lin, Z., & Ge, Y. (2019) · Analytical Chemistry · doi:10.1021/acs.analchem.8b04082 · Cited by 49 · see citing works
Ugrin, S. A., English, A. M., Syka, J. E. P., Bai, D. L., Anderson, L. C., Shabanowitz, J., & Hunt, D. F. (2019) · Journal of the American Society for Mass Spectrometry · doi:10.1007/s13361-019-02290-8 · Cited by 44 · see citing works
Intact-Mass Analysis Facilitating the Identification of Large Human Heart Proteoforms
Schaffer, L. V., Tucholski, T., Shortreed, M. R., Ge, Y., & Smith, L. M. (2019) · Analytical Chemistry · doi:10.1021/acs.analchem.9b02343 · Cited by 17 · see citing works
- Authors
- Compton, P. D., Zamdborg, L., Thomas, P. M., & Kelleher, N. L.
- Affiliations
- Northwestern University (US)
- Corresponding author
- Kelleher, Neil L.
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2011-07-29
- Volume
- 83
- Issue
- 17
- Pages
- 6868-6874
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC3165072
- OpenAlex
- W2010033716
- Cited by
- 243
- References
- 36
- Citations by year
- 2026: 14 · 2025: 20 · 2024: 27 · 2023: 21 · 2022: 18 · 2021: 11 · 2020: 22 · 2019: 16 · 2018: 17 · 2017: 17 · 2016: 18 · 2015: 7 · 2014: 12 · 2013: 10 · 2012: 9
- FWCI
- 5.08
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institute on Drug Abuse (DA026672, F30DA026672, P30DA018310)
- National Institute of General Medical Sciences (GM067193-09, R01GM067193)
- 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 › Metabolomics and Mass Spectrometry Studies
- Keywords
- top-down proteomics, electrospray ionization, charge state distribution, isotope depletion, supercharging, detection sensitivity, scalability
- MeSH terms
- Carbon Isotopes, Isotope Labeling, Nitrogen Isotopes, Proteins, Spectrometry, Mass, Electrospray Ionization, Proteomics
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
