Skip to main content

Protein Quantitation Using Mass Spectrometry

  • Protocol
  • First Online:
Computational Biology

Abstract

Mass spectrometry is a method of choice for quantifying low-abundance proteins and peptides in many biological studies. Here, we describe a range of computational aspects of protein and peptide quantitation, including methods for finding and integrating mass spectrometric peptide peaks, and detecting interference to obtain a robust measure of the amount of proteins present in samples.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Y. Oda, K. Huang, F.R. Cross, D. Cowburn, and B.T. Chait (1999) Accurate quantitation of protein expression and site-specific phosphorylation, Proc Natl Acad Sci USA, 96, 6591ā€“6.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  2. S.E. Ong, B. Blagoev, I. Kratchmarova, D.B. Kristensen, H. Steen, A. Pandey, and M. Mann (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics, Mol Cell Proteomics, 1, 376ā€“86.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  3. B. Schwanhausser, M. Gossen, G. Dittmar, and M. Selbach (2009) Global analysis of cellular protein translation by pulsed SILAC, Proteomics, 9, 205ā€“9.

    ArticleĀ  PubMedĀ  Google ScholarĀ 

  4. S.P. Gygi, B. Rist, S.A. Gerber, F. Turecek, M.H. Gelb, and R. Aebersold (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags, Nat Biotechnol, 17, 994ā€“9.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  5. O.A. Mirgorodskaya, Y.P. Kozmin, M.I. Titov, R. Korner, C.P. Sonksen, and P. Roepstorff (2000) Quantitation of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry using (18)O-labeled internal standards, Rapid Commun Mass Spectrom, 14, 1226ā€“32.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  6. S.A. Gerber, J. Rush, O. Stemman, M.W. Kirschner, and S.P. Gygi (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS, Proc Natl Acad Sci USA, 100, 6940ā€“5.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  7. P.L. Ross, Y.N. Huang, J.N. Marchese, B. Williamson, K. Parker, S. Hattan, N. Khainovski, S. Pillai, S. Dey, S. Daniels, S. Purkayastha, P. Juhasz, S. Martin, M. Bartlet-Jones, F. He, A. Jacobson, and D.J. Pappin (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents, Mol Cell Proteomics, 3, 1154ā€“69.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  8. R.J. Beynon, M.K. Doherty, J.M. Pratt, and S.J. Gaskell (2005) Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides, Nat Methods, 2, 587ā€“9.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  9. L. Anderson and C.L. Hunter (2006) Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins, Mol Cell Proteomics, 5, 573ā€“88.

    PubMedĀ  CASĀ  Google ScholarĀ 

  10. E.C. Yi, X.J. Li, K. Cooke, H. Lee, B. Raught, A. Page, V. Aneliunas, P. Hieter, D.R. Goodlett, and R. Aebersold (2005) Increased quantitative proteome coverage with (13)C/(12)C-based, acid-cleavable isotope-coded affinity tag reagent and modified data acquisition scheme, Proteomics, 5, 380ā€“7.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  11. P. Schulz-Knappe, H.D. Zucht, G. Heine, M. Jurgens, R. Hess, and M. Schrader (2001) Peptidomics: the comprehensive analysis of peptides in complex biological mixtures, Comb Chem High Throughput Screen, 4, 207ā€“17.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  12. W. Wang, H. Zhou, H. Lin, S. Roy, T.A. Shaler, L.R. Hill, S. Norton, P. Kumar, M. Anderle, and C.H. Becker (2003) QuanĀ­tification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards, Anal Chem, 75, 4818ā€“26.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  13. M.C. Wiener, J.R. Sachs, E.G. Deyanova, and N.A. Yates (2004) Differential mass spectrometry: a label-free LC-MS method for finding significant differences in complex peptide and protein mixtures, Anal Chem, 76, 6085ā€“96.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  14. T.A. Addona, S.E. Abbatiello, B. Schilling, S.J. Skates, D.R. Mani, D.M. Bunk, C.H. Spiegelman, L.J. Zimmerman, A.J. Ham, H. Keshishian, S.C. Hall, S. Allen, R.K. Blackman, C.H. Borchers, C. Buck, H.L. Cardasis, M.P. Cusack, N.G. Dodder, B.W. Gibson, J.M. Held, T. Hiltke, A. Jackson, E.B. Johansen, C.R. Kinsinger, J. Li, M. Mesri, T.A. Neubert, R.K. Niles, T.C. Pulsipher, D. Ransohoff, H. Rodriguez, P.A. Rudnick, D. Smith, D.L. Tabb, T.J. Tegeler, A.M. Variyath, L.J. Vega-Montoto, A. Wahlander, S. Waldemarson, M. Wang, J.R. Whiteaker, L. Zhao, N.L. Anderson, S.J. Fisher, D.C. Liebler, A.G. Paulovich, F.E. Regnier, P. Tempst, and S.A. Carr (2009) Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma, Nat Biotechnol, 27, 633ā€“41.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  15. H. Liu, R.G. Sadygov, and J.R. Yates, 3rd (2004) A model for random sampling and estimation of relative protein abundance in shotgun proteomics, Anal Chem, 76, 4193ā€“201.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  16. Y. Ishihama, Y. Oda, T. Tabata, T. Sato, T. Nagasu, J. Rappsilber, and M. Mann (2005) Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein, Mol Cell Proteomics, 4, 1265ā€“72.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  17. X.J. Li, H. Zhang, J.A. Ranish, and R. Aebersold (2003) Automated statistical analysis of protein abundance ratios from data generated by stable-isotope dilution and tandem mass spectrometry, Anal Chem, 75, 6648ā€“57.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  18. J. Cox and M. Mann (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification, Nat Biotechnol, 26, 1367ā€“72.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  19. J. Cox, I. Matic, M. Hilger, N. Nagaraj, M. Selbach, J.V. Olsen, and M. Mann (2009) A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics, Nat Protoc, 4, 698ā€“705.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  20. P. Mortensen, J.W. Gouw, J.V. Olsen, S.E. Ong, K.T. Rigbolt, J. Bunkenborg, J. Cox, L.J. Foster, A.J. Heck, B. Blagoev, J.S. Andersen, and M. Mann (2010) MSQuant, an open source platform for mass spectrometry-based quantitative proteomics, J Proteome Res, 9(1):393ā€“403.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  21. Z. Khan, J.S. Bloom, B.A. Garcia, M. Singh, and L. Kruglyak (2009) Protein quantification across hundreds of experimental conditions, Proc Natl Acad Sci USA, 106, 15544ā€“8.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  22. A.M. Boehm, S. Putz, D. Altenhofer, A. Sickmann, and M. Falk (2007) Precise protein quantification based on peptide quantification using iTRAQ, BMC Bioinformatics, 8, 214.

    ArticleĀ  PubMedĀ  Google ScholarĀ 

  23. C.J. Mason, T.M. Therneau, J.E. Eckel-Passow, K.L. Johnson, A.L. Oberg, J.E. Olson, K.S. Nair, D.C. Muddiman, and H.R. Bergen, 3rd (2007) A method for automatically interpreting mass spectra of 18O-labeled isotopic clusters, Mol Cell Proteomics, 6, 305ā€“18.

    PubMedĀ  CASĀ  Google ScholarĀ 

  24. B. MacLean, D.M. Tomazela, N. Shulman, M. Chambers, G. Finney, B. Frewen, R. Kern, D.L. Tabb, D.C. Liebler and M.J. Maccoss (2010) Skyline: an open source document editor for creating and analyzing targeted proteomics experiments, Bioinformatics, 26, 966ā€“8.

    Google ScholarĀ 

  25. E.M. Woo, D. Fenyo, B.H. Kwok, H. Funabiki, and B.T. Chait (2008) Efficient identification of phosphorylation by mass spectrometric phosphopeptide fingerprinting, Anal Chem, 80, 2419ā€“25.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

  26. G. Zhang, D. Fenyo, and T.A. Neubert (2009) Evaluation of the variation in sample preparation for comparative proteomics using stable isotope labeling by amino acids in cell culture, J Proteome Res, 8, 1285ā€“92.

    ArticleĀ  PubMedĀ  CASĀ  Google ScholarĀ 

Download references

Acknowledgments

This work was supported by funding provided by the National Institutes of Health Grants RR00862, RR022220, NS050276, and CA126485, the Carl Trygger foundation, and the Swedish research council.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to David Fenyƶ .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

Ā© 2010 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Zhang, G. et al. (2010). Protein Quantitation Using Mass Spectrometry. In: Fenyƶ, D. (eds) Computational Biology. Methods in Molecular Biology, vol 673. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-842-3_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-842-3_13

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-841-6

  • Online ISBN: 978-1-60761-842-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics