Mass Spectrometry for Protein Quantification in Biomarker Discovery

  • Mu WangEmail author
  • Jinsam You
Part of the Methods in Molecular Biology book series (MIMB, volume 815)


Major technological advances have made proteomics an extremely active field for biomarker discovery in recent years due primarily to the development of newer mass spectrometric technologies and the explosion in genomic and protein bioinformatics. This leads to an increased emphasis on larger scale, faster, and more efficient methods for detecting protein biomarkers in human tissues, cells, and biofluids. Most current proteomic methodologies for biomarker discovery, however, are not highly automated and are generally labor-intensive and expensive. More automation and improved software programs capable of handling a large amount of data are essential to reduce the cost of discovery and to increase throughput. In this chapter, we discuss and describe mass spectrometry-based proteomic methods for quantitative protein analysis.

Key words

Biomarkers Proteomics Mass spectrometry Stable isotope labeling Label-free protein quantification 



The authors would like to thank Ms. Heather Sahm for critical reading of this book chapter.


  1. 1.
    Blackstock, W. P. and Weir, M. P. (1999) Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotechnol. 17, 121–127.PubMedCrossRefGoogle Scholar
  2. 2.
    Gygi, S. P., Rist, B., and Aebersold, R. (2000) Measuring gene expression by quantitative proteome analysis. Curr. Opin. Biotechnol. 11, 396–401.PubMedCrossRefGoogle Scholar
  3. 3.
    Rabilloud, T. (2002) Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains. Proteomics 2, 3–10.PubMedCrossRefGoogle Scholar
  4. 4.
    Conrads, T. P., Issaq, H. J., and Veenstra, T. D. (2002) New tools for quantitative phosphoproteome analysis. Biochem. Biophys. Res. Commun. 290, 885–890.PubMedCrossRefGoogle Scholar
  5. 5.
    Ong, S. E., Foster, L. J., and Mann, M. (2003) Mass spectrometric-based approaches in quantitative proteomics. Methods 29, 124–130.PubMedCrossRefGoogle Scholar
  6. 6.
    Tao, W. A. and Aebersold, R. (2003) Advances in quantitative proteomics via stable isotope tagging and mass spectrometry. Curr. Opin. Biotechnol. 14, 110–118.PubMedCrossRefGoogle Scholar
  7. 7.
    McDonald, W. H. and Yates, J. R., 3 rd. (2002) Shotgun proteomics and biomarker discovery. Dis. Markers 18, 99–105.PubMedGoogle Scholar
  8. 8.
    Wu, C. C., MacCoss, M. J., Howell, K. E., and Yates, J. R., 3 rd. (2003) A method for the comprehensive proteomic analysis of membrane proteins. Nat. Biotechnol. 21, 532–538.PubMedCrossRefGoogle Scholar
  9. 9.
    Washburn, M. P., Ulaszek, R., Deciu, C., Schieltz, D. M., and Yates, J. R., 3 rd. (2002) Analysis of quantitative proteomic data generated via multidimensional protein identification technology. Anal. Chem. 74, 1650–1657.PubMedCrossRefGoogle Scholar
  10. 10.
    Washburn, M. P., Wolters, D., and Yates, J. R., 3 rd. (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247.PubMedCrossRefGoogle Scholar
  11. 11.
    Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R. (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999.PubMedCrossRefGoogle Scholar
  12. 12.
    Yan, W. and Chen, S. S. (2005) Mass spectrometry-based quantitative proteomic profiling. Brief. Funct. Genomic. Proteomics 4, 27–38.CrossRefGoogle Scholar
  13. 13.
    Zhang, B., VerBerkmoes, N. C., Langston, M. A., Uberbacher, E., Hettich, R. L., and Samatova, N. F. (2006) Detecting differential and correlated protein expression in label-free shotgun proteomics. J. Proteome Res. 5, 2909–2918.PubMedCrossRefGoogle Scholar
  14. 14.
    Wang, G., Wu, W. W., Zeng, W., Chou, C-L., and Shen, R-F. (2006) Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: reproducibility, linearity, and application with complex proteomes. J. Proteome Res. 5, 1214–1223.PubMedCrossRefGoogle Scholar
  15. 15.
    Ono, M., Shitashige, M., Honda, K., Isobe, T., Kuwabara, H., Matsuzuki, H., et al. (2006) Label-free quantitative proteomics using large peptide data sets generated by nanoflow liquid chromatography and mass spectrometry. Mol. Cell. Proteomics 5, 1338–1347.PubMedCrossRefGoogle Scholar
  16. 16.
    Li, J., Steen, H., and Gygi, S. P. (2003) Protein Profiling with Cleavable Isotope-coded Affinity Tag (cICAT) Reagents: The Yeast Salinity Stress Response. Mol. Cell. Proteomics 2, 1198–1204.PubMedCrossRefGoogle Scholar
  17. 17.
    Ross, P. L., Huang, Y. N., Marchese, J. N., Williamson, B., Parker, K., Hattan, S., et al. (2004) Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents. Mol. Cell. Proteomics 3, 1154–1169.PubMedCrossRefGoogle Scholar
  18. 18.
    Oda, Y., Huang, K., Cross, F. R., Cowburn, D., and Chait, B. T. (1999) Accurate Quantitation of Protein Expression and Site-Specific Phosphorylation. Proc. Natl. Acad. Sci. USA. 96, 6591–6596.PubMedCrossRefGoogle Scholar
  19. 19.
    Ong, S., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A., et al. (2002) Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics. Mol. Cell. Proteomics 1, 376–386.PubMedCrossRefGoogle Scholar
  20. 20.
    Ong, S., Kratchmarova, I., and Mann, M. (2003) Properties of 13C-substituted Arginine in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC). J. Proteome Res. 2, 173–181.PubMedCrossRefGoogle Scholar
  21. 21.
    Moulder, R., Lonnberg, T., Elo, L. L., Filen, J. J., Rainio, E., Corthals, G., et al. (2010) Quantitative proteomics analysis of the nuclear fraction of human CD4+ cells in the early phases of IL-4-induced Th2 differentiation. Mol. Cell. Proteomics 9, 1937–1953.PubMedCrossRefGoogle Scholar
  22. 22.
    Collier, T. S., Sarkar, P., Rao, B., and Muddiman, D. C. (2010) Quantitative Top-down Proteomics of SILAC Labeled Human Embryonic Stem Cells. J. Am. Soc. Mass. Spectrom. 21, 879–889.PubMedCrossRefGoogle Scholar
  23. 23.
    Imami, K., Sugiyama, N., Tomita, M., and Ishihama, Y. (2010) Quantitative proteome and phosphoproteome analyses of cultured cells based on SILAC labeling without requirement of serum dialysis. Mol. Biosyst. 6, 594–602.PubMedCrossRefGoogle Scholar
  24. 24.
    Gerber, S. A., Rush, J., Stemman, O., Kirschner, M. W., and Gygi, S. P. (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc. Natl. Acad. Sci. USA. 100, 6940–6945.PubMedCrossRefGoogle Scholar
  25. 25.
    Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res. 12, 1231–1245.PubMedCrossRefGoogle Scholar
  26. 26.
    Ishihama, Y., Oda, Y., Tabata, T., Sato, T., Nagasu, T., Rappsilber, J., et al. (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, 1265–1272.PubMedCrossRefGoogle Scholar
  27. 27.
    Lu, P., Vogel, C., Wang, R., Yao, X., and Marcotte, E. M. (2007) Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation. Nat. Biotechnol. 25, 117–124.PubMedCrossRefGoogle Scholar
  28. 28.
    Griffin, T. J., Lock, C. M., Li, X. J., Patel, A., Chervetsova, I., Lee, H., et al. (2003) Abundance ratio-dependent proteomic analysis by mass spectrometry. Anal. Chem. 75, 867–874.PubMedCrossRefGoogle Scholar
  29. 29.
    Bondarenko, P. V., Chelius, D., and Shaler, T. A. (2002) Identification and relative quantitation of protein mixtures by enzymatic digestion followed by capillary reversed-phase liquid chromatography-tandem mass spectrometry. Anal. Chem. 74, 4741–4749.PubMedCrossRefGoogle Scholar
  30. 30.
    Chelius, D., and Bondarenko, P. V. (2002) Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry. J. Proteome Res. 1, 317–323.PubMedCrossRefGoogle Scholar
  31. 31.
    Wang, W., Zhou, H., Lin, H., Roy, S., Shaler, T., Hill, L., et al. (2003) Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards. Anal. Chem. 75, 4818–4826.PubMedCrossRefGoogle Scholar
  32. 32.
    Bantscheff, M. and Schirle, M. (2007) Quantitative mass spectrometry in proteomics: a critical review. Anal. Bioanal. Chem. 389, 1017–1031.PubMedCrossRefGoogle Scholar
  33. 33.
    Old, W. M., Meyer-Arendt, K., Aveline-Wolf, L., Pierce, K. G., Mendoza, A., Sevinsky, J. R., et al. (2005) Comparison of label-free methods for quantifying human proteins by shotgun proteomics. Mol. Cell. Proteomics 4, 1487–1502.PubMedCrossRefGoogle Scholar
  34. 34.
    Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254.PubMedCrossRefGoogle Scholar
  35. 35.
    Hill, H. D. and Straka, J. G. (1988) Protein determination using bicinchoninic acid in the presence of surfhydryl reagents. Anal. Biochem. 170, 203–208.PubMedCrossRefGoogle Scholar
  36. 36.
    Hale, J. E., Butler, J. P., Gelfanova, V., You, J. S., and Knierman, M. D. (2004) A simplified procedure for the reduction and alkylation of cysteine residues in proteins prior to proteolytic digestion and mass spectral analysis. Anal. Biochem. 333, 174–181.PubMedCrossRefGoogle Scholar
  37. 37.
    Higgs, R. E., Knierman, M. D., Freeman, A. B., Gelbert, L. M., Patil, S. T., and Hale, J. E. (2007) Estimating the statistical significance of peptide identifications from shotgun proteomics experiments. J. Proteome Res. 6, 1758–1767.PubMedCrossRefGoogle Scholar
  38. 38.
    Higgs, R.E., Knierman, M.D., Gelfanova, V., Butler, J.P., and Hale, J.E. (2005) Comprehensive label-free method for the relative quantification of proteins from biological samples. J. Proteome Res. 4, 1442–1450.PubMedCrossRefGoogle Scholar
  39. 39.
    Carr, S., Aebersold, R., Baldwin, M., Burlingame, A., Clauser, K., and Nesvizhskii, A. (2004) The need for guidelines in publication of peptide and protein identification data: Working Group on Publication Guidelines for Peptide and Protein Identification Data. Mol. Cell. Proteomics 3, 531–533.PubMedCrossRefGoogle Scholar
  40. 40.
    Shadforth, I. P., Dunkley, T. P., Lilley, K. S., and Bessant, C. (2005) i-Tracker: for quantitative proteomics using iTRAQ. BMC Genomics 6, 145–150.PubMedCrossRefGoogle Scholar
  41. 41.
    Bolstad, B. M., Irizarry, R. A., Astrand, M., and Speed, T. P. (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19, 185–193.PubMedCrossRefGoogle Scholar
  42. 42.
    Limpert, E., Stahel, W. A., Abbt, M. (2001) Log-normal Distributions across the Sciences: Keys and Clues. BioScience 51, 341–352.CrossRefGoogle Scholar
  43. 43.
    Zybailov, B., Coleman, M. K., Florens, L., and Washburn, M. P. (2005) Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum counting for quantitative proteomic analysis using stable isotope labeling. Anal. Chem. 77, 6218–6224.PubMedCrossRefGoogle Scholar
  44. 44.
    Florens, L., Carozza, M. J., Swanson, S. K., Fournier, M., Coleman. M. K., Workman, J. L., et al. (2006) Analyzing chromatin remodeling complexes using shotgun proteomics and normalized spectral abundance factors. Methods 40, 303–311.PubMedCrossRefGoogle Scholar
  45. 45.
    Pang, J. X., Ginanni, N., Dongre, A. R., Hefta, S. A., and Opiteck, G. J. (2002) Biomarker discovery in urine by proteomics. J. Proteome Res. 1, 161–169.PubMedCrossRefGoogle Scholar
  46. 46.
    Rao, P. V., Reddy, A. P., Lu, X., Dasari, S., Krishnaprasad, A., Biggs, E., et al. (2009) Proteomic identification of salivary biomarkers of type-2 diabetes. J. Proteome Res. 8, 239–245.PubMedCrossRefGoogle Scholar
  47. 47.
    Pan, J., Chen, H. Q., Sun, Y. H., Zhang, J. H., and Luo, X. Y. (2008) Comparative proteomic analysis of non-small-cell lung cancer and normal controls using serum label-free quantitative shotgun technology. Lung 186, 255–261.PubMedCrossRefGoogle Scholar
  48. 48.
    Asara, J. M., Christofk, H. R., Freimark, L. M., and Cantley, L. C. (2008) A label-free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen. Proteomics 8, 994–999.PubMedCrossRefGoogle Scholar
  49. 49.
    Seyfried, N. T., Huysentruyt, L. C., Atwood III, J. A., Xia, Q., Seyfried, T. N., and Orlando, R. (2008) Up-regulation of NG2 proteoglycan and interferon-induced transmembrane proteins 1 and 3 in mouse astrocytoma: a membrane proteomics approach. Cancer Letters 263, 243–252.PubMedCrossRefGoogle Scholar
  50. 50.
    Liu, H., Sadygov, R. G., and Yates, J. R., III (2004) A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal. Chem. 76, 4193–4201.PubMedCrossRefGoogle Scholar
  51. 51.
    Dong, M. Q., Venable, J. D., Au, N., Xu, T., Park, S. K., Cociorva, D., et al. (2007) Quantitative mass spectrometry identifies insulin signaling targets in C. elegans. Science 317 (5838), 660–663.Google Scholar
  52. 52.
    Zhang, B., VerBerkmoes, N. C., Langston, M. A., Uberbacher, E., Hettich, R. L., and Samatova, N. F. (2006) Detecting differential and correlated protein expression in label-free shotgun proteomics. J. Proteome Res. 5, 2909–2918.PubMedCrossRefGoogle Scholar
  53. 53.
    Reiner, A., Yekutieli, D., and Benjamini, Y. (2003) Identifying differentially expressed genes using false discovery rate controlling procedures. Bioinformatics 19, 368–375.PubMedCrossRefGoogle Scholar
  54. 54.
    Zhang, R., Sioma, C. S., Wang, S., and Regnier, F. E. (2001) Fractionation of isotopically labeled peptides in quantitative proteomics. Anal. Chem. 73, 5142–5149.PubMedCrossRefGoogle Scholar
  55. 55.
    Yang, Y. H. and Speed, T. (2003) Design issues for cDNA microarray experiments. Nat. Rev. Genet. 19, 649–659.Google Scholar
  56. 56.
    Simon, R., Radmacher, M. D., and Dobbin, K. (200) Design of studies using DNA microarrays. Genet Epidemilo. 23, 21–36.Google Scholar
  57. 57.
    Zybailov, B., Mosley, A. L., Sardiu, M. E., Coleman, M. K., Florens, L., and Washburn, M. P. (2006) Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae. J. Proteome Res. 5, 2339–2347.PubMedCrossRefGoogle Scholar
  58. 58.
    Paoletti, A. C., Parmely, T. J., Tomomori-Sato, C., Sato, S., Zhu, D., Conaway, R. C., et al. (2006) Quantitative proteomic analysis of distinct mammalian Mediator complexes using normalized spectral abundance factors. Proc. Natl. Acad. Sci. USA. 103, 18928–18933.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyIndiana University School of MedicineIndianapolisUSA

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