Abstract
Protein quantification represents an important extension to identification proteomics, enabling the comparison of protein expression across different samples or treatments. Comparative protein quantification by mass spectrometry typically employs stable isotope incorporation, but recently, comparative quantification of label-free LCn-MS proteomics data has emerged as an alternative approach. In this chapter, we provide an overview of the different approaches for extracting quantitative data from label-free LCn-MS experiments. The computational procedure for recovering the quantitative information is outlined. Examples of statistical tests used to evaluate the relevance of results are also provided.
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References
Aebersold, R., and Mann, M. (2003) Mass spectrometry-based proteomics, Nature 422, 198-207.
Link, A. J., Eng, J., Schieltz, D. M., Carmack, E., Mize, G. J., Morris, D. R., Garvik, B. M., and Yates, J. R. (1999) Direct analysis of protein complexes using mass spectrometry, Nat. Biotechnol. 17, 676-82.
Washburn, M. P., Wolters, D., and Yates, J. R. (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology, Nat. Biotechnol. 19, 242-47.
Wolters, D. A., Washburn, M. P., and Yates, J. R., III (2001) An automated multidimensional protein identification technology for shotgun proteomics, Anal. Chem. 73, 5683-90.
Annesley, T. M. (2003) Ion suppression in mass spectrometry, Clin. Chem. 49, 1041-44.
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, 994-99.
Ong, S. E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A., and Mann, M. (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics, Mol. Cell. Proteomics 1, 376-86.
Ong, S. E., Mittler, G., and Mann, M. (2004) Identifying and quantifying in vivo methylation sites by heavy methyl SILAC, Nat. Methods 1, 119-26.
Ong, S. E., 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, 173-81.
Cagney, G., and Emili, A. (2002) De novo peptide sequencing and quantitative profiling of complex protein mixtures using mass-coded abundance tagging, Nat. Biotechnol. 20, 163-70.
Ross, P. L., Huang, Y., Marchese, J., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., Purkayastha, S., Juhasz, P., Martin, S., Bartlet-Jones, M., He, F., Jacobson, A., and Pappin, D. (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents, Mol. Cell. Proteomics 3, 1154-69.
Mirgorodskaya, O., Kozmin, Y., Titov, M., Körner, R., Sönksen, C., and Roepstorff, P. (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, 1226-32.
Bondarenko, P., Chelius, D., and Shaler, T. (2002) Identification and relative quantitation of protein mixtures by enzymatic digestion followed by capillary reversed-phase liquid chromatography-tandem mass spectrometry, Anal. Chem. 74, 4741-49.
Chelius, D., and Bondarenko, P. (2002) Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry, J. Proteome Res. 1, 317-23.
Chelius, D., Zhang, T., Wang, G., and Shen, R. (2003) Global protein identification and quantification technology using two-dimensional liquid chromatography nanospray mass spectrometry, Anal. Chem. 75, 6658-65.
Wang, W., Zhou, H., Lin, H., Roy, S., Shaler, T. A., Hill, L. R., Norton, S., Kumar, P., Anderle, M., and Becker, C. H. (2003) Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards, Anal. Chem. 75, 4818-26.
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, 6218-24.
Gravett, M. G., Thomas, A., Schneider, K. A., Reddy, A. P., Dasari, S., Jacob, T., Lu, X. F., Rodland, M., Pereira, L., Sadowsky, D. W., Roberts, C. T., Novy, M. J., and Nagalla, S. R. (2007) Proteomic analysis of cervical-vaginal fluid: Identification of novel biomarkers for detection of intra-amniotic infection, J. Proteome Res. 6, 89-96.
Wienkoop, S., Larrainzar, E., Niemann, M., Gonzalez, E., Lehmann, U., and Weckwerth, W. (2006) Stable isotope-free quantitative shotgun proteomics combined with sample pattern recognition for rapid diagnositics, J. Sep. Sci. 29, 2793-801.
Wang, H. X., Qian, W. J., Chin, M. H., Petyuk, V. A., Barry, R. C., Liu, T., Gritsenko, M. A., Mottaz, H. M., Moore, R. J., Camp, D. G., Khan, A. H., Smith, D. J., and Smith, R. D. (2006) Characterization of the mouse brain proteome using global proteomic analysis complemented with cysteinyl-peptide enrichment, J. Proteome Res. 5, 361-69.
Ruth, M. C., Old, W. M., Emrick, M. A., Meyer-Arendt, K., Aveline-Wolf, L. D., Pierce, K. G., Mendoza, A. M., Sevinsky, J. R., Hamady, M., Knight, R. D., Resing, K. A., and Ahn, N. G. (2006) Analysis of membrane proteins from human chronic myelogenous leukemia cells: Comparison of extraction methods for multidimensional LC-MS/MS, J. Proteome Res. 5, 709-19.
Le Bihan, T., Goh, T., Stewart, II, Salter, A. M., Bukhman, Y. V., Dharsee, M., Ewing, R., and Wisniewski, J. R. (2006) Differential analysis of membrane proteins in mouse fore- and hindbrain using a label-free approach, J. Proteome Res. 5, 2701-10.
Kislinger, T., Cox, B., Kannan, A., Chung, C., Hu, P. Z., Ignatchenko, A., Scott, M. S., Gramolini, A. O., Morris, Q., Hallett, M. T., Rossant, J., Hughes, T. R., Frey, B., and Emili, A. (2006) Global survey of organ and organelle protein expression in mouse: Combined proteomic and transcriptomic profiling, Cell 125, 173-86.
Fang, R. H., Elias, D. A., Monroe, M. E., Shen, Y. F., McIntosh, M., Wang, P., Goddard, C. D., Callister, S. J., Moore, R. J., Gorby, Y. A., Adkins, J. N., Fredrickson, J. K., Lipton, M. S., and Smith, R. D. (2006) Differential label-free quantitative proteomic analysis of Shewanella oneidensis cultured under aerobic and suboxic conditions by accurate mass and time tag approach, Mol. Cell. Proteomics 5, 714-25.
Cao, R., Li, X. W., Liu, Z., Peng, X., Hu, W. J., Wang, X. C., Chen, P., Xie, J. Y., and Liang, S. P. (2006) Integration of a two-phase partition method into proteomics research on rat liver plasma membrane proteins, J. Proteome Res. 5, 634-42.
Lu, P., Vogel, C., Wang, R., Yao, X., and Marcotte, E. (2007) Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation, Nat. Biotechnol. 25, 117-24.
Mallick, P., Schirle, M., Chen, S., Flory, M., Lee, H., Martin, D., Ranish, J., Raught, B., Schmitt, R., Werner, T., Kuster, B., and Aebersold, R. (2007) Computational prediction of proteotypic peptides for quantitative proteomics, Nat. Biotechnol. 25, 125-31.
Liu, H. B., Sadygov, R. G., and Yates, J. R. (2004) A model for random sampling and estimation of relative protein abundance in shotgun proteomics, Anal. Chem. 76, 4193-201.
Zhang, B., VerBerkmoes, N., Langston, M., Uberbacher, E., Hettich, R., and Samatova, N. F. (2006) Detecting differential and correlated protein expression in label-free shotgun proteomics, J. Proteome Res. 5, 2909-18.
Anderson, L., and Hunter, C. L. (2006) Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins, Mol. Cell. Proteomics 5, 573-88.
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. U. S. A. 100, 6940-45.
Keller, A., Nesvizhskii, A. I., Kolker, E., and Aebersold, R. (2002) Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search, Anal. Chem. 74, 5383-92.
Eng, J. K., McCormack, A. L., and Yates, J. R. (1994) An approach to correlate tandem mass spectra data of peptides with amino acid sequences in a protein database, J. Am. Soc. Mass Spectrom. 5, 976.
Perkins, D. N., Pappin, D. J., Creasy, D. M., and Cottrell, J. S. (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data, Electrophoresis 20, 3551-67.
Fenyo, D. (1999) The biopolymer markup language, Bioinformatics 15, 339-40.
Geer, L. Y., Markey, S. P., Kowalak, J. A., Wagner, L., Xu, M., Maynard, D. M., Yang, X. Y., Shi, W. Y., and Bryant, S. H. (2004) Open mass spectrometry search algorithm, J. Proteome Res. 3, 958-64.
Tanner, S., Shu, H. J., Frank, A., Wang, L. C., Zandi, E., Mumby, M., Pevzner, P. A., and Bafna, V. (2005) InsPecT: Identification of posttranslationally modified peptides from tandem mass spectra, Anal. Chem. 77, 4626-39.
Li, X. J., Zhang, H., Ranish, J. A., and Aebersold, R. (2003) Automated statistical analysis of protein abundance ratios from data generated by stable-isotope dilution and tandem mass spectrometry, Anal. Chem. 75, 6648-57.
Nesvizhskii, A. I., Keller, A., Kolker, E., and Aebersold, R. (2003) A statistical model for identifying proteins by tandem mass spectrometry, Anal. Chem. 75, 4646-58.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the folin phenol reagent, J. Biol. Chem. 193, 265-75.
Bradford, M. M. (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding, Anal. Biochem. 72, 248-54.
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Wong, J.W.H., Cagney, G. (2010). An Overview of Label-Free Quantitation Methods in Proteomics by Mass Spectrometry. In: Hubbard, S., Jones, A. (eds) Proteome Bioinformatics. Methods in Molecular Biology™, vol 604. Humana Press. https://doi.org/10.1007/978-1-60761-444-9_18
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DOI: https://doi.org/10.1007/978-1-60761-444-9_18
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