Modern biologists have at their disposal a large array of techniques used to assess the existence and relative or absolute quantity of any molecule of interest in a sample. However, implementing most of these procedures can be a daunting task for the first time, even in a lab with experienced researchers. Just choosing a protocol to follow can take weeks while all of the nuances are examined and it is determined whether a protocol will (a) give the desired results, (b) result in interpretable and unbiased data, and (c) be amenable to the sample of interest. We detail here a robust procedure for labeling proteins in a complex lysate for the ultimate differential quantification of protein abundance following experimental manipulations. Following a successful outcome of the labeling procedure, the sample is submitted for mass spectrometric analysis, resulting in peptide quantification and protein identification. While we will concentrate on cells in culture, we will point out procedures that can be used for labeling lysates generated from other tissues, along with any minor modifications required for such samples. We will also outline, but not fully document, other strategies used in our lab to label proteins prior to mass spectrometric analysis, and describe under which conditions each procedure may be desirable. What is not covered in this chapter is anything but the most brief introduction to mass spectrometry (instrumentation, theory, etc.), nor do we attempt to cover much in the way of software used for post hoc analysis. These two topics are dependant upon one’s resources, and where applicable, one’s collaborators. We strongly encourage the reader to seek out expert advice on topics not covered here.
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References
Anderson, L. and Seilhamer, J. (1997) A comparison of selected mRNA and protein abundances in human liver. Electrophoresis 18, 533–537.
Gygi, S., Rochon, Y., Franza, B., and Aebersold, R. (1999) Correlation between protein and mRNA abundance in yeast. Mol. Cell Biol. 19, 1720–1730.
Grant, S. and Blackstock, W. (2001) Proteomics in neuroscience: From protein to network. J. Neurosci. 21, 8315–8318.
Gavin, A., Aloy, P., Grandi, P., Krause, R., Boesche, M., Marzioch, M., et al. (2006) Proteome survey reveals modularity of the yeast cell machinery. Nature 440, 631–636.
Coughenour, H., Spaulding, R., and Thompson, C. (2004) The synaptic vesicle proteome: A comparative study in membrane protein identification. Proteomics 4, 3141–3155.
Andersen, J., Lam, Y., Leung, A., Ong, S., Lyon, C., Lamond, A., and Mann, M. (2005) Nucleolar proteome dynamics. Nature 433, 77–83.
Yates, J., Gilchrist, A., Howell, K., and Bergeron, J. (2005) Proteomics of organelles and large cellular structures. Nat. Rev. Mol. Cell Biol. 6, 702–714.
Langnaese K., Seidenbecher C., Wex H., Seidel B., Hartung K., Appeltauer U., Garner A., Voss B., Mueller B., Garner C. C., and Gundelfinger E. D. (1996) Protein components of a rat brain synaptic junctional protein preparation. Brain Res. Mol. Brain Res. 42, 118–122.
Yates, J. (1998) Mass spectrometry and the age of the proteome. J. Mass Spectrometry 33, 1–19.
Yates, J. (2000) Mass spectrometry. From genomics to proteomics. Trends Genet. 16, 5–8.
Fountoulakis, M. (2004) Application of proteomics technologies in the investigation of the brain. Mass Spectrometry Rev 23, 231–258.
Ferguson, P. and Smith, R. (2003) Proteome analysis by mass spectrometry. Ann. Rev. Biophys. Biomol. Struct. 32, 399–424.
Mann, M., Hendrickson, R., and Pandey, A. (2001) Analysis of proteins and proteomes by mass spectrometry. Ann. Rev. Biochem. 70, 437–473.
Gygi, S., Rist, B., Gerber, S., Turecek, F., Gelb, M., and Aebersold, R. (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nature Biotechnol. 17, 994–999.
Shiio, Y. and Aebersold, R. (2006) Quantitative proteome analysis using isotope-coded affinity tags and mass spectrometry. Nature Prot. 1, 139–145.
Ong, S. and Mann, M. (2006) A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nature Prot. 1, 2650–2660.
Amanchy, R., Kalume, D., and Pandey, A. (2005) Stable isotope labeling with amino acids in cell culture (SILAC) for studying dynamics of protein abundance and posttranslational modifications. Sci. STKE 267, p12.
Blagoev, B., Ong, S., Kratchmarova, I., and Mann, M. (2004) Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nature Biotechnol. 22, 1139–1145.
Krijgsveld, J., Ketting, R. F., Mahmoudi, T., Johansen, J., Artal-Sanz, M., Verrijzer, C. P., Plasterk, R. H., and Heck, A. J. (2003) Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics. Nat. Biotechnol. 21, 927–931.
Julka, S. and Regnier, F. (2004) Quantification in Proteomics through Stable Isotope Coding: A Review. J. Proteome Res. 3, 350–363.
Rivolta, M., Grix, N., Lawlor, P., Ashmore, J., Jagger, D., and Holley, M. (1998) Auditory hair cell precursors immortalized from the mammalian inner ear. Proc. Biol. Sci. 265, 1595–1603.
Rivolta, M. and Holley, M. (2002) Cell lines in inner ear research. J. Neurobiol. 53, 306–318.
Bork, P., Jensen, L. J., von Mering, C., Ramani, A. K., Lee, I., and Marcotte, E. M. (2004) Protein interaction networks from yeast to human. Curr. Opin. Struct. Biol. 14, 292–299.
Sharan, R., Suthram, S., Kelley, R. M., Kuhn, T., McCuine, S., Uetz, P., Sittler, T., Karp, R. M., and Ideker, T. (2005) Conserved Patterns of Protein Interaction in Multiple Species. Proc. Natl. Acad. Sci. U.S.A. 102, 1974–1979.
Sharan, R., Ulitsky, I., and Shamir, R. (2007) Network-Based Prediction of Protein Function. Mol. Systems Biol. 3, 88.
Fernandez-Ballester, G. and Serrano, L. (2006) Prediction of Protein-Protein Interaction Based on Structure. Meth. Mol. Biol. 340, 207–234.
Iragne, F., Nikolski, M., Mathieu, B., Auber, D., and Sherman, D. (2005) Proviz: Protein interaction visualization and exploration. Bioinformatics 21, 272–274.
Yan, W., Lee, H., Yi, E. C., Reiss, D., Shannon, P., Kwieciszewski, B. K., Coito, C., Li, X. J., Keller, A., Eng, J., Galitski, T., Goodlett, D. R., Aebersold, R., and Katze, M. G. (2004) System-based proteomic analysis of the interferon response in human liver cells. Genome Biol. 5, R54.
Suderman, M. and Hallett, M. (2007) Tools for Visually Exploring Biological Networks. Bioinformatics 23, 2651–2659.
Han, K., Park, B., Kim, H., Hong, J., and Park, J. (2004) Hpid: The human protein interaction database. Bioinformatics 20, 2466–2470.
Chatr-aryamontri, A., Ceol, A., Palazzi, L. M., Nardelli, G., Schneider, M. V., Castagnoli, L., and Cesareni, G. (2007) Mint: The molecular interaction database. Nucleic Acids Res. 35, D572–574.
Jones, P., Cote, R. G., Cho, S. Y., Klie, S., Martens, L., Quinn, A. F., Thorneycroft, D., and Hermjakob, H. (2008) Pride: New developments and new datasets. Nucleic Acids Res. 36, D878–D883.
Jones, P., Cote, R. G., Martens, L., Quinn, A. F., Taylor, C. F., Derache, W., Hermjakob, H., and Apweiler, R. (2006) Pride: A public repository of protein and peptide identifications for the proteomics community. Nucleic Acids Res. 34, D659–D663.
Shannon, P., Markiel, A., Ozier, O., Baliga, N. S., Wang, J. T., Ramage, D., Amin, N., Schwikowski, B., and Ideker, T. (2003) Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504.
Krüger, M., Moser, M., Ussar, S., Thievessen, I., Luber, C.A., Fomer, F., Schmidt, S., Zanivan, S., Fössler, R., Mann, M. (2008) SILAC mouse for quantitative proteomics uncovers Kindlin-3 as an essential factor for red blood cell function. Cell 134, 353–364.
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Vetter, D.E., Basappa, J., Turcan, S. (2009). Multiplexed Isobaric Tagging Protocols for Quantitative Mass Spectrometry Approaches to Auditory Research. In: Sokolowski, B. (eds) Auditory and Vestibular Research. Methods in Molecular Biology™, vol 493. Humana Press. https://doi.org/10.1007/978-1-59745-523-7_21
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DOI: https://doi.org/10.1007/978-1-59745-523-7_21
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