Abstract
We describe a new method for quantitative tissue proteomics using culture-derived isotope tags (CDIT), which are cells grown in stable isotope-enriched medium and added to each tissue sample to provide internal standards. After protein identification by mass spectrometry (MS), each peak derived from tissue protein is quantified relative to the corresponding CDIT peak. The amounts of each peak in different tissue samples can be compared relative to CDIT. Even if the corresponding peak from CDIT can not be detected, a peak with a similar scan number, but different sequence on liquid chromatography (LC)-MS, can be used to obtain semiquantitative values. Absolute quantification is possible by determining the protein amount in CDIT in advance using unlabeled synthetic peptides; this is less costly than other methods, such as AQUA.
For identification of specific components in a protein complex, target proteins are enriched or isolated by affinity techniques using bait-conjugated matrix, but many non-specific binders are often found. Stable isotope labeling strategies have proven particularly advantageous for the discrimination of proteins specifically associated with the target population from nonspecifically, copurified contaminants. We also describe a protocol for efficient in-gel digestion and high-performance nano-LC column preparation, which makes it possible to quantify larger numbers of proteins.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
Sechi, S. and Oda, Y. (2003) Quantitative proteomics using mass spectrometry. Curr. Opin. Chem. Biol. 7, 70–77.
Wu, C. C., MacCoss, M. J., Howell, K. E., Matthews, D. E., and Yates, J. R. 3rd. (2004) Metabolic labeling of mammalian organisms with stable isotopes for quantitative proteomic analysis. Anal. Chem. 76, 4951–4959.
Ishihama, Y., Sato, T., Tabata, T., et al. (2005) Quantitative mouse brain proteomics using culture-derived isotope tags asinternal standards. Nat. Biotechnol. 23, 617–621.
Barr, J. R., Maggio, V. L., Patterson, D., et al. (1996) Isotope dilution—mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I. Clin. Chem. 42, 1676–1682.
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.
Havlis, J. and Shevchenko, A. (2004) Absolute quantification of proteins in solutions and in polyacrylamide gels by mass spectrometry. Anal. Chem. 76, 3029–3036.
Oda, Y., Owa, T., Sato, T., et al. (2003) Quantitative chemical proteomics for identifying candidate drug targets. Anal. Chem. 75, 2159–2165.
Ranish, J. A., Yi, E. C., Leslie, D. M., et al. (2003) The study of macromolecular complexes by quantitative proteomics. Nat. Genet. 33, 349–355.
Blagoev, B., Kratchmarova, I., Ong, S. E., Nielsen, M., Foster, L. J., and Mann, M. (2003) A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat. Biotechnol. 21, 315–318.
Katayama, H., Nagasu, T., and Oda, Y. (2001) Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix assisted laser desorption/ionization-time of flight mass spectrometry. Rapid Commun. Mass Spectrom. 15, 1416–1421.
Katayama, H., Sato K., Takeuchi M., Deguchi-Tawarada M., Oda Y., and Nagasu, T. (2003) Optimization of in-gel digestion system in combination with thin gel separation and negative staining in 96-well plate format. Rapid Commun. Mass Spectrom. 17, 1071–1078.
Katayama, H., Tabata, T., Ishihama, Y., Sato, T., Oda, Y., and Nagasu, T. (2004) Efficient in-gel digestion procedure using 5-cyclohexyl-1-pentyl-beta-D-maltoside as an additive for gel-based membrane proteomics. Rapid Commun. Mass Spectrom. 18, 2388–2394.
Rappsilber, J., Ishihama, Y., and Mann, M. (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670.
Ishihama, Y., Rappsilber, J., Andersen, J. S., and Mann, M. (2002) Microcolumns with self-assembled particle frits for proteomics. J. Chromatogr. A 979, 233–239.
Gill, S. C. and von Hipple, P. H. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319–326.
Ong, S. E., Blagoev, B., Kratchmarova, I., et al. (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as simple and accurate approach to expression proteomics. Mol. Cell Proteomics 1, 376–386.
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, 173–181.
Everley, P. A., Krijgsveld, J., Zetter, B. R., and Gygi, S. P. (2004) Quantitative cancer proteomics: stable isotope labeling with amino acids in cell culture (SILAC) as a tool for prostate cancer research. Mol. Cell Proteomics 3, 729–735.
Hathout, Y., Flippin, J., Fan, C., Liu, P., and Csaky, K. (2005) Metabolic labeling of human primary retinal pigment epithelial cells for accurate comparative proteomics. J. Proteome Res. 4, 620–627.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Sato, T., Ishihama, Y., Oda, Y. (2007). Quantitative Proteomics of Mouse Brain and Specific Protein-Interaction Studies Using Stable Isotope Labeling. In: Sechi, S. (eds) Quantitative Proteomics by Mass Spectrometry. Methods in Molecular Biology, vol 359. Humana Press. https://doi.org/10.1007/978-1-59745-255-7_4
Download citation
DOI: https://doi.org/10.1007/978-1-59745-255-7_4
Publisher Name: Humana Press
Print ISBN: 978-1-58829-571-2
Online ISBN: 978-1-59745-255-7
eBook Packages: Springer Protocols