Abstract
To date, large-scale protein quantitation mainly relies on high-resolution separation of complex mixtures using two-dimensional gel electrophoresis (2-DE). In comparison with 2-DE-based protein intensity displays for quantitating differentially expressed proteins, mass spectrometry (MS)-based quantitation of protein expression remains a challenging task because a mass spectrometer itself is a poor quantitative analyzer, as a result of the uneven ionization efficiency of different peptides. Recently, several techniques coupling stable isotope labeling (SIL) to MS have emerged as the primary approach for rapid, large-scale protein quantitation (1–4). There are two major strategies to quantitate proteins through SIL—chemically introducing SIL tags either after cell lysis, or in vivo/in vitro during cell growth (5–7). Chemical SIL approaches usually target at a particular residue of tryptic peptides through chemical reactions after cell lysis, thus reducing the complexity of a sample. The most representative methods are isotope-coded affinity tags (ICAT) (1) and mass-coded abundance tagging (MCAT) (4). Disadvantages include the relatively low efficiency of these chemical modification reactions and the limited abundance of target residues. On the other hand, during cell growth, for example, Oda et al. used 15N uniformly labeled medium to label all nitrogen atoms in the whole proteome, and applied this strategy to quantitate protein expression and to identify modifications (5). Our laboratory originally developed a different isotope-tagging strategy, using SIL amino acids as tag precursors that can be incorporated into cellular proteins in a residue-specific manner. These amino acidcoded mass tags (AACTs) then provide a signature for each individual protein or modification for quantitation and concurrent identification (7–13).
Keywords
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
Geng, M., Ji, J., and Regnier, F. E. (2000) Signature-peptide approach to detecting proteins in complex mixtures. J Chromatogr. A 870, 295–313.
Shevchenko, A., Chemushevich, I., Ens, W., et al. (1997) Rapid “de novo” peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/timeof-flight mass spectrometer. Rapid Commun. Mass Spectrom. 11, 1015–1024.
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, 163–170.
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.
Veenstra, T. D., Martinovic, S., Anderson, G. A., Pasa-Tolic, L., and Smith, R. D. (2000) Proteome analysis using selective incorporation of isotopically labeled amino acids. J. Am. Soc. Mass Spectrom. 11, 78–82.
Chen, X., Smith, L. M., and Bradbury, E. M. (2000) Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification. Anal. Chem. 72, 1134–1143.
Gu, S., Pan, S., Bradbury, E. M., and Chen, X. (2002) Use of deuterium-labeled lysine for efficient protein identification and peptide de novo sequencing. Anal. Chem. 74, 5774–5785.
Gu, S., Pan, S., Bradbury, E. M., and Chen, X. (2003) Precise peptide sequencing and protein quantification in the human proteome through in vivo lysine-specific mass tagging. J. Am. Soc. Mass Spectrom. 14, 1–7.
Zhu, H., Pan, S., Gu, S., Bradbury, E. M., and Chen, X. (2002) Amino acid residue specific stable isotope labeling for quantitative proteomics. Rapid Commun. Mass Spectrom. 16, 2115–2123.
Zhu, H., Hunter, T. C., Pan, S., Yau, P. M., Bradbury, E. M., and Chen, X. (2002) Residuespecific mass signatures for the efficient detection of protein modifications by mass spectrometry. Anal. Chem. 74, 1687–1694.
Pan, S., Gu, S., Bradbury, E. M., and Chen, X. (2003) Single peptide-based protein identification in human proteome through MALDI-TOF MS coupled with amino acids coded mass tagging. Anal. Chem. 75, 1316–1324.
Hunter, T. C., Yang, L., Zhu, H., Majidi, V., Bradbury, E. M., and Chen, X. (2001) Peptide mass mapping constrained with stable isotope-tagged peptides for identification of protein mixtures. Anal. Chem. 73, 4891–4902.
Donald, S. P., Sun, X. Y., Hu, C. A., et al. (2001) Proline oxidase, encoded by p53-induced gene-6, catalyzes the generation of proline-dependent reactive oxygen species. Cancer Res. 61, 1810–1815.
Yu, J., Zhang, L., Hwang, P. M., Kinzler, K. W., and Vogelstein, B. (2001) PUMA induces the rapid apoptosis of colorectal cancer cells.Mol. Cell 7, 673–682.
Bae, W. and Chen, X. (2004) Proteomic study for the cellular responses to cadmium in Schizosaccharomyces pombe through amino acid-coded mass tagging and LC-MS/MS. Mol. Cell. Proteomics 3(6), 596–607.
Zhu, H., Hunter, T. C., Pan, S., Yau, P. M., Bradbury, E. M., and Chen, X. (2002) Residuespecific mass signatures for the efficient detection of protein modifications by mass spectrometry. Anal. Chem. 74, 1687–1694.
Gu, S. et al. (2004) Global investigation of p53-induced apoptosis through quantitative profiling regulatory proteins using comparative amino acid-coded tagging proteomics. Mol. Cell Proteomics 3, 998–1008.
Gu, S., Chen J., Dobos K. M., Bradbury E. M., Belisle J. T., and Chen X. (2003) Comprehensive proteomic profling of the membrane constituents of a Mycobacterium tuberculosis strain. Mol. Cell. Proteomics 1, 1284–1296.
Mortz, E., Krogh, T. N., Vorum, H., and and Gorg, A. (2001) Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ ionization-time of flight analysis. Proteomics 1, 1359–1363.
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, 3551–3567.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Chen, X. (2005). Amino Acid-Coded Mass Tagging for Quantitative Profiling of Differentially Expressed Proteins and Modifications in Cells. In: Walker, J.M. (eds) The Proteomics Protocols Handbook. Springer Protocols Handbooks. Humana Press. https://doi.org/10.1385/1-59259-890-0:393
Download citation
DOI: https://doi.org/10.1385/1-59259-890-0:393
Publisher Name: Humana Press
Print ISBN: 978-1-58829-343-5
Online ISBN: 978-1-59259-890-8
eBook Packages: Springer Protocols