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Mass-Coded Abundance Tagging for Protein Identification and Relative Abundance Determination in Proteomic Experiments

  • Gerard Cagney
  • Andrew Emili
Protocol
  • 2.2k Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Advances in mass spectrometry have led to the emergence of the distinct field of proteomics. One aim of proteomics, the identification of the protein components of complex biological mixtures, is now routinely realized, typically by peptide mass mapping following matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) or by peptide sequence determination from tandem mass spectra obtained by electrospray ionization followed by collision-induced dissociation (CID) (1). Both approaches rely on the identified proteins being present in DNA or protein sequence databases. This is because the behavior of ionized peptides in MS experiments is somewhat unpredictable and the resulting spectra are searched against “idealized” spectra generated from the sequence databases to find the nearest match. Nevertheless, both approaches have been highly successful, with thousands of proteins identified in a single large-scale analysis (reviewed in ref. 2). A method that is independent of databases would be useful in certain cases, however, especially for protein samples deriving from organisms whose genomes remain unsequenced, proteins with erroneous sequences deposited in the databases, or proteins whose splicing patterns or modification states are unknown. Another partially fulfilled goal of proteomics is to determine the quantities of each protein present in a mixture, or at least the relative abundance of proteins present in two different samples, such as a test sample and a reference control. Several approaches for determining relative abundance in proteomic experiments have involved differential incorporation of stable isotopes into one of the samples, using either labeled growth media (3) or postexperimental chemical labeling (4,5). At least two methods using nonisotopic reagents for purposes of proteomic quantification have recently been reported (6,7)

Keywords

Tandem Mass Spectrum Protein Sequence Database Ethyleneglycol Tetraacetic Acid Proteomic Experiment Strong Cation Exchange 
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.

References

  1. 1.
    Aebersold, R. and Mann, M. (2003) Mass spectrometry-based proteomics. Nature 422(6928), 198–207.PubMedCrossRefGoogle Scholar
  2. 2.
    Kislinger, T. and Emili, A. (2003) Going global: protein expression profiling using shotgun mass spectrometry. Curr. Opin. Mol. Ther. 5(3), 285–293.Google Scholar
  3. 3.
    Ong, S-E., Blagoev, B., Kratchmarova, I., 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(5), 376–386.Google Scholar
  4. 4.
    Munchback, M., Quadroni, M., Miotto, G., and James, P. (2000) Quantitation and facilitated de novo sequencing of proteins by isotopic N-terminal labeling of peptides with a fragmentation-directing moiety. Anal. Chem. 72, 4047–4057.CrossRefGoogle Scholar
  5. 5.
    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
  6. 6.
    Cagney, G. and Emili, A. E. (2002) De novo peptide sequencing and quantitative profiling of complex protein mixtures using mass-coded abundance tagging. Nat. Biotechnol. 20(2), 163–170.CrossRefGoogle Scholar
  7. 7.
    Beardsley, R. L. and Reilly, J. P. (2003) Quantitation using enhanced signal tags: a technique for comparative proteomics. J. Proteome Res. 2, 15–21.PubMedCrossRefGoogle Scholar
  8. 8.
    Beardsley, R. L., Karty, J. A., and Reilly, J. P. (2000) Enhancing the intensities of lysineterminated tryptic peptide ions in matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 14, 2147–2153.PubMedCrossRefGoogle Scholar
  9. 9.
    Brancia, F. L., Oliver, S. G., and Gaskell, S. J. (2000) Improved matrix-assisted laser desorption/ ionization mass spectrometric analysis of tryptic hydrolysates of proteins following guanidination of lysine-containing peptides. Rapid Commun. Mass Spectrom. 14, 2070–2073.PubMedCrossRefGoogle Scholar
  10. 10.
    Hale, J. E., Butler, J. P., Knierman, M. D., and Becker, G. W. (2000) Increased sensitivity of tryptic peptide detection by MALDI-TOF mass spectrometry is achieved by conversion of lysine to homoarginine. Anal. Biochem. 287, 110–117.PubMedCrossRefGoogle Scholar
  11. 11.
    Keough, T., Lacey, M. P., and Youngquist, R. S. (2000) Derivatization procedures to facilitate de novo sequencing of lysine-terminated tryptic peptides using postsource decay matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 14, 2348–2356.PubMedCrossRefGoogle Scholar
  12. 12.
    Link, A., Eng, J., Schieltz, D. M., et al. (1999) Direct analysis of proteins complexes using mass spectrometry. Nat. Biotechnol. 17, 676–682.PubMedCrossRefGoogle Scholar
  13. 13.
    Washburn, M. P., Wolters, D., and Yates, J. R. III. (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247.PubMedCrossRefGoogle Scholar
  14. 14.
    Eng, J. K., McCormack, A. L., and Yates, J. R. I. (1994) An approach to correlate tandem mass-spectral data of peptides with amino-acid-sequences in a protein database. J. Am. Soc. Mass Spectrom. 11, 976–989.CrossRefGoogle Scholar
  15. 15.
    Kislinger, T., Rahman, K., Radulovic, D., Cox, B., Rossant, J., and Emili, A. (2003) PRISM, a generic large scale proteomic investigation strategy for mammals. Mol. Cell. Proteomics 2(2), 96–106.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • Gerard Cagney
    • 1
  • Andrew Emili
    • 2
  1. 1.Department of Medical ResearchUniversity of TorontoCanada
  2. 2.Banting and Best Departments of Medical ResearchUniversity of TorontoCanada

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