Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry


The adaptation of sequences of chemical reactions to a solid-phase format has been essential to the automation, reproducibility, and efficiency of a number of biotechnological processes including peptide and oligonucleotide synthesis and sequencing1,2,3,4. Here we describe a method for the site-specific, stable isotopic labeling of cysteinyl peptides in complex peptide mixtures through a solid-phase capture and release process, and the concomitant isolation of the labeled peptides. The recovered peptides were analyzed by microcapillary liquid chromatography and tandem mass spectrometry (μLC-MS/MS) to determine their sequences and relative quantities. The method was used to detect galactose-induced changes in protein abundance in the yeast Saccharomyces cerevisiae. A side-by-side comparison with the isotope-coded affinity tag (ICAT) method5 demonstrated that the solid-phase method for stable isotope tagging of peptides is comparatively simpler, more efficient, and more sensitive.

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Figure 1: Schematic representation of the solid-phase isotope tagging method.
Figure 2: Validation of the solid-phase capture and release method.
Figure 3: Summary of the number of proteins identified and quantified by the solid-phase and ICAT methods.


  1. 1

    Merrifield, B. Solid phase synthesis. Science 232, 341–347 (1986).

    CAS  Article  Google Scholar 

  2. 2

    Caruthers, M.H. et al. Chemical synthesis of deoxyoligonucleotides by the phosphoramidite method. Methods Enzymol. 154, 287–313 (1987).

    CAS  Article  Google Scholar 

  3. 3

    Hunkapiller, M. et al. Microchemical facility for the analysis and synthesis of genes and proteins. Nature 310, 105–111 (1984).

    CAS  Article  Google Scholar 

  4. 4

    Edman, P. & Begg, G. A protein sequenator. Eur. J. Biochem. 1, 80–91 (1967).

    CAS  Article  Google Scholar 

  5. 5

    Gygi, S.P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 (1999).

    CAS  Article  Google Scholar 

  6. 6

    Holmes, C.P. & Jones, D.G. Reagents for combinatorial organic synthesis: development of a new o-nitrobenzyl photolabile linker for solid phase synthesis. J. Org. Chem. 60, 2318–2319 (1995).

    CAS  Article  Google Scholar 

  7. 7

    Oda, Y., Huang, K., Cross, F.R., Cowburn, D. & Chait, B.T. Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. USA 96, 6591–6596 (1999).

    CAS  Article  Google Scholar 

  8. 8

    Ideker, T. et al. Integrated genomic and proteomic analyses of a systematically perturbed metabolic network. Science 292, 929–934 (2001).

    CAS  Article  Google Scholar 

  9. 9

    Han, D., Eng, J., Zhou, H. & Aebersold, R. Quantitative profiling of differentiation-induced membrane associated proteins using isotope-coded affinity tags and mass spectrometry. Nat. Biotechnol. 19, 946–951 (2001).

    CAS  Article  Google Scholar 

  10. 10

    Washburn, M.P., Wolters, D. & Yates, J.R. 3rd. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247 (2001).

    CAS  Article  Google Scholar 

  11. 11

    Johnston, M. & Carlson, M. In The molecular and cellular biology of the yeast Saccharomyces (eds. Jones, E.W., Pringle, J.R. & Broach, J.R.) 193–281 (Cold Spring Harbor Press, Cold Spring Harbor, NY; 1992).

    Google Scholar 

  12. 12

    Zhou, H., Watts, J.D. & Aebersold, R. A systematic approach to the analysis of protein phosphorylation. Nat. Biotechnol. 19, 375–378 (2001).

    CAS  Article  Google Scholar 

  13. 13

    Lapatsanis, L., Milias, G., Froussios, K. & Kolovos, M. Synthesis of N-2,2,2-(trichloroethoxycarbonyl)-l-amino acids and N-(9-fluorenylmethoxycarbonyl)-l-amino acids involving succinimidoxy anion as a leaving group in amino acid protection. Synthesis 671–673 (1983).

  14. 14

    Ausubel, F.M. et al. (eds.) Current protocols in molecular biology (Wiley, New York; 1992).

    Google Scholar 

  15. 15

    Eng, J., McCormack, A.L. & Yates, J.R. 3rd. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994).

    CAS  Article  Google Scholar 

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This work was supported in part by the US National Cancer Institute grant (CA84698), National Institutes of Health (NIH) Research Resource Center (RR11823), NIH grant (GM 41109) to R.A., and NIH postdoctoral fellowship (GM19884) to J.A.R.

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Correspondence to Ruedi Aebersold.

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The authors declare no competing financial interests.

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Zhou, H., Ranish, J., Watts, J. et al. Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry. Nat Biotechnol 20, 512–515 (2002). https://doi.org/10.1038/nbt0502-512

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