This chapter describes the approaches used to prepare protein samples for MS analysis. At this stage of proteomic analysis, we must do two things (Fig. 1). First, we must convert proteins to peptides. This is generally done with proteolytic enzymes. Second, we must separate very complex mixtures of proteins or peptides into somewhat less complex mixtures. This gives the MS instruments a better opportunity to obtain useful data on the components of the mixture. There is no obligatory order for these two steps. We can first separate proteins, then digest them and analyze the peptides. Alternatively, we can first digest a complex mixture of proteins to peptides, and then resolve the peptides. Each approach has advantages and drawbacks, which will be discussed here.
KeywordsPeptide Mixture Intact Protein Separation Mode Strong Cation Exchange Peptide Separation
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- Link, A. J. (1998) 2-D Proteome Analysis Protocols. Humana Press, Totowa, NJ.Google Scholar
- Walker, J. M. (1996) Protein Protocols Handbook. Humana Press, Totowa, NJ.Google Scholar
- Wall, D. B., Kachman, M. T., Gong, S., Hinderer, R., Parus, S., Misek, D. E., et al. (2000) Isoelectric focusing nonporous RP HPLC: a two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry. Anal. Chem. 72, 1099–1111.PubMedCrossRefGoogle Scholar