Comparative protein profiling is a key approach to understanding the human and other proteomes. Systems-level profiling technologies, such as differential fluorescence two-dimensional gel electrophoresis (DIGE), often require the identification of the proteins that are contained within 50 or more spots per gel. A major focus of this chapter therefore is devoted to a general approach for high throughput protein identification that is based on liquid chromatography (LC)/tandem mass spectrometry (MS/MS) analysis of tryptic digests of individual proteins or mixtures of only a few proteins (i.e., as are usually obtained from individual DIGE spots), and that is also applicable to the analysis of complex protein extracts. Additionally, multiple techniques will be described for identifying sites of protein posttranslational modification, with emphasis on phosphorylation and Arg methylation.
In an era when large scale gene expression analysis and MS/proteomics are playing increasingly important roles, our review of 148 samples sequenced last year reveals that Edman degradation has evolved from being used to elucidate complete protein sequences to fulfilling several roles for which it is uniquely well suited. These include determining N‐termini of limited cleavage products and constructs used in structure/function and biophysical studies, and of proteins whose start sequences are not well predicted; and for QC of recombinant proteins (e.g., of antigens used to produce vaccines against tuberculosis). The demand for Edman sequencing may expand as emphasis shifts from completing genome sequences to identifying and understanding protein functions, interactions, and post‐translational modifications. In addition, our review of 1,558 amino acid analysis (AAA) samples indicates that hydrolysis/AAA is used to determine the accurate protein/peptide concentrations needed for quantitative biophysical studies and to match protein amounts for comparative proteome analyses such as iTRAQ, to verify the frequency of incorporation of modifications, and the compositions of synthetic and naturally occurring peptides (e.g., differentiating between isobaric Ile/Leu in peptides sequenced by de novo MS/MS). We also will report on preliminary LC‐MRM AAA studies.
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