Protein Enrichment

In some cases, column chromatography is the only step used to fractionate proteins or peptides prior to mass spec analysis, for example, MudPIT analysis (30). Continuous data acquisition generates considerable lists of protein annotations and may be used to create nonquantitative expression profiles; otherwise its value in discovery is limited. Nevertheless, the discovery of proteins in unexpected contexts can be readily validated by more conventional techniques and is a powerful means of target identification, particularly if the protein in question is already well characterized. Enrichment of subcellular fractions, organelles, and protein functional complexes considerably improves the annotation efficiency. Recent publications have described large scale analyses of protein sequences from isolated spliceosome complexes (31,32), proteasomes (33), and yeast protein complexes binding to selected "bait" proteins (34). The expression by genetic engineering of affinity tags, which are exposed on the surface of protein complexes without disrupting normal protein function, has been exploited to enrich active complexes from living cells, in a process termed tandem affinity purification (35). Mass spectrometric sequencing of proteins captured in the complex reveals interaction pathways linking protein activities.

Thus, proteomics can be used to identify differentially expressed proteins en masse, considerably broadening the scope for biomarker discovery. The process in its current form is not yet sufficiently sensitive or rapid to replace clinical diagnostic technologies; what it provides is a rapid discovery platform yielding new potential diagnostic, prognostic, and pharmacologic indicators.

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