SAMPLE HANDLING FOR PROTEOME ANALYSIS

The main factor limiting the sensitivity range for the identification of proteins isolated by two-dimensional (2-D) electrophoresis is sampl e handling: protein detection limits on the gel, losses during extract ion and digestion, as well as interference of gel contaminants and det ergents with the mass spectrometry (MS) detection increasing backgroun d noise. At the one hundred picomole level, losses are fairly negligib le but when the amounts drop below 1 picomole (and subfemtomole peptid e detection limits have been reported recently by MS), the losses beco me a critical point. In order to extend proteome analysis to include v ery low copy number proteins, methods must be developed to minimize lo sses and handling steps, maximize digestion and extraction yields, as well as to lower chemical noise. We present several methods that we ha ve developed in our laboratory to: (i) increase the amount of material available in a sodium dodecyl sulfate (SDS)-free form which does not require staining, (ii) increase protein extraction and digestion yield s and lower the contamination by autoproteolytic products, and (iii) a llow direct modification of the peptide mixture to generate sequence t ags.