Systematic analysis of peptide recoveries from in-gel digestions for protein identifications in proteome studies

摘要: Metabolically radiolabeled recombinant proteins were used to systematically evaluate peptide recoveries from in-gel trypsin digestion.At least 80% of the labeled tryptic peptides could be readily extracted gel bands containing 1 10 pmol, and at 70% 200- 500-fmol levels using a 52-kd protein. Alkylation before electrophoresis or digestion had minimal effects on recovery; although alkylation, especially analysis,may reduce heterogeneity resulting cysteines. Comparison different thicknesses unminced suggested that 1.0-mm gels optimal. Surprisingly, 0.5-mm low variable, primarily because increased diffusion protein out thin during fixing staining. Although 85% typically over range conditions concentrations, further processing extracts resulted in substantial additional losses. Even handling loss about 10% 15% by adsorption plastic surfaces. Adsorptive losses particularly high, sometimes exceeding 50%, variable if partially dried Speedvac concentrate sample remove acetonitrile. High acetonitrile extraction and/or concentration appear detrimental, their elimination simplifies automation. SYPRO Ruby Red, sensitive noncovalent fluorescent stain appears an attractive alternative Coomassie blue for digestion. These results suggest optimized strategy which 1.0-mm-thick are stained with digested overnight modified trypsin, one two times small volumes aqueous buffer. It is critical subsequent surface exposure minimized, vacuum drying should avoided.