Examination of micro-tip reversed-phase liquid chromatographic extraction of peptide pools for mass spectrometric analysis

Mass spectrometry occupies a central position in most current protein ident ification schemes. So-called 'mass fingerprinting' techniques rely on compo site mass patterns of proteolytic fragments, or dissociation products there of, to query databases. Keys to successful analysis of ever smaller amounts are sensitivity and complete spectral information, both of which depend fo r a large part on proper sample preparation. Clean-up and concentration of peptide mixtures over eppendorf gel loading tips filled with chromatographi c media (i.e. 'micro-tips') are believed to be quite useful in this regard. We have studied quantitative and qualitative aspects of polypeptide extrac tion using these small manual devices. Optimization of sample volume and ad ditives, micro-tip bed volume, and eluent composition and volume, all contr ibute to effective recovery (similar to 65-70%, on average). Improper diges t conditions can, in fact, lead to far bigger losses, suggesting the need f or at least trace amounts of Zwittergent 3-16. Of particular interest is ou r finding that partial fractionation, obtained by two-step micro-tip elutio n, generally results in more and better signals during subsequent mass anal ysis. Thus, by using optimized micro-tips, in combination with adequate sam ple handling and instrumentation, direct mass spectrometric identification can be routinely and successfully done in any resource facility type settin g. (C) 1998 Published by Elsevier Science B.V. All rights reserved.