Use of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) for routine identification of enzymatically digested proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Automated liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis of >100 tryptic digests carried out on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separated, Coomassie Blue-stained proteins that were prepared by >50 different laboratories demonstrates that a commercial electrospray/quadrupole ion trap mass spectrometer and the tandem mass correlation algorithm developed by Eng et al. (Am. Soc. Mass Spectrom. 1994, 5, 976-989) provide an extremely robust and facile approach to routine protein identification. By requiring a minimum of two significant matches to peptides that would be predicted to be produced by the protease that was used, low pmol levels of proteins can be identified with high confidence while minimizing the probability of identifying the protease itself and/or the ubiquitous contaminant, keratin. Hence, in only 7% of the digests analyzed was keratin identified and in only 5% of the digests analyzed was the protease itself identified. In contrast, 58% of the analyzed samples were identified and, in many instances, multiple proteins were identified in the same sample. Although the median amount of digest analyzed was 6.1 pmol, the limit of sensitivity (as the instrument is configured with a flow rate of 4 microL/min) appears to be at the 500 fmol level. Since one of the primary reasons for not identifying a sample is that its sequence is not yet in the database searched, the utility of an LC MS/MS approach to protein identification will certainly increase in the future as the sequences of more genomes are completed.