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-GELELECTROPHORESIS/

Automated liquid chromatography-electrospray ionization-tandem mass sp ectrometry (LC-ESI-MS/MS) analysis of >100 tryptic digests carried out on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAG E) separated, Coomassie Blue-stained proteins that were prepared by >5 0 different laboratories demonstrates that a commercial electrospray/q uadrupole ion trap mass spectrometer and the tandem mass correlation a lgorithm developed by Eng et al. (Am. Sec. Mass Spectrom. 1994, 5, 976 -989) provide an extremely robust and facile approach to routine prote in identification. By requiring a minimum of two significant matches t o peptides that would be predicted to be produced by the protease that was used, low pmol levels of proteins can be identified with high con fidence while minimizing the probability of identifying the protease i tself and/or the ubiquitous contaminant, keratin. Hence, in only 7% of the digests analyzed was keratin identified and in only 5% of the dig ests 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 amoun t of digest analyzed was 6.1 pmol, the limit of sensitivity las the in strument is configured with a flow rate of 4 mu L/min) appears to be a t the 500 fmol level. Since one of the primary reasons for not identif ying 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 c ertainly increase in the future as the sequences of more genomes are c ompleted.