RAPID IDENTIFICATION OF COMIGRATING GEL-ISOLATED PROTEINS BY ION-TRAPMASS-SPECTROMETRY

In the search for novel nuclear binding proteins, two bands from a sod ium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were analyzed and each was found to contain a number of proteins that subsequently were identified by tandem mass spectrometry (MS/MS) on a quadrupole ion trap instrument. The bands were digested with trypsin i n situ on a polyvinylidene difluoride (PVDF) membrane following electr oblot transfer. Analysis of a 2.5% aliquot of each peptide mixture by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-M S) followed by an initial database search with the peptide masses fail ed to identify the proteins. The peptides were separated by reversed-p hase capillary high performance liquid chromatography (HPLC) in antici pation of subsequent Edman degradation, but mass analysis of the chrom atographic fractions by MALDI-MS revealed multiple, coeluting peptides that precluded this approach. Selected fractions were analyzed by cap illary HPLC-electrospray ionization-ion trap mass spectrometry. Tandem mass spectrometry provided significant fragmentation from which full or partial sequence was deduced for a number of peptides. Two stages o f fragmentation (MS3) were used in one case to determine additional se quence. Database searches, each using a single peptide mass plus parti al sequence, identified four proteins from a single electrophoretic ba nd at 45 kDa, and four proteins from a second band at 60 kDa. Many of these proteins were derived from human keratin. The protein identifica tions were corroborated by the presence of multiple matching peptide m asses in the MALDI-MS spectra. In addition, a novel sequence, not foun d in protein or DNA databases, was determined by interpretation of the MS/MS data. These results demonstrate the power of the quadrupole ion trap for the identification of multiple proteins in a mixture, and fo r de novo determination of peptide sequence. Reanalysis of the fragmen tation data with a modified database searching algorithm showed that t he same sets of proteins were identified from a limited number of frag ment ion masses, in the absence of mass spectral interpretation or ami no acid sequence. The implications for protein identification solely f rom fragment ion masses are discussed, including advantages for low si gnal levels, for a reduction of the necessary interpretation expertise , and for increased speed.