Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS MS and database searching

We describe the impact of advances in mass measurement accuracy, +/-10 ppm (internally calibrated), on protein identification experiments. This capabi lity was brought about by delayed extraction techniques used in conjunction with matrix-assisted laser desorption ionization (MALDI) on a reflectron t ime-of-flight (TOF) mass spectrometer. This work explores the advantage of using accurate mass measurement (and thus constraint on the possible elemen tal composition of components in a protein digest) in strategies for search ing protein, gene, and EST databases that employ (a) mass values alone, (b) fragment-ion tagging derived from MS/MS spectra, and (c) de novo interpret ation of MS/MS spectra. Significant improvement in the discriminating power of database searches has been found using only molecular weight values (i. e., measured mass) of >10 peptide masses. When MALDI-TOF instruments are ab le to achieve the +/-0.5-5 ppm mass accuracy necessary to distinguish pepti de elemental compositions, it is possible to match homologous proteins havi ng >70% sequence identity to the protein being analyzed. The combination of a +/-10 ppm measured parent mass of a single tryptic peptide and the near- complete amino acid (AA) composition information from immonium ions generat ed by MS/MS is capable of tagging a peptide in a database because only a fe w sequence permutations >11 AA's in length for an AA composition can ever b e found in a proteome, Dc novo interpretation of peptide MS/MS spectra may be accomplished by altering our MS-Tag program to replace an entire databas e with calculation of only the sequence permutations possible from the accu rate parent mass and immonium ion limited AA compositions. A hybrid strateg y is employed using de novo MS/MS interpretation followed by text-based seq uence similarity searching of a database.