Role of Accurate Mass Measurement (±10 ppm) in Protein Identification Strategies Employing MS or MS/MS and Database Searching

摘要

We descrfoe the impact of advances in mass measurement accuracy, ±10 ppm (intemally ealibrated), on protein identification experimente. This capabilRy was bronght about by delayed extraction techniques used in conjuncfion with matrix-asslsted laser desorption ionization (MALDI) on a refiectron time-of-fiight (TOF) mass spectrometer. This work explores the advantage of using accurato mass measurement (and thus constraint on the possible elemental composition of components in a pro. tein digest) in stmtegies for searchlng proteln, gene, and EST databases that employ (a) mass raines alone, (b) fragment-ion tagging derived from MS/MS spectra, and (c) de novo interpretation of MS/MS spectra. Significant improvement in the discriminating power of database searches has been found uslng only molecular weight values (i.e., measured mass) of> 10 peptide masses. When MALDI-TOF instruments are able to achieve the ±0.5-5 ppm mass accuracy necessary to distinguish pepfide elemental compositions, it is possible to match homologous proteins having>70% sequence identity to the protein helng analyzed. The combinatlon of a ±10 ppm measured patent mass of a single trypfic peptide and the near-complete amino acid (AA) composition information from immoninm ions genemted by MS/MS is capable of tagging a peptide in a database because only a few sequence permutations> 11 AA's in !ength for an AA compositlon can erer be found in a proteome. De novo interpretation of peptide MS/MS spectm may be accomplished by altering our MS-Tag program to replace an entire database with calculafion of only the sequence permutations possible from the accurate parent mass and immonium ion limited AA compositions. A hybrid strategy is employed using de novo MS/MS interpretation followed by text-based sequence similarity searching of a database.