Delayed extraction improves specificity in database searches by matrix-assisted laser desorption/ionization peptide maps.

Peptide mass maps obtained by matrix-assisted laser desorption ionization (MALDI) are an attractive means to identify proteins by searches in sequence databases. Here we demonstrate that the recently introduced delayed ion-extraction technique, when coupled to reflectron MALDI time-of-flight mass spectrometry, leads to dramatically improved search specificity. Routine resolution in the range of 6,000 to 12,000 allows assignment of monoisotopic masses throughout the peptide mass range. Database searches can be performed with high precision by use of a mass accuracy which is currently better than 30 ppm over a wide mass range and better than 5 ppm for a narrow mass range. This high performance makes it possible to identify proteins with fewer peptide masses than before. Additional low intensity peaks can be assigned after a search because of the improved signal-to-noise ratio of delayed-extraction peptide mass spectra, increasing sequence coverage of matched proteins. The improvements in database search specificity can be used to identify the components of simple protein mixtures. In combination with advanced sample preparation and automation techniques, delayed-extraction MALDI time-of-flight mass spectrometry is now an extremely powerful tool for the database identification of proteins.