OPTIMIZATION OF CAPILLARY CHROMATOGRAPHY ION-TRAP MASS-SPECTROMETRY FOR IDENTIFICATION OF GEL-SEPARATED PROTEINS

The current paradigm for protein identification using mass spectrometr ic derived peptide-mass and fragment-ion data employs computer algorit hms which match uninterpreted or partially interpreted fragment-ion da ta to sequence databases, both protein and translated nucleotide seque nce databases. Nucleotide sequence databases continue to grow at a rap id rate for some species, providing an unsurpassed resource for protei n identification in those species. Ion-trap mass spectrometers with th eir ability to rapidly gene-rate fragment-ion spectra in a data-depend ent manner with high sensitivity and accuracy has led to their increas ed use for protein identification. We have investigated various parame ters on a commercial ion trap-mass spectrometer to enhance our ability to identify peptides separated by capillary reversed phase-high perfo rmance liquid chromatography (RP-HPLC) coupled on-line to the mass spe ctrometer. By systematically evaluating the standard parameters (ion i njection time and number of microscans) together with selection of mul tiple ions from the full mass range, improved tandem mass spectrometry (MS/MS) spectra were generated, facilitating identification of protei ns at a low pmol level. Application of this technology to the identifi cation of a standard protein and an unknown from an affinity-enriched mixture are shown.