Optimization of capillary chromatography ion trap‐mass spectrometry for identification of gel‐separated proteins

The current paradigm for protein identification using mass spectrometric derived peptide‐mass and fragment‐ion data employs computer algorithms which match uninterpreted or partially interpreted fragment‐ion data to sequence databases, both protein and translated nucleotide sequence databases. Nucleotide sequence databases continue to grow at a rapid rate for some species, providing an unsurpassed resource for protein identification in those species. Ion‐trap mass spectrometers with their ability to rapidly generate fragment‐ion spectra in a data‐dependent manner with high sensitivity and accuracy has led to their increased use for protein identification. We have investigated various parameters on a commercial ion trap‐mass spectrometer to enhance our ability to identify peptides separated by capillary reversed phase‐high performance liquid chromatography (RP‐HPLC) coupled on‐line to the mass spectrometer. By systematically evaluating the standard parameters (ion injection time and number of microscans) together with selection of multiple ions from the full mass range, improved tandem mass spectrometry (MS/MS) spectra were generated, facilitating identification of proteins at a low pmol level. Application of this technology to the identification of a standard protein and an unknown from an affinity‐enriched mixture are shown.