Technology used for protein identification

Nano-LC/MS/MS technique has become the standard method for protein identification when sensitivity and accuracy of the analysis are needed, especially when many proteins are present in the sample.

The processing of the sample starts with proteolytic digestion, typically with trypsin. The resulting peptide mixture is concentrated on a peptide trap column and washed to get rid of salts and other impurities. Then the peptides are separated on a microcapillary C18 reverse-phase chromatography column. We use PicoFrit columns that enable direct spray of the eluting peptides from the tip of the column into the mass spectrometer, eliminating post-column losses.

Full MS and MS/MS spectra are acquired by the LCQ Deca XP Plus ion trap mass spectrometer (ThermoFinnigan). MS/MS spectra are obtained after precursor peptide ions are isolated and fragmented inside the ion trap of the mass spectrometer and the resulting series of daughter ions are detected in a tandem mass spectrometry experiment. In a typical 90 min analysis of a sample, about 400 MS and 1200 MS/MS spectra can be acquired, which is sufficient for identification of hundreds of peptides. The detection of low-abundance peptides is facilitated by the dynamic exclusion function of the instrument. It temporarily puts a parental mass on the exclusion list after its MS/MS spectra are acquired and lets the instrument collect MS/MS spectra of the less intense components that otherwise would not be examined. The sequences of the parent peptides are inferred by matching the MS/MS spectra to protein sequence databases. This analysis is performed by the TURBOSEQUEST software.