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 a 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 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 analytical 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 is 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.