We offer protein identification by tandem mass spectrometry. We utilize nano-LC/MS/MS technique, which is one of the most sensitive and reliable methods currently available. It allows the identification of proteins at low fmol levels and enables identification of numerous proteins in protein mixtures.
Protein identification in gel slices.
Our protein identification services include: sample preparation, digestion, peptide extraction, nano-LC/MS/MS data acquisition, and data analysis (including automated NCBI nr database search by SEQUEST-based software and data quality control by a human expert). The analysis results are e-mailed to the customer in Excel format (example) or PDF format (example). In addition, we can provide the customer with the raw data in .raw, .dta or .mzXML formats.The turnaround time is 4-5 business days.
Below is an outline of our analysis protocol:
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 remove salts and other impurities. The peptides are then separated on a microcapillary C18 reverse-phase chromatography column. We use PicoFrit columns that enable a 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 event. In the typical 90 min analysis of a sample, about 400 MS and 1200 MS/MS spectra can be acquired, which is sufficient for the 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 abundant 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 BioWorks software that utilizes the TurboSEQUEST algorithm.