Direct deconvolution of multiply charged ion spectra determines a protein mass. Identification of proteins is made, in part, from searching protein databases with this found mass. Further confirmation results from searching with the masses of enzymatically cleaved peptides from the protein and from partical sequences of one or more peptides. Obtaining masses and sequencing information from peptide mixtures is aided by separation methods such as HPLC and CE, and an electrospray-ion trap instrument is ideally suited to on-line MS and MS/MS analysis. The fast scanning gives more scans across an HPLC or CE peak for improved detection and quantitation. If required, a slower scan results in resolution of up to 0.2Da across the full mass range, for determination of the charge state of multiply charged parent and product ion spectra.
For on-line LC/MS/MS the automated detection and isolation of one or more ions from each LC peak without user intervention is important. An autoMS/MS facility allows for complete fragment ion spectra from each peak in an LC/MS/MS run, such as a peptide map, giving more sequence confirming information from each experiment. An example will be given of the LC/MS/MS of a peptide mixture. Software automates the isolation and fragmentation of each significant ion in turn during the elution of a peak, to give alternate parent ion and fragment ion scans. Control of both the fragmentation energy and time are important for optimal fragment ion intensity.
Identification and confirmation of the LC components is aided by the MS/MS spectral patterns. An example will be given of exporting a peaklist to a separate software package for comparision with theoretical peptides from a protein digest, and also of identifying a protein from a partial sequence of one of the peptides in a peptide map, using the MS/MS spectrum to give sequence information.