Protein Identification by LC-MS/MS (<50 proteins)

Benefit description

Triptic digestion and separation of simple protein mixtures (less than 50 proteins) by liquid chromatography and identification by triple quadrupole mass spectrometry and time of flight.

Benefit description

Liquid chromatography coupled with tandem mass spectrometry, or LC-MS/MS, is a highly effective technique for the detailed characterization of a protein sequence and for the global analysis of the proteins present in a protein extract or a complete proteome. The process begins with tryptic digestion of the sample. The resulting peptides are introduced into a capillary chromatography system connected online to the mass spectrometer through a microelectrospray or nanospray interface. The peptides are then separated by reversed-phase chromatography using an acetonitrile gradient. Following separation, they are eluted and ionized directly into the mass spectrometer. The instrument performs acquisition cycles that include a full scan, in which the mass-to-charge ratios of the peptides eluting at a given time are determined, followed by between 10 and 30 MS/MS analyses of the most abundant ions detected. The resulting fragmentation spectra enable the identification of the peptides present in the sample and, consequently, of their corresponding proteins. Identification is carried out by comparing the MS/MS data against sequence databases using different search engines. De novo analysis: When the data cannot be adequately matched against a reference database—for example, when working with partially annotated species or in the presence of unexpected modifications—de novo sequencing can be performed using PEAKS software.