Peptide Mass Fingerprinting by MS-MALDI TOF

Service description

Peptide mass fingerprinting (PMF) is an analytical technique for protein identification. Basically, the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately measured with MALDI-TOF mass spectrometer. Then these masses are compared to a database containing known protein sequences. Then the absolute masses of the peptides from each protein are calculated theoretically for mass comparison between the peptides of the unknown protein and the theoretical peptide masses of each protein to find the best match. In sample preparation for PMF, protein samples can be derived from SDS-PAGE or in protein solution and then subject to some chemical modifications. Then the proteins are cut into several fragments using proteolytic enzymes to generate peptides for mass spectrometric analysis. A small fraction of the peptide is pipetted onto a MALDI target with a suitable matrix and then the matrix and peptide molecules co-crystallize on the MALDI target and are ready to be analyzed. The target is inserted into the vacuum chamber of the mass spectrometer and the desorption and ionisation of the polypeptide fragments is initiated by a pulsed laser beam. The ions are accelerated in the electric field of the mass spectrometer and fly towards an ion detector where their arrival is detected as an electric signal. The mass spectrometric analysis produces a list of molecular weights of the fragments. The peptide masses are compared to protein databases such as Swissprot, NCBInr, which contain protein sequence information. The results are statistically analyzed and possible matches are returned. The mass spectrometer that we use for the analysis is an Autoflex III MALDI-TOF/TOF. A suite of software is used with this instrument. Acquisition of mass spectra is done in Flex Control, spectrum processing and annotation in Flex Analysis, and additional data processing and data searching is done in BioTools and Sequence Editor (Bruker). The search engine used for the protein identification is MASCOT (MatrixScience, UK).