MALDItof MS analysis of tryptic digests

• Protein identification by peptide fingerprinting requires the controlled fragmentation of the isolated protein by a proteolytic enzyme, customarily modified trypsin. The individual peptide masses (m/z values) from the resultant digest are then determined with a high degree of accuracy by MS. For peptide fingerprinting,  a MALDItof (Matrix Assisted Laser Desorption Ionisation time of flight) instrument is used to generate a profile of proteolytic fragments. The collective peptide masses are then submitted to a protein databank search engine, such as MASCOT or PEPTIDENT.
• Identification of the unknown protein is based on matching the experimentally generated peptide masses (m/z values) with the predicted peptide fragments generated from theoretical digests of proteins found in the databank. This approach depends on the sequence of the protein already existing in the databank.
• Samples can be sent to us for analysis. Typically the sample can be sent to us frozen in buffer or lyophilised, which should be noted on the submission form. The buffer should be compatible with SDS PAGE buffers (e.g. salt concentrations should not exceed 500 mM and not contain chaotropic agents, such as Guanidine Hydrochloride
• Tryptic digestion: Proteins are digested with sequencing grade trypsin, which cuts at the C-terminal side of lysine (lys, K) or arginine (arg, R). The trypsin has been modified to remove certain internal lys and arg residues which lead to autolysis. In gel tryptic digestions are performed overnight at 37°C after removal of SDS by several acetonitrile washes.

           Enquire for protein ID services