Question

Discuss how proteins are identified by Peptide Mass Fingerprinting (PMF) using Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDITOF) mass spectrometry.

Answer 1

One of the main uses of MALDI-TOF-MS is in the identification of proteins, by peptide mass fingerprinting (PMF). The unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately measured with a mass spectrometer such as MALDI-TOF.

The procedure involves excision of the band from the gel, washing and destaining, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF-MS of the tryptic peptides and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method. It has been found the most successful method for identification of peptides so far. The peptide masses are compared to either a database containing known protein sequences or even the genome. This is achieved by using computer programs that translate the known genome of the organism into proteins, then theoretically cut the proteins into peptides, and calculate the absolute masses of the peptides from each protein.