PMF is an analytical technique for protein identification using data from intact peptide masses. A protease such as Trypsin is used to cleave a protein of interest. The masses of the proteins are measured with a mass spectrometer. Each protein can be uniquely identified by the masses of its constituent peptides since protein masses are extremely discriminatory. The Accuracy of PMF depends on quality and relative intensities of the peaks, mass accuracy of the instrument, and interfering factors such are PTMs. PMF can only be used to identify proteins which are sequenced. Therefore PMF is best suited to those organisms whose cDNA protein sequence data is available in a database. It must be noted that even small differences in mass can result in faulty results since PMF accuracy depends entirely on the accurate correlation of determined and predicted masses.

PMF relies on proteases to digest the protein into smaller peptides. Different proteases cut the proteins at different amino acids. An enzyme of low specificity, which digests a protein into too many peptides or results in many missed cleavages, should not be used. A complex mixture result is overlapping peaks. A missed cleavage is when a protease doesn’t cleave where it is supposed to. Trypsin or an enzyme with similar or higher specificity is a good choice. One should expect 1 or 2 missed cleavages from Trypsin per protein. The mass of a peptide, regardless of the protease used to cleave it, is the sum of the amino acids present in the peptide. The effects of modification which might be made on the amino acids must also be taken into account. For example, a phosphorylation results in an addition of a phosphate group, an addition of about 80 Daltons.

Mascot is tool which uses protein mass spectrometry data to identify proteins from primary sequence databases. This tool allows peptide mass fingerprinting. It allows you to specify the enzyme used, missed cleavages expected.