Peptide mass fingerprinting

[7][8] Due to the long, tedious process of analyzing proteins, peptide mass fingerprinting was developed.

Disulfide bridges in proteins are reduced and cysteine amino acids are carbamidomethylated chemically or acrylamidated during the gel electrophoresis.

Then the proteins are cut into several fragments using proteolytic enzymes such as trypsin, chymotrypsin or Glu-C. A typical sample:protease ratio is 50:1.

The peptides are then dissolved in a small amount of distilled water or further concentrated and purified and are ready for mass spectrometric analysis.

MALDI-TOF is often the preferred instrument because it allows a high sample throughput and several proteins can be analyzed in a single experiment, if complemented by MS/MS analysis.

[14] 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 which transfers high amounts of energy into the matrix molecules.

Coupling ESI with capillary LC can separate peptides from protein digests, while obtaining their molecular masses at the same time.

[15] Capillary electrophoresis coupled with ESI-MS is another technique; however, it works best when analyzing small amounts of proteins.

Software performs in silico digests on proteins in the database with the same enzyme (e.g. trypsin) used in the chemical cleavage reaction.