Use in peptide mapping

PeptideMass (ExPASy Proteomic tools), which is designed for use in peptide mapping experiments, determines the cleavage products of a protein after exposure to a specific protease or chemical reagent. The enzymes and reagents available for cleavage via PeptideMass are trypsin, chymotrypsin, Lys C, cyanogen bromide, Arg C, Asp N, and Glu C. 

Fig. 6. Protein identification using a peptide map measured with a matrix-assisted laser desorption time-of-flight mass spectrometer. All the peptide extracted from the gel is measured and the set of masses is used in the database search. The mass resolution is in the order of 10,000. Individual isotopes of a 2.5 kDa peptide are clearly resolved.

A variety of proteases are used for peptide mapping, but trypsin is the most popular because it is stable and available in high purity with little contamination... 

The introduction of "fast HPLC" has proven to be particularly valuable in protein analysis. As stated earlier, assay time in RP-HPLC analysis of proteins is typically long compared to that for smaller organic molecules. We have evaluated the use of 0.6-cm ID x 4-cm columns packed with 3-um particles in the analysis of insulin by RP-HPLC for potency determination, related substances, and in peptide mapping (7). The use of the "fast column" allows considerable savings (40-60%) in analysis time, compared to the regular (0.46-cm ID x 25-cm) columns, without loss in resolving power. 

Peptide mapping has been widely used for characterization of protein products, and is almost always used for confirmation of identity. Generally, peptide mapping is not a very sensitive technique for detection of impurities in our experience, derivatives present at levels below a few percent are not normally detected in peptide maps, whereas HPLC of the intact protein can generally detect 0.1% of such derivatives. However, in relatively simple maps such as the V-8 protease digest of insulin, relatively good limits of detection can be achieved (5). 

O Connor, J.V. (1993) The use of peptide mapping for the detection of heterogeneity in recombinant DNA-derived proteins. Biologicals 21 111-117. 

Continuous epitope, a peptide sequence used in peptide epitope mapping where the amino acids involved occur consecutively in the sequence. 

To detect the presence of glycosylation, two approaches are in common use comparative peptide mapping and selective detection of glycopeptides. 

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