Peptide mapping is the analysis of peptides generated from the digestion of a protein by mass spectrometry. It is a comparative procedure and can be used as an identity test for proteins. It requires a reference, either a reference sequence, reference standard or a reference material, to compare and contrast with the target of interest.
Peptide mapping is particularly useful to find differences between two or more samples or conditions when visualized in the liquid chromatogram. It can also be used to confirm the sequence of the target protein against a known sequence and discover point mutations. The technology utilizes more information than intact mass measurement, and therefore is more reliable. However, it still has difficulty in detecting some subtle mutations such as the swap of two amino acids.
The general steps of peptide mapping is as follows,
- Digestion of the monoclonal antibody protein into peptides using one or more enzymes with diverse cleavage sites
- Separation of the peptides using chromatographic technologies such as reverse-phase HPLC
- Identification of the peptides (when MS/MS is used for the peptide identification, a database search engine such as Mascot and SEQUEST may be used)
- Reporting and visualization of the analysis by mapping the peptides to the LC Chromatogram or the known protein sequence
The image below illustrates a tryptic peptide mapping comparing three monoclonal antibodies.
(Image credit: http://www.slideshare.net/pscad123/usp-biotherapeutics-biological-medicines)
The image below shows how mapping peptides to a known protein sequence can help confirm the sequence information.
(Image credit: http://ms.biomed.cas.cz/MSTools/)