Overview: Peptide mapping is used to confirm the identity of a protein therapeutic by examining the primary structure of proteins. It involves proteolytic digestion of the protein, the separation, and identification of each resulting peptide. The separation is done with liquid chromatography, and the peptides are analyzed with mass spectrometry. Unlike intact protein analysis, peptide mapping has the advantage of being able to provide site-specific information on post-translational and chemical modifications.
Sample preparation: The protein sample is usually purified and then denatured, reduced, and alkylated to break and blocked disulfide bonds. The protein sample is then digested with a protease enzyme (such as trypsin, chymotrypsin) to produce a mixture of peptides.
High-performance liquid chromatography – mass spectrometry (HPLC-MS) analysis: The resulting peptide mixture is separated using liquid chromatography (LC) to reduce sample complexity and improve sensitivity. The peptides are then ionized and introduced into the mass spectrometer. Peptides are separated on MS1 mass spectra based on their mass-to-charge ratio (m/z). Each peptide is further fragmented, and the resulting fragments presenting on MS2 mass spectra carry the sequence information of the peptide.
Data analysis: The resulting data is searched against the protein sequence using certain algorithm to identify each peptide and map them to the protein(s) of interest.