Peptide Mapping
An analytical technique where a peptide or protein is enzymatically digested into smaller fragments that are separated and identified by HPLC and mass spectrometry. Peptide mapping provides detailed sequence coverage and can detect modifications, degradation, or errors in the primary structure.
Technical Context
Peptide mapping (fingerprinting) procedure: (1) denature and reduce the peptide/protein (unfolding and breaking disulphide bonds with DTT or TCEP under denaturing conditions), (2) alkylate free cysteines (preventing disulphide re-formation with iodoacetamide or N-ethylmaleimide), (3) enzymatic digestion (trypsin is standard — cleaves after Arg and Lys with high specificity; Glu-C, Asp-N, chymotrypsin provide complementary cleavage for complete sequence coverage), (4) LC-MS analysis (RP-HPLC separation coupled to high-resolution MS), and (5) data analysis (matching observed fragment masses to theoretical digest, confirming sequence coverage, identifying modifications). Peptide mapping provides: sequence confirmation (>95% coverage expected), post-translational modification identification (oxidation, deamidation, glycosylation sites), disulphide bond assignment, and detection of sequence variants or modifications. It is the most informative single analytical method for peptide/protein characterisation and is essential for biosimilar comparability assessment.