INTRODUCTION Peptide mapping or fingerprinting of proteins is a technique widely used in academic and industrial laboratories. Methods for analyzing peptides resulting from proteolytic digestion include gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high-performance liquid chromatography(HPLC), and capillary electrophoresis (CE) among others. HPLC has become the preferred method due to its proven ability to provide high-resolution separations that are more reproducible and less labor-intensive than other analysis techniques.This application note describes a detailed procedure for digestion of reduced and alkylated protein with trypsin, and separation of the resulting peptides by reversed-phase HPLC. The method uses a wide-pore (300Å) C8 column designed specifically for the separation of proteins and peptides. The wider pore size allows free diffusion of larger peptides, thus ensuring maximum selectivity.