20] Trace analysis peptide mapping by high-performance displacement chromatography

Abstract

Publisher Summary Peptide mapping, by high-performance liquid chromatography (HPLC), is an indispensable tool for the structural characterization of proteins that is also widely used as a sensitive identity test in the biotechnology industry. This chapter describes the use of high-performance displacement chromatography (HPDC) for high-sensitivity peptide mapping of a recombinant protein and the application of the technique to liquid chromatography-mass spectrometry. The rationale, for adopting high-performance displacement chromatography, for mapping the peptides, formed by tryptic digestion of a protein, is to characterize the minor components of the mixture, whose presence conveys information about the composition of the protein preparation digested as well as about the specificity of the enzymatic digestion and side reactions, occurring in the digestion. Chief among the objectives of examining the “fine structure” in the enzymatic map is to identify the tell-tale peptides, indicating the presence of protein variants in the preparation. These variants can arise from degradative processes, acting on the protein, such as deamidation, oxidation, and proteolysis; from incorrect folding and disulfide rearrangement, from errors in translation by the host cell, from chemical modifications to the protein, caused by processing conditions used, for example, to cleave the target protein from a larger fusion construct, or from the heterogeneity conferred on a glycoprotein, by the covalently linked carbohydrate structures.