Sample preparation for peptide mapping--A pharmaceutical quality-control perspective

In quality control of therapeutic proteins peptide mapping is used for confirmation of primary structure and detection of posttranslational modifications. The demands put on the experimental procedure are therefore different than in the case of determination of an unknown protein structure. It is here recognized that a peptide-mapping method for quality control of proteins should be inert (not induce or revert modifications), general, robust, and allow a high sample throughput. The steps prior to the separation of the generated peptides are identified as crucial for meeting these demands. This includes denaturation, reduction, alkylation, buffer exchange, solubilization, and digestion. A critical review of the literature regarding these steps is presented. Relevant options in all steps are experimentally evaluated. Novel approaches are developed for many of the steps. The result is a sample preparation procedure that essentially meets the stated demands.