Micro-scale Sample Preparation for C-terminal Protein Characterization by Mass Spectrometry Using Combined Liquid- and Solid Phase Derivatization

摘要

The isolation of C-terminal fragments and subsequent structural characterization is of general interest because of the high sequence specificity at the protein's C-terminal region resulting in an increased success rate of protein identification. In addition, the isolation of C-terminal fragments from peptide mixtures may provide for a10-fold reduction sample complexity. Drawbacks associated with current C-terminal peptide enrichment methods include mainly non-specific peptide-isolation and modest detectability of the isolates.¹ To address these shortcomings, we developed a reaction scheme that involves protein carboxyl groups protection by glycinamidation followed by trypsination. The digests are then adsorbed onto C18 reversed-phase supports used as venue for subsequent, sequential peptide α-amine acetylation and EDC-mediated carboxylate condensation using ethylenediamine as nucleophile. The amino group-functionalized N-terminal and internal peptides are then depleted on N-hydroxysuccinimide (NHS)-activated Sepharose leaving the C-terminal peptides since rendered impervious to amination in the flow-through fractions. Examples of the successful application of the method to low-level quantities of protein digests will be presented.¹Nakazowa T, Yamaguchi M, Okamura T, Ando E, Nishimura O, Tsunasawa S. Terminal proteomics: N-and C-terminal analyses for high-fidelity identification of proteins using MS. Proteomics 2008;8:673-685. College of Medicine, Bronx, NY