One-Step Isolation of Protein C-Terminal Peptides from V8 Protease-Digested Proteins by Metal Oxide-Based Ligand-Exchange Chromatography.

We have developed a one-step method to isolate protein C-terminal peptides from V8 protease-digested proteins by metal oxide-based ligand-exchange (MOLEX) chromatography. V8 protease cleaves the C-terminal side of Asp and Glu, affording a digested peptide with two carboxy groups at the C-terminus, whereas the protein C-terminal peptide has only one α-carboxy group. In MOLEX chromatography, a stable chelate is formed between dicarboxylates and metal atoms, so that the nonterminal (i.e., internal) peptide is retained, whereas the protein C-terminal peptide flows through the MOLEX column. After the optimization of the MOLEX chromatographic conditions, 1619 protein C-termini were identified from 30 μg of peptides (10 μg each, in triplicate) derived from human HeLa cells by means of nanoLC/MS/MS. When the MOLEX-isolated sample from 200 μg of HeLa peptides was further divided into six fractions by high-pH reversed-phase liquid chromatography (LC) prior to nanoLC/MS/MS, 2203 protein C-termini were identified with less than 3% contamination with internal peptides. We believe that this is the largest coverage with the highest purity reported to date in human protein C-terminomics. This fast, simple, sensitive, and selective method to isolate protein C-terminal peptides should be useful for profiling protein C-termini on a proteome-wide scale.