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 -carboxy group. In MOLEX chromatography, a stable chelate is formed between dicarboxylates and metal atoms, so that the non-terminal (i.e., internal) peptide is retained, whereas the protein C-terminal peptide flows through the MOLEX column. After optimization of the MOLEX chromatographic conditions, 1619 protein C-termini were identified from 30 g of peptides (10 g each, in triplicate) derived from human HeLa cells by means of nanoLC/MS/MS. When the MOLEX-isolated sample from 200 {micro}g of HeLa peptides was further divided into six fractions by high-pH reversed-phase LC prior to nanoLC/MS/MS, 2202 protein C-termini were identified with less than 3% contamination with internal peptides. We believe 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.