Proteolytic cleavages generate active precursor proteins by creating new N-termini in a protein. Several Post Translation Modifications (PTMs) occurring at the N-terminus also regulate cellular localization and protein-protein interactions. Because of the importance of this region, a significant amount of effort has been placed on the analysis of their amino acid compositions. Recent publications on N-terminal peptide enrichment methods involve the modification of lysine residues to homoarginine via a guanidination reaction. The capture of N-terminal peptides is then achieved by tryptic digestion, followed by direct or reverse purification of original and newly formed N-terminal peptides by amine reactive reagents. In order to efficiently capture N-terminal peptides, trypsin hydrolysis in guanidinated proteins must be understood. Furthermore, evaluations of current methods must be carried out as well as the development of efficient novel capture reagents. In this report, we present a detailed Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometric study of the efficiency of the enzyme trypsin on a guanidinated protein. In addition to this, we evaluated the direct and reverse N-terminal peptide purification strategies utilizing performic acid oxidation of the disulfide bonds as well as reduction and alkylation with dithiothreitol and iodoacetamide. For large-scale determination and quantitation of proteolytic cleavage sites by mass spectrometry, two novel N-terminal capture reagents were developed using acid-cleavable and gas-phase mass spectrometry-cleavable properties. We found the enzyme trypsin to be active in guanidinated proteins depending on the enzyme to protein concentration ratios. Furthermore, our performic acid oxidation and reverse purification strategy revealed efficient capture of methionine containing N-terminal peptide from a mixture of four standard proteins. Evaluations of the newly designed solid-phase acid cleavable and gas-phase mass spectrometry-cleavable reagents are currently under study for large-scale application.

Evaluation of mass spectrometric strategies for enrichment of protein N-terminal peptides

247th National Meeting (2014)

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