ProAlanase is an effective alternative to trypsin for proteomics applications and disulfide bond mapping_part3

ABSTRACT: Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid composition of target proteins and the pH of the digestion solution. In this study we characterize ProAlanase, a protease from the fungus Aspergillus niger that cleaves primarily on the C-terminal side of proline and alanine residues. ProAlanase achieves high proteolytic activity and specificity when digestion is carried out at acidic pH (1.5) for relatively short (2 h) time periods. To elucidate the potential of ProAlanase in proteomics applications, we conducted a series of investigations comprising comparative multi-enzymatic profiling of a human cell line proteome, histone PTM analysis, ancient bone protein identification, phosphosite mapping and de novo sequencing of a proline-rich protein and disulfide bond mapping in monoclonal antibody. The results demonstrate that ProAlanase is highly suitable for proteomics analysis of the arginine- and lysine-rich histones, enabling high sequence coverage of multiple histone family members. It also facilitates an efficient digestion of bone collagen thanks to the cleavage at the C-terminus of hydroxyproline which is highly prevalent in collagen. This allows to identify complementary proteins in ProAlanase- and trypsin-digested ancient bone samples, as well as to increase sequence coverage of non-collagenous proteins. Moreover, digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain (N3ICD). Furthermore, we achieve a nearly complete coverage of N3ICD protein by de novo sequencing using the combination of ProAlanase and tryptic peptides. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-containing regions in a non-reduced monoclonal antibody.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture, Hela Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Diana Samodova  

LAB HEAD: Jesper Velgaard Olsen

PROVIDER: PXD021703 | Pride | 2020-10-19

REPOSITORIES: Pride