ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping
peer-reviewed · Molecular & Cellular Proteomics · 2020
| Date | 2020-12-01 |
| Type | peer-reviewed |
| Venue | Molecular & Cellular Proteomics |
| Publisher | Elsevier BV |
| Contribution | downstream-application |
| DOI | 10.1074/mcp.tir120.002129 |
| Citations (OpenAlex) | 45 |
| Venue 2-year citedness | 4.69 |
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 mAb. 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 noncollagenous 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 nonreduced mAb.
Methods and tools
- ProAlanase protease for proteomics and de novo sequencing: Characterises the proline/alanine-specific protease ProAlanase and shows that combining its peptides with tryptic ones gives nearly complete de novo sequencing coverage of the proline-rich Notch3 intracellular domain with PEAKS.
Methods it uses
- PEAKS: Commercial DP-based de novo
Data deposited
- ProAlanase is an effective alternative to trypsin for proteomics applications and disulfide bond mapping — as deposited · PXD019039
- ProAlanase is an effective alternative to trypsin for proteomics applications and disulfide bond mapping_part2 — as deposited · PXD021191
- ProAlanase is an effective alternative to trypsin for proteomics applications and disulfide bond mapping_part3 — as deposited · PXD021703
Cites (7)
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- PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry (2003) crossref