ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping

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. 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. In most proteomics investigations, proteins are enzymatically digested into shorter peptides, which are more amenable for sequencing by tandem MS (MS/MS) and identification via peptide-spectrum matching (PSM) (1Zhang Y. Fonslow B.R. Shan B. Baek M.-C. Yates J.R. Protein analysis by shotgun/bottom-up proteomics.Chem. Rev. 2013; 113: 2343-2394Crossref PubMed Scopus (741) Google Scholar). This digestion is typically conducted using a sequence-specific protease that cuts proteins after or before specific amino acids. Cleavage specificity, in turn, determines the precursor charge and peptide length distribution, as well as charge localization within the sequence. Trypsin is the most widely used protease in proteomics (2Tabb D.L. Huang Y. Wysocki V.H. Yates J.R. Influence of basic residue content on fragment ion peak intensities in low-energy collision-induced dissociation spectra of peptides.Anal. Chem. 2004; 76: 1243-1248Crossref PubMed Scopus (131) Google Scholar, 3Cristobal A. Marino F. Post H. van den Toorn H.W.P. Mohammed S. Heck A.J.R. Toward an Optimized Workflow for Middle-Down Proteomics.Anal. Chem. 2017; 89: 3318-3325Crossref PubMed Scopus (49) Google Scholar). Sequencing grade trypsin is readily available, stable, and extremely specific with cleavage exclusively after the basic amino acids, arginine (R) and lysine (K) under normal experimental conditions (4Olsen J.V. Ong S.-E. Mann M. Trypsin cleaves exclusively C-terminal to arginine and lysine residues.Mol. Cell. Proteomics. 2004; 3: 608-614Abstract PubMed Scopus Google Scholar). of the content of lysine and arginine in most tryptic are typically relatively short to sequence a arginine or lysine in C terminus and an charge on its tryptic for by and fragment spectra by collision-induced dissociation by fragment ion series (2Tabb D.L. Huang Y. Wysocki V.H. Yates J.R. Influence of basic residue content on fragment ion peak intensities in low-energy collision-induced dissociation spectra of peptides.Anal. Chem. 2004; 76: 1243-1248Crossref PubMed Scopus (131) Google Scholar, A. A. using PubMed Scopus Google Scholar). However, is widely that tryptic are to by or regions in proteins can to short or tryptic peptides, which are to by Heck A.J.R. for proteomics PubMed Scopus Google Scholar, D.L. of using multiple for PubMed Scopus Google Scholar). In of tryptic from the human are shorter amino and be used to identify specific proteins via MS Heck A.J.R. PubMed Scopus Google Scholar). the histone protein family is by a high content of lysine and arginine H. The amino acid composition and of PubMed Scopus Google Scholar). are the most proteins in the cell to in a as PubMed Scopus Google Scholar). is by of by histone PubMed Scopus Google on histones, as the histone The of histone PubMed Scopus Google which are to and analysis of histone is a of with a the of is by the collagen protein family S. The collagen PubMed Scopus Google proteins are of the amino proline and hydroxyproline amino a which of collagen proteolytic is a of which collagen H. protease specificity profiling of as of Proteomics. PubMed Scopus Google Scholar, M. in the of protein in ancient PubMed Scopus Google Scholar, Scopus Google Scholar). a of bone with collagen M. in the of protein in ancient PubMed Scopus Google be after M. in the of protein in ancient PubMed Scopus Google Scholar). Furthermore, trypsin is in the pH from to J.R. of sequencing grade PubMed Scopus Google in pH digestion is of this is disulfide bond mapping, conditions with pH disulfide bond which can in the of disulfide H. and for disulfide bond in Chem. PubMed Scopus Google Scholar). protein digestion with that are at acidic pH H. and for disulfide bond in Chem. PubMed Scopus Google disulfide bond by J.R. of and PubMed Scopus Google Scholar). in of proteins The at pH Y. S. J.R. in used for Chem. PubMed Scopus Google Scholar). to the and MS analysis be at pH Y. S. J.R. in used for Chem. PubMed Scopus Google Scholar). more with trypsin is the cleavage of basic for or or which can in phosphosite localization trypsin for protein C-terminal and PubMed Scopus Google Scholar). It is that can the of target proteins by and J.V. B. F. B. Mann M. in and in PubMed Scopus Google Scholar). mapping of is In bottom-up proteomics are typically to identify proteins from A. to the of peptide by and Chem. PubMed Scopus Google Scholar). However, in are To this a sequence can be used to peptide from spectra B. M. A. for peptide de novo sequencing by tandem PubMed Scopus Google Scholar). of a protein sequence a is highly for analysis of from or samples, and are and for which the are A. Y. M. A. Y. protein sequencing de novo protein and sequence Cell. Proteomics. 2017; PubMed Scopus Google Scholar). Finally, de novo sequencing can be to analysis of or peptides, or amino novo sequencing of by PubMed Scopus Google Scholar). To an efficient de novo protein sequencing by tandem and analysis of is typically This can be by a multi-enzymatic protein digestion by MS A. de novo analysis of from 2013; PubMed Scopus Google that tryptic digestion is for de novo sequencing of the proteins. most of the used in as and also proteins at amino Heck A.J.R. for proteomics PubMed Scopus Google Scholar). with cleavage specificity for proteomics the are to from regions in the In an acidic from Aspergillus niger by for applications, showing potential for protein digestion M. H. M. of in from Aspergillus niger and of the for its application in PubMed Scopus Google Scholar). also in and protein analysis in and high in that application M. M. Heck A.J.R. Aspergillus niger for and protein Chem. 2017; 89: PubMed Scopus Google Scholar). In the also by M. M. 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