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 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.

Native Structural and Functional Proteoform Characterization of the Prolyl-Alanyl-Specific Endoprotease EndoPro from Aspergillus niger

“…After incubation, the digestion was quenched by loading and cleaning the peptides using Sep-Pak cartridges. 10 The obtained BSA peptide mixtures were analyzed with RPLC-MS/MS using both EThcD and HCD for peptide fragmentation, similar to the work of van der Laarse et al 11 Table S6 shows the search outcome, which was used for data interpretation. For both pH values, high sequence coverage was obtained for all samples (≥91% for pH 2 and ≥92% for pH 4).…”

“…Our results agree with previous studies showing that EndoPro acts primarily on the C-terminus of proline and alanine residues and minor cleavage can occur at glycine, leucine, arginine, serine, and tyrosine. 10 − 13 Relatively broad digestion specificity may have several origins. However, our data suggest that the high number of nonspecific peptides results from the action of EndoPro.…”

“…However, our data suggest that the high number of nonspecific peptides results from the action of EndoPro. Moreover, Samadova et al 10 demonstrated that EndoPro is less specific with increasing digestion time. We evaluated more in detail a subset of BSA peptide mixtures generated by EndoPro, revealing many C-terminally truncated peptides detected as ladder-like peptide series.…”

Single-chain tandem macrocyclic peptides as a scaffold for growth factor and cytokine mimetics

“…3 ), we analyzed the patterns of disulfide bonds of aMD4dY-PA22 and aMD4dY-PA49. Two STaMPtides were digested by thermolysin 24 and ProAlanase 25 , respectively, without reduction, and the molecular masses of digested fragments were subsequently analyzed using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The molecular masses of the fragments resulting from the enzymatic digestion of the STaMPtides with the desired disulfide pattern were detected (Fig.…”

Targeted liquid chromatography‐tandem mass spectrometry analysis of proteins: Basic principles, applications, and perspectives

“…In addition to trypsin, other proteases can also be used, such as chymotrypsin, ArgC, and LysC [84]. The use of proAlanase [85] or the sequential digest by two proteases (e.g., trypsin and AspN [86]) is promising. Both approaches can be used in experiments, as shown in Soldi's analysis of post-translational histone modifications [87].…”