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Link to original content: https://pubmed.ncbi.nlm.nih.gov/33020190
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. 2020 Dec;19(12):2139-2157.
doi: 10.1074/mcp.TIR120.002129. Epub 2020 Oct 5.

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

Diana Samodova  1 , Christopher M Hosfield  2 , Christian N Cramer  3 , Maria V Giuli  4 , Enrico Cappellini  5 , Giulia Franciosa  1 , Michael M Rosenblatt  2 , Christian D Kelstrup  1 , Jesper V Olsen  6
Affiliations

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

Diana Samodova et al. Mol Cell Proteomics. 2020 Dec.

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.

Keywords: De novo sequencing; Disulfides*; Histones*; PTMs; Phosphoproteins*; ProAlanase; Proline-rich Proteins; Proteases*; Proteolysis*.

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Conflict of interest statement

Conflict of interest—The authors have declared a conflict of interest. Christopher Hosfield and Michael Rosenblatt are employees of Promega Corporation. Promega plans to sell sequencing grade ProAlanase for proteomics. Christian N. Cramer is an employee of Novo Nordisk a/s.

Figures

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Graphical abstract
Fig. 1.
Fig. 1.
Determination of optimal digestion conditions for ProAlanase, using HeLa cell lysate. Specificity plots reflecting the average C-terminal amino acid frequency in ProAlanase-digested HeLa cell lysates (n = 2) at the different pH (B) and time (A) of digestion. Unspecific MaxQuant search settings were used for specificity estimation. C-terminal amino acid frequency on the y axis was calculated as a relative count of identified peptides, containing the specific C-terminal residue. Average proportion of unique peptides identified in ProAlanase digests of HeLa cell lysates (n = 2) carrying 0, 1, 2, 3 or 4 missed cleavage sites, using enzyme-specific settings for peptide-to-sequence matching. The comparison is done for the different pH (D) and time (C) of digestion. Digestion conditions, considered as optimal, are marked with the *. Unique peptides sequences identi_ed in ProAlanase digests of HeLa cell lysates (n = 4) at the optimal digestion conditions (E).
Fig. 2.
Fig. 2.
Characterization of missed cleavage patterns in ProAlanase-digested HeLa cell lysate. Frequency distribution plots of cleavage sites identified in human proteome samples, digested with ProAlanase. The unique peptide sequences from the two experimental replicates were concatenated to evaluate sequence context of missed proline and alanine cleavages. Sequences were aligned at cleavage sites between P1 and P1'. The significant differences in amino acid occurrence are compared with the natural abundance in human proteome (P < 0.05).
Fig. 3.
Fig. 3.
Multi-enzymatic histone profiling in human proteome. Protein family enrichment analysis Pfam (A) was performed using protein groups, obtained from ProAlanase, tryptic, rAsp-N and Glu-C digests of HeLa cell lysates (n = 4 for ProAlanase and trypsin; n = 3 for rAsp-N and Glu-C). Protein groups were concatenated across the experimental replicates and duplicate entries were removed. All proteins were ranked by sequence coverage in descending order and Top120 entries were used for Pfam analysis. The Pfam was performed in STRING. The hierarchical clustering was done using the Manhattan distance metric within Perseus with relative enrichment on gene level and an FDR of 0.05. A sequence coverage plot (B) showing the coverage of histone H2A type 2-A protein sequence by petide sequences obtained from ProAlanase (n = 4), tryptic (n = 4) and Glu-C (n = 3) digests of HeLa cell lysate. The peptides used for alignment were concatenated across the experimental replicates and duplicate entries were removed. The medications mapped on the sequence were identified only in ProAlanase-digested samples.
Fig. 4.
Fig. 4.
Palaeoproteomic characterization of Pleistocene mammoth bone, using ProAlanase and trypsin. Sample preparation workflow (A), including mechanical crushing and demineralization of the bone sample, followed by protein extraction, manual fractionation and digestion either with Pro- Alanase, or with trypsin. The Venn diagrams are done based on the proteins concatenated from the 2 experimental replicates. Only leading proteins in each protein group were used to do the overlap. Noncollagenous proteins are marked in black, but collagen isoforms - in gray. Proline, alanine and hydroxyproline cleavage sites (B) in ProAlanase-digested (all fractions merged for the 2 replicates) mammoth bone sample. Multiple sequence alignment (C) of fetuin-A protein sequences between the Pleistocene mammoth fetuin and the five closely related mammalian species. The signal peptide is marked with the pink rectangle, whereas the phylogenetically-informative amino acid substitutions are marked with the *. The alignment was performed using ClustalX2, and complete version, including the alignment of all species with BLASTpidentity ≥ 50% is shown in supplemental Fig. S5. Protein family enrichment analysis Pfam (D) was performed using protein groups, obtained from ProAlanase and tryptic digests of Pleistocene mammoth bone sample (all fractions merged for the 2 replicates). The Pfam was performed in STRING. The hierarchical clustering was done using the Manhattan distance metric within Perseus with relative enrichment on gene level and an FDR of 0.05.
Fig. 5.
Fig. 5.
Phosphorylation profiling in immunoprecipitated N3ICD protein. A sequence coverage plot (A) showing the coverage of N3ICD protein sequence by concatenated ProAlanase and tryptic peptides obtained from in-gel digestion of N3ICD protein immunoprecipitates (n = 2). Average number of unique phosphopeptide sequences (B) identified in ProAlanase (n = 3) and tryptic (n = 2) digests of N3ICD immunoprecipitates. A Venn Diagram (C) showing an overlap of class I (localization probability ≥ 75%) phosphorylation sites in ProAlanase- and trypsin digested samples of N3ICD protein immunoprecipitates (n = 2). A scatter plot (D) of all phosphorylation sites identi_ed in ProAlanase and tryptic digests of N3ICD protein (n = 2). The variances were estimated using the two-sample Fisher's exact test and the significance was determined with the one-tailed two-sample t test (α = 0.05) with equal variance. The mean value is highlighted as a dash and n corresponds to the number of unique phosphorylation sites identified with each of the proteases.
Fig. 6.
Fig. 6.
Disulfide bond mapping in NIST mAb, using ProAlanase and trypsin. Total ion current (TIC) chromatograms of ProAlanase-and trypsin-digested mAb. Both constant and variable regions (except for the hinge region) are covered by ProAlanase peptides (A); only constant region is covered by the tryptic peptides (B).
Fig. 7.
Fig. 7.
Database-independent sequence reconstruction of N3ICD protein (Q61982), using ProAlanase and trypsin. De novo sequencing plot showing a 89% coverage of N3ICD protein (Q61982) by unique ProAlanase and tryptic de novo peptides, obtained from in-gel digested N3ICD immunoprecipitates. The two biological replicates of ProAlanase- and trypsin-digested N3ICD were merged in as single data search, by protease. PEAKS X+ Studio was used to generate ProAlanase and tryptic de novo peptide sequences. The generated peptides were _filtered using a 90% De Novo score threshold and duplicates were removed. Sequence alignment was performed using PepExplorer within PatternLab for Proteomics.
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