ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping
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ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping. / Samodova, Diana; Hosfield, Christopher M.; Cramer, Christian N.; Giuli, Maria V.; Cappellini, Enrico; Franciosa, Giulia; Rosenblatt, Michael M.; Kelstrup, Christian D.; Olsen, Jesper V.
In: Molecular & Cellular Proteomics, Vol. 19, No. 12, 2020.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping
AU - Samodova, Diana
AU - Hosfield, Christopher M.
AU - Cramer, Christian N.
AU - Giuli, Maria V.
AU - Cappellini, Enrico
AU - Franciosa, Giulia
AU - Rosenblatt, Michael M.
AU - Kelstrup, Christian D.
AU - Olsen, Jesper V.
PY - 2020
Y1 - 2020
N2 - The ProAlanase protease cleaves after proline and alanine residues in highly acidic conditions and provides efficient digestion of proline-rich proteins allowing for accurate phosphorylation site profiling. Because of its activity at low pH, it facilitates efficient disulfide bond mapping in monoclonal antibodies and is highly suitable for analysis of histone family members and their PTMs. The protease is complementary to trypsin and allows for increased protein sequence coverage and nearly complete de novo sequencing of exogenously expressed proteins when combined with trypsin.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.
AB - The ProAlanase protease cleaves after proline and alanine residues in highly acidic conditions and provides efficient digestion of proline-rich proteins allowing for accurate phosphorylation site profiling. Because of its activity at low pH, it facilitates efficient disulfide bond mapping in monoclonal antibodies and is highly suitable for analysis of histone family members and their PTMs. The protease is complementary to trypsin and allows for increased protein sequence coverage and nearly complete de novo sequencing of exogenously expressed proteins when combined with trypsin.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.
KW - Proteases
KW - Phosphoproteins
KW - De novo sequencing
KW - Disulfides
KW - Proteolysis
KW - Histones
KW - ProAlanase
KW - Proline-rich Proteins
KW - PTMs
KW - MASS-SPECTROMETRY
KW - COLLAGEN STRUCTURE
KW - PROTEIN
KW - DISSOCIATION
KW - ENERGY
KW - IDENTIFICATION
KW - ENDOPROTEASE
KW - DIGESTION
KW - PEPTIDES
KW - EXCHANGE
U2 - 10.1074/mcp.TIR120.002129
DO - 10.1074/mcp.TIR120.002129
M3 - Journal article
C2 - 33020190
VL - 19
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
SN - 1535-9476
IS - 12
ER -
ID: 253732609