Dataset Information

ProAlanase is an effective alternative to trypsin for proteomics applications and disulfide bond mapping_part2

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 digestion solution. In this study we characterized 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 digestions are carried out at acidic pH (1.5) for relatively short (2 hr) time periods. To elucidate the potential of ProAlanase in proteomic applications, we conducted a series of investigations comprising digestion of proline-rich proteins, PTM analysis, de novo protein sequencing and disulfide bond mapping. The results demonstrated that digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain and improves non-collagenous bone protein identification. Notably, cleavage also occurs at the C-terminus of hydroxyproline, facilitating efficient digestion of bone collagen. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-containing regions in monoclonal antibodies, as well as achieving nearly complete database-independent sequence reconstruction of endogenous protein by de novo sequencing.

INSTRUMENT(S): Q Exactive HF-X, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Leukocyte, Epithelial Cell, Cell Culture, Hela Cell

DISEASE(S): Cervix Carcinoma,Myeloid Leukemia

SUBMITTER: Diana Samodova

LAB HEAD: Jesper Velgaard Olsen

PROVIDER: PXD021191 | Pride | 2020-10-19

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20180926_QE3_nLC3_DS_SA_AD1_01.raw	Raw
	20180926_QE3_nLC3_DS_SA_AD2_01.raw	Raw
	20180926_QE3_nLC3_DS_SA_AD3_01.raw	Raw
	20181209_QE5_nLC5_DS_SA_GB1_01.raw	Raw
	20181209_QE5_nLC5_DS_SA_GB2_01.raw	Raw

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

Samodova Diana D Hosfield Christopher M CM Cramer Christian N CN Giuli Maria V MV Cappellini Enrico E Franciosa Giulia G Rosenblatt Michael M MM Kelstrup Christian D CD Olsen Jesper V JV

Molecular & cellular proteomics : MCP 20201005 12

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 <i>Aspergillus niger</i> 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) tim ...[more]

PMID: 33020190

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