Comparative Analysis of Cleavage Specificities of Immobilized Porcine Pepsin and Nepenthesin II under Hydrogen/Deuterium Exchange Conditions.

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  • Additional Information
    • Source:
      Publisher: American Chemical Society Country of Publication: United States NLM ID: 0370536 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1520-6882 (Electronic) Linking ISSN: 00032700 NLM ISO Abbreviation: Anal Chem Subsets: MEDLINE
    • Publication Information:
      Original Publication: Washington, American Chemical Society.
    • Subject Terms:
      Proteolysis*; Enzymes, Immobilized/*chemistry ; Pepsin A/*chemistry; Animals ; Biocatalysis ; Deuterium/chemistry ; Deuterium Exchange Measurement ; Mass Spectrometry ; Sarraceniaceae/enzymology ; Substrate Specificity ; Swine
    • Abstract:
      Hydrogen/Deuterium Exchange (HDX) coupled with Mass Spectrometry (HDX-MS) is a sensitive and robust method to probe protein conformational changes and protein-ligand interactions. HDX-MS relies on successful proteolytic digestion of target proteins under acidic conditions to localize perturbations in exchange behavior to protein structure. The ability of the protease to produce small peptides and overlapping fragments and provide sufficient coverage of the protein sequence is essential for localizing regions of interest. While the acid protease pepsin has been the enzyme of choice for HDX-MS studies, recently, it was shown that aspartic proteases from carnivorous pitcher plants of the genus Nepenthes are active under low-pH conditions and cleave at basic residues that are "forbidden" in peptic digests. In this report, we describe the utility of one of these enzymes, Nepenthesin II (NepII), in a HDX-MS workflow. A systematic and statistical analysis of data from 11 proteins (6391 amino acid residues) digested with immobilized porcine pepsin or NepII under conditions compatible with HDX-MS was performed to examine protease cleavage specificities. The cleavage of pepsin was most influenced by the amino acid residue at position P1. Phe, Leu, and Met are favored residues, each with a cleavage probability of greater than 40%. His, Lys, Arg, or Pro residues prohibit cleavage when found at the P1 position. In contrast, NepII offers advantageous cleavage to all basic residues and produces shortened peptides that could improve the spatial resolution in HDX-MS studies.
    • Grant Information:
      F31 GM126842 United States GM NIGMS NIH HHS
    • Accession Number:
      0 (Enzymes, Immobilized)
      AR09D82C7G (Deuterium)
      EC 3.4.23.1 (Pepsin A)
    • Publication Date:
      Date Created: 20200714 Date Completed: 20210225 Latest Revision: 20230717
    • Publication Date:
      20231215
    • Accession Number:
      10.1021/acs.analchem.9b05694
    • Accession Number:
      32658454