ArticleAnalytical chemistry2017
Aspergillus niger Prolyl Endoprotease for Hydrogen-Deuterium Exchange Mass Spectrometry and Protein Structural Studies.
Article in Analytical chemistry, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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Who cites it
22 citing papers in PubMed, 39 citations in OpenAlex.
- DigDig: A Software for In-Depth Analysis and Comparison of Proteolytic Digestion.Analytical chemistry · 2025Article
- Identification of a direct interaction between the Fab domains of IgG antibodies and human FcRn upon IgG-FcRn complex formation.Communications biology · 2025Article
- Activation mechanism of small heat shock protein HSPB5 revealed by disease-associated mutants.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- Structural insights into the role of the proline rich region in tau function.Structure (London, England : 1993) · 2025Article
- Postproline Cleaving Enzymes also Show Specificity to Reduced Cysteine.Analytical chemistry · 2024Article
- Computational Tools for Hydrogen-Deuterium Exchange Mass Spectrometry Data Analysis.Chemical reviews · 2024Review
- Cyclic Ion Mobility for Hydrogen/Deuterium Exchange-Mass Spectrometry Applications.Analytical chemistry · 2024Article
- Structural and time-resolved mechanistic investigations of protein hydrolysis by the acidic proline-specific endoprotease from Aspergillus niger.Protein science : a publication of the Protein Society · 2024Article
- Hydrogen/Deuterium Exchange Mass Spectrometry of Heme-Based Oxygen Sensor Proteins.Methods in molecular biology (Clifton, N.J.) · 2023Article
- Study of Protein Conformational Dynamics Using Hydrogen/Deuterium Exchange Mass Spectrometry.Methods in molecular biology (Clifton, N.J.) · 2023Article
- Post-Proline Cleaving Enzymes (PPCEs): Classification, Structure, Molecular Properties, and Applications.Plants (Basel, Switzerland) · 2022Review
- Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems.Chemical reviews · 2022Review
- Native Structural and Functional Proteoform Characterization of the Prolyl-Alanyl-Specific Endoprotease EndoPro fromJournal of proteome research · 2021Review
- Mass Spectrometry-Based Structural Virology.Analytical chemistry · 2021Review
- Relative Rates of Gluten Digestion by Nine Commercial Dietary Digestive Supplements.Frontiers in nutrition · 2021Article
- ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping.Molecular & cellular proteomics : MCP · 2020Article
- Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes.Life (Basel, Switzerland) · 2020Review
- Organic Solvents for Enhanced Proteolysis of Stable Proteins for Hydrogen-Deuterium Exchange Mass Spectrometry.Analytical chemistry · 2020Article
- HDX-MS: An Analytical Tool to Capture Protein Motion in Action.Biomedicines · 2020Review
- Targeting proline in (phospho)proteomics.The FEBS journal · 2020Article
Corrections and comments
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
To monitor the structural integrity of therapeutic proteins, hydrogen-deuterium exchange mass spectrometry (HDX-MS) is increasingly utilized in the pharmaceutical industry. The successful outcome of HDX-MS analyses depends on the sample preparation conditions, which involve the rapid digestion of proteins at 0 °C and pH 2.5. Very few proteases are able to withstand such harsh conditions, with pepsin being the best-known exception, even though its activity is also strongly reduced at 0 °C. Here, we evaluate the usage of a prolyl endopeptidase from Aspergillus niger (An-PEP) for HDX-MS. What makes this protease very attractive is that it cleaves preferentially the hardest to digest amino acid, proline. To our surprise, and in contrast to previous reports, An-PEP activity was found optimal around pH 2.5 and could be further enhanced by urea up to 40%. Under typical HDX-MS conditions and using small amounts of enzyme, An-PEP generated an equivalent number of peptides as pepsin, as exemplified by using the two model systems tetrameric human hemoglobin (Hb) and human IgG4. Interestingly, because An-PEP peptides are shorter than pepsin-generated peptides, higher sequence resolution could be achieved, especially for Pro-containing protein regions in the alpha subunit of Hb, revealing new protected Hb regions that were not observed with pepsin. Due to its Pro-preference and resistance to low pH, we conclude that An-PEP is an archetype enzyme for HDX-MS, highly complementary to pepsin, and especially promising for structural studies on Pro-rich proteins or proteins containing Pro-rich binding domains involved in cellular signaling.
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