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PXD006766

PXD006766 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleAspergillus niger Prolyl endoprotease (An-PEP) for Hydrogen-Deuterium Exchange Mass Spectrometry and Protein Structural Studies
DescriptionTo 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 known 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 its ability to cleave 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 (PRPs) or proteins containing Pro-rich binding domains involved in cellular signaling.
HostingRepositoryPRIDE
AnnounceDate2024-10-22
AnnouncementXMLSubmission_2024-10-22_04:38:36.026.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterLiana Tsiatsiani
SpeciesList scientific name: Escherichia coli; NCBI TaxID: 562;
ModificationListiodoacetamide derivatized residue
InstrumentLTQ Orbitrap Elite
Dataset History
RevisionDatetimeStatusChangeLog Entry
02017-06-21 01:53:14ID requested
12017-07-12 05:38:10announced
22017-07-13 03:54:11announcedUpdated publication reference for PubMed record(s): 28657298.
32024-10-22 04:38:44announced2024-10-22: Updated project metadata.
Publication List
10.1021/acs.analchem.7b01161;
Tsiatsiani L, Akeroyd M, Olsthoorn M, Heck AJR, Aspergillus niger Prolyl Endoprotease for Hydrogen-Deuterium Exchange Mass Spectrometry and Protein Structural Studies. Anal Chem, 89(15):7966-7973(2017) [pubmed]
Keyword List
curator keyword: Technical
submitter keyword: hydrogen-deuterium exchange, hemoglobin,Protease, mass spectrometry, Aspergillus niger, shorgun proteomics, IgG4
Contact List
Albert JR Heck
contact affiliationBiomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, and Netherlands Proteomics Centre, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
contact emailA.J.R.Heck@uu.nl
lab head
Liana Tsiatsiani
contact affiliationDr. Ir.
contact emaill.tsiatsiani@uu.nl
dataset submitter
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Dataset FTP location
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