PXD006766

PXD006766 is an original dataset announced via ProteomeXchange.

Title	Aspergillus niger Prolyl endoprotease (An-PEP) for Hydrogen-Deuterium Exchange Mass Spectrometry and Protein Structural Studies
Description	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 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.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_04:38:36.026.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Liana Tsiatsiani
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: 562;
ModificationList	iodoacetamide derivatized residue
Instrument	LTQ Orbitrap Elite

Revision	Datetime	Status	ChangeLog Entry
0	2017-06-21 01:53:14	ID requested
1	2017-07-12 05:38:10	announced
2	2017-07-13 03:54:11	announced	Updated publication reference for PubMed record(s): 28657298.
⏵ 3	2024-10-22 04:38:44	announced	2024-10-22: Updated project metadata.

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]

curator keyword: Technical

submitter keyword: hydrogen-deuterium exchange, hemoglobin,Protease, mass spectrometry, Aspergillus niger, shorgun proteomics, IgG4

Albert JR Heck
contact affiliation	Biomolecular 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 email	A.J.R.Heck@uu.nl
lab head
Liana Tsiatsiani
contact affiliation	Dr. Ir.
contact email	l.tsiatsiani@uu.nl
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2017/07/PXD006766

PRIDE project URI

• PRIDE
  1. PXD006766
     1. Label: PRIDE project
     2. Name: Aspergillus niger Prolyl endoprotease (An-PEP) for Hydrogen-Deuterium Exchange Mass Spectrometry and Protein Structural Studies