PXD008068

PXD008068 is an original dataset announced via ProteomeXchange.

Title	Quantifying missing (phospho)proteome regions with the broad-specificity protease subtilisin
Description	Despite huge efforts to map the human proteome using mass spectrometry the overall sequence coverage achieved to date is still below 50%. Reasons for missing areas of the proteome comprise protease-resistant domains including the lack/excess of enzymatic cleavage sites, non-unique peptide sequences, impaired peptide ionization/separation and low expression levels. To access novel areas of the proteome the beneficial use of enzymes complementary to trypsin, such as GluC, AspN, LysN, ArgC, LysArginase has been reported. Here, we present how the broad-specificity protease subtilisin enables mapping of previously hidden areas of the proteome. We systematically evaluated its digestion efficiency and reproducibility and compared it to the gold standard in the field, trypsin. Notably, subtilisin allows reproducible near-complete digestion of cells lysates in 1 minute. As expected from its broad specificity the generation of overlapping peptide sequences reduces the number of identified proteins compared to trypsin (8,363 vs. 6,807; 1% protein FDR). However, subtilisin considerably improved the coverage of missing and particularly proline-rich areas of the proteome. Along 14,628 high confidence phosphorylation sites identified in total, only 33% were shared between both enzymes, while 37% were exclusive to subtilisin. Notably, 926 of these were not even accessible by additional in silico digestion with either AspN, ArgC, GluC, LysC, or LysN. Thus, subtilisin might be particularly beneficial for system-wide profiling of post-translational modification sites. Finally, we demonstrate that subtilisin can be used for reporter-ion based in-depth quantification, providing a precision comparable to trypsin – despite broad specificity and fast digestion that may increase technical variance.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_04:41:22.905.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Humberto Jorge
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue; iTRAQ8plex-116 reporter+balance reagent acylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	LTQ Orbitrap Velos; Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2017-10-30 02:29:01	ID requested
1	2017-11-29 17:05:03	announced
⏵ 2	2024-10-22 04:41:23	announced	2024-10-22: Updated project metadata.

10.1021/acs.analchem.7b02395;

Gonczarowska-Jorge H, Loroch S, Dell'Aica M, Sickmann A, Roos A, Zahedi RP, Quantifying Missing (Phospho)Proteome Regions with the Broad-Specificity Protease Subtilisin. Anal Chem, 89(24):13137-13145(2017) [pubmed]

curator keyword: Technical, Biological

submitter keyword: novel phosphorylation sites, proteome coverage, Pro-rich regions, alternative enzymes, trypsin,sample preparation, missing proteins

René Peiman Zahedi
contact affiliation	Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., Dortmund, Germany Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, Canada. Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, Canada.
contact email	rene.zahedi@isas.de
lab head
Humberto Jorge
contact affiliation	Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V.
contact email	humgj87@gmail.com
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/2017/11/PXD008068

PRIDE project URI

• PRIDE
  1. PXD008068
     1. Label: PRIDE project
     2. Name: Quantifying missing (phospho)proteome regions with the broad-specificity protease subtilisin