PXD072165-1

PXD072165 is an original dataset announced via ProteomeXchange.

Title	Enhanced Proteomics Analysis with a Novel Recombinant Chymotrypsin Analogue En-gineered for High Cleavage Specificity
Description	Chymotrypsin is widely used in shotgun proteomics owing to its orthogonal cleavage specificity relative to trypsin, which enhances sequence coverage of hydrophobic protein regions. However, commercial preparations often display variable cleavage specificity, trypsin contamination, and elevated missed-cleavage rates, which collectively can reduce proteome coverage and data reproducibility. To address these limitations, we present a novel recombinant chymotrypsin (rChymoSelect) engineered for improved cleavage specificity and robustness in proteomics workflows. Benchmarking against standard bovine chymotrypsin revealed 97 % C-terminal cleavages after tyrosine (Y), phenylalanine (F), and leucine (L) for rChymoSelect, compared with 72 % for the standard enzyme. This enhanced cleavage specificity re-duced missed cleavages and increased peptide-spectrum matches across charge states. Across 3,638 identified proteins, rChymoSelect yielded 22.2 % unique identifications compared with 8.2 % for stand-ard chymotrypsin, while maintaining similar peptide length, m/z, and hydrophobicity distributions. No-tably, rChymoSelect showed enriched recovery of mitochondrial proteins, consistent with its improved digestion of hydrophobic targets. The enzyme remained active in up to 6 M urea and achieved near-maximal proteome coverage within 2 hours (only a 2.4 % gain after overnight digestion). Integration with data-independent acquisition (DIA) increased total protein identifications from approximately 2,200 (DDA) to 3,200 (DIA), a 45 % gain, with rChymoSelect outperforming standard chymotrypsin by 16.6–22.4 % in peptide-spectrum matches and 4.6–6.2 % in protein identifications. These results estab-lish rChymoSelect as an advanced tool with improved cleavage specificity that reduces analytical com-plexity, and enhances the reliability of proteomic analysis, while expanding chymotryptic digestion to hydrophobic and high-denaturant proteomics applications.
HostingRepository	PRIDE
AnnounceDate	2026-06-08
AnnouncementXML	Submission_2026-06-07_20:26:29.656.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Kish Adoni
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Eclipse

Revision	Datetime	Status	ChangeLog Entry
0	2025-12-18 15:26:26	ID requested
⏵ 1	2026-06-07 20:26:30	announced

10.1021/acs.jproteome.5c01262;

Adoni KR, Ditcham JE, Gonz, á, lez Rivera AK, Charlton GH, Saveliev SV, Thalassinos K, Zenezini Chiozzi R, Enhanced Proteomics Analysis with a Novel Recombinant Chymotrypsin Analogue Engineered for High Cleavage Specificity. J Proteome Res, 25(5):2511-2519(2026) [pubmed]

submitter keyword: Chymotrypsin, Missed Cleavages, Sample Preparation, Proteomics, Protein Digestion

Konstantinos Thalassinos
contact affiliation	UCL
contact email	k.thalassinos@ucl.acuk
lab head
Kish Adoni
contact affiliation	University College London
contact email	kish.95@live.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/2026/06/PXD072165

PRIDE project URI

• PRIDE
  1. PXD072165
     1. Label: PRIDE project
     2. Name: Enhanced Proteomics Analysis with a Novel Recombinant Chymotrypsin Analogue En-gineered for High Cleavage Specificity