PXD063988

PXD063988 is an original dataset announced via ProteomeXchange.

Title	Deep Coverage and Extended Sequence Reads Obtained with a Single Archaeal Protease Expedite de novo Protein Sequencing by Mass Spectrometry
Description	Label-free protein sequencing is critically enabled by bottom-up, mass spectrometry-based proteomics workflows. Applications such as antibody sequencing or antigen discovery require de novo reconstruction of peptide and protein sequences. While trypsin has long served as the gold-standard protease in proteomics, its restricted C-terminal cleavage specificity constrains peptide diversity, particularly limiting coverage in antibody hypervariable complementarity-determining regions (CDRs). As a result, current workflows yield sparse reads and sequence gaps. Although multi-protease and hybrid-fragmentation strategies can notably improve coverage, they add complexity and compromise scalability and reproducibility. Here, we present a novel approach using HyperThermoacidic Archaeal (HTA) proteases Krakatoa or Vesuvius as powerful single-enzyme solutions for de novo antibody sequencing. Each protease generated over five times more unique peptide reads than trypsin or chymotrypsin with high redundancy across CDRs. Combined with EAciD fragmentation on a ZenoTOF 7600 system, this workflow enabled complete, unambiguous antibody sequencing. Despite most de novo tools being optimized for CID/HCD-tryptic data, analysis using PEAKS/DeepNovo and Stitch softwares showed that HTA-Proteases yielded up to fourfold higher alignment scores and fewer sequence mistakes across variable regions. Redundant reads increased more than threefold compared to standard proteases, boosting confidence in amino acid assignment and reducing ambiguity in final assemblies. Our alternative HTA-EAciD approach offers short digestion times, eliminates extensive cleanup, and enables analysis in a single LC-MS/MS run. This single-protease strategy delivers sequencing performance comparable to multi-enzyme workflows, providing a scalable, efficient, and highly confident approach for de novo sequencing in antibody discovery and beyond.
HostingRepository	PRIDE
AnnounceDate	2026-03-17
AnnouncementXML	Submission_2026-03-17_10:44:21.705.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Tereza Kadavá
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	iodoacetamide derivatized residue
Instrument	ZenoTOF 7600

Revision	Datetime	Status	ChangeLog Entry
0	2025-05-15 10:02:28	ID requested
⏵ 1	2026-03-17 10:44:22	announced

P, é, rez Pa, ñ, eda L, Kadav, á T, Shamorkina TM, Schulte D, Pribil P, Heidelberger S, Narlock-Brand AM, Yannone SM, Snijder J, Heck AJR, Deep coverage and extended sequence reads obtained with a single archaeal protease expedite de novo protein sequencing by mass spectrometry. Cell Syst, ():101536(2026) [pubmed]

10.1016/j.cels.2026.101536;

submitter keyword: HTA-Proteases, Hybrid fragmentation by EAciD,De novo antibody sequencing, Bottom-up proteomics

Albert JR Heck
contact affiliation	Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, Utrecht 3584 CH, the Netherlands and Netherlands Proteomics Center, Padualaan 8, Utrecht 3584 CH, the Netherlands
contact email	A.J.R.Heck@uu.nl
lab head
Tereza Kadavá
contact affiliation	Utrecht University, Biomolecular Mass Spectrometry and Proteomics
contact email	t.kadava@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/2026/03/PXD063988

PRIDE project URI

• PRIDE
  1. PXD063988
     1. Label: PRIDE project
     2. Name: Deep Coverage and Extended Sequence Reads Obtained with a Single Archaeal Protease Expedite de novo Protein Sequencing by Mass Spectrometry