PXD069252-1

PXD069252 is an original dataset announced via ProteomeXchange.

Title	NHS Carbamate Cross-Linkers Provide Complementary XL-MS Restraints via Preferential N-Terminal Targeting
Description	Cross-linking mass spectrometry (XL-MS) has become a valuable tool for investigating the structural morphology and plasticity of proteins. Traditional cross-linkers contain two N-hydroxy succinimide (NHS) esters that mainly react with lysine residues. In this work, we optimized the in-solution reactivity of the cross-linker disuccinimidyl sulfoxide (DSSO) carbamate. DSSO carbamate is a DSSO analogue bearing two NHS carbamate groups instead of NHS esters as reactive moiety. The higher stability of the carbamate function mitigates the degradation of DSSO via retro-ene sulfoxide elimination in standard XL-MS buffers which limits the cross-linking yield. Additionally, we elucidated its characteristic gas-phase dissociation behavior and optimized the collision energy (CE) for automated analysis with XL-MS search engines. Cross-link site analysis of bovine serum albumin highlighted an unexpected abundance of cross-link species involving the N-terminus of the protein. We attributed this to a higher N-terminal reactivity of the NHS carbamate group compared to NHS esters. We confirmed our hypothesis by cross-linking non-acetylated and N-terminally acetylated α-asynuclein with DSSO carbamate and the NHS ester-based disuccinimidyl dibutyric urea (DSBU) cross-linker. Finally, we applied the NHS carbamate-based cross-linker NNP9 and observed the same chemical propensity. NHS carbamate-based reagents give complementary XL-MS restraints to NHS ester-based cross-linkers and can be useful for studying systems where N-terminal binding plays a significant role. We anticipate that this unexpected selectivity of NHS carbamates to protein N-termini may have applications in bioconjugation and chemical proteomics in general.
HostingRepository	PRIDE
AnnounceDate	2026-03-30
AnnouncementXML	Submission_2026-03-29_17:36:38.109.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Alessio Di Ianni
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606; scientific name: Bos taurus (Bovine); NCBI TaxID: NEWT:9913;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion Lumos; Q Exactive Plus

Revision	Datetime	Status	ChangeLog Entry
0	2025-10-09 06:42:49	ID requested
⏵ 1	2026-03-29 17:36:39	announced

Di Ianni A, Leischner TF, Brutiu BR, Saridakis I, Di Ianni A, Krolle H, Ihling CH, Shaaban S, Maulide N, Sinz A, Iacobucci C, Intrinsic N-Terminal Reactivity and Improved Analysis of DSSO-Carbamate and Carbamate-Based Cross-Linkers. Anal Chem, 98(11):8167-8178(2026) [pubmed]

10.1021/acs.analchem.5c06834;

submitter keyword: Crosslinking MS, Cross-linking MS, DSSO carbamate, NHS carbamate cross-linkers, N-terminal reactivity, XL-MS, Cross-linking mass spectrometry, MS-cleavable cross-linkers

Andrea Sinz
contact affiliation	Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, Halle/Saale D-01620, Germany Center for Structural Mass Spectrometry, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, Halle/Saale D-01620, Germany
contact email	andrea.sinz@pharmazie.uni-halle.de
lab head
Alessio Di Ianni
contact affiliation	Human Technopole
contact email	alessio.diianni@fht.org
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/PXD069252

PRIDE project URI

• PRIDE
  1. PXD069252
     1. Label: PRIDE project
     2. Name: NHS Carbamate Cross-Linkers Provide Complementary XL-MS Restraints via Preferential N-Terminal Targeting