PXD056949-1

PXD056949 is an original dataset announced via ProteomeXchange.

Title	NHS esters are non-innocent protein acylating reagents
Description	N-Hydroxysuccinimide (NHS)-ester derivatives represent one of the most widely used reagents in biological chemistry and chemical biology. Their perceived utility as acylating agents –intermediates that are sufficiently stable to be isolated (even in commercial form) yet active enough to allow direct and acylation simply upon addition – has seen their widespread application. Their efficacy relies critically on the exclusive chemoselectivity of activated acyl over that of the imidic acyl moieties in the succinimide, such that NHS acts simply as an innocent nucleofuge ‘leaving group’. Here, through systematic structural variation that modulates acyl reactivity, coupled with a statistically controlled ultra-rapid screen for unknown modifications in tandem mass spectra and lysine profiling within the native E. coli proteome, which presents complex lysine environments, we reveal that ring-opening reaction of the succinimide to afford several N-succinamide derivatives is a present, sometimes dominant, side-reaction of certain NHS esters. Moreover, the extent of side-reaction is shown to be lysine nucleophile and therefore site-dependent, with both side-reaction and desired reaction occurring within the same protein substrate. The resulting formation of bioconjugates with unintended, unstable linkages and modifications – that may have previously gone undetected – not only suggests re-evaluation of i) use of potentially several hundred of the many-thousand, known commercial reagents; and ii) the functional conclusions previously drawn using NHS esters where these hinge crucially on the precise identity of conjugates in areas as diverse as antibody-drug biotherapy, vaccination to cross-link-enabled structural analyses.
HostingRepository	PRIDE
AnnounceDate	2025-04-27
AnnouncementXML	Submission_2025-04-27_09:06:09.211.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Weibing Liu
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: 562; scientific name: Bacteria; NCBI TaxID: NCBITaxon:2;
ModificationList	carbamoylated residue; acetylated residue; monohydroxylated residue
Instrument	Orbitrap Fusion Lumos; Orbitrap Exploris 480; Orbitrap Ascend

Revision	Datetime	Status	ChangeLog Entry
0	2024-10-18 06:59:51	ID requested
⏵ 1	2025-04-27 09:06:09	announced

Dataset with its publication pending

submitter keyword: ring-opening reaction,N-Hydroxysuccinimide (NHS)-ester derivatives, N-succinamide derivatives

Benjamin G. Davis
contact affiliation	The Rosalind Franklin Institute, Oxfordshire, United Kingdom Department of Pharmacology, University of Oxford
contact email	Ben.Davis@rfi.ac.uk
lab head
Weibing Liu
contact affiliation	The Rosalind Franklin Institute, Oxfordshire, OX11 0FA, UK Department of Pharmacology, University of Oxford, OX1 3QT, UK
contact email	weibingliu@outlook.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD056949
     1. Label: PRIDE project
     2. Name: NHS esters are non-innocent protein acylating reagents