PXD067389-1

PXD067389 is an original dataset announced via ProteomeXchange.

Title	An evaluation of high-field asymmetric-waveform ion mobility spectrometry coupled to electron-transfer/higher-energy collision dissociation for ADP-ribosylation proteomics
Description	Objective: ADP-ribosylation is a post-translational modification that plays an important role in cellular processes. Our previous work implemented multiple gas-phase separation strategies (e.g., FAIMS) and in-source CID on the quadrupole-Orbitrap (Exploris 480) to increase the yield and acceptor site confidence scores of HCD-dependent ADP-ribosyl (ADPr) peptide identifications. We evaluated whether FAIMS coupled on the quadruple-ion trap-Orbitrap (Fusion Lumos) also improves EThcD-dependent ADP-ribosyl peptide sequencing. Methods: ADPr peptides derived from the human macrophage-like cell line THP-1 (THP-1-Mφ) were analyzed on the Lumos fronted with a FAIMS Pro and EASY-Spray Source, coupled to an Easy-nLC1200 HPLC pump. Gas-phase segmentation (GPS) for the MS1 scan range, and single and multiple combined compensation voltages (CVs) for FAIMS were applied for HCD and EThcD properties. ADP-ribosyl peptide spectra were analyzed using Proteome Discoverer 2.5. The ptmRS function was used for calculating ADP-ribosyl site probabilities. Results: We evaluated the number of ADPr and non-ADPr PSMs using ADPr peptides pooled from PBS and IFN-γ treated THP-1-Mφ across a range of CVs (-40V to -85V) using FAIMS with HCD and EThcD. The peak CVs for ADPr and non-ADPr PSMs were shifted for HCD, while they represented similar distributions for EThcD. The net number of unique ADPr and non-ADPr peptides across the CVs increased by 3.2- and 3.8-fold more respectively for HCD, and 2.0- and 3.6-fold respectively for EThcD, compared to no FAIMS. We then tested 4 distinct MS methods: (method 1: m/z 400-900, method 2: m/z 400-655 and m/z 650-900, method 3: three CVs combination (-50V, -60V, -70V) with m/z 400-900, method 4: three different acquisitions using distinct CVs (-50V, -60V, -70V) with m/z 400-900) for identifying the maximum number of ADPr acceptor sites with high (>95%) confidence using EThcD. Method 4 was best with 562 ADPr PSMs, but only 54% were high confidence ADPr sites. Due to sample volume limitation, we used method 3 for analyzing ADPr peptides enriched from THP-1-Mφ treated with PBS or IFN-γ separately. We identified 324 and 196 unique ADPr peptides from PBS and IFN-γ, of which 257 and 139 unique acceptor sites were identified with high confidence. The most frequent ADPr acceptor site was lysine (>90%), followed by serine. Conclusion: Our data demonstrated that while FAIMS is valuable for EThcD-dependent sequencing of ADPr peptides, the gains are less for ADPr peptides or contaminant non-ADPr peptides when using HCD.
HostingRepository	PRIDE
AnnounceDate	2026-04-13
AnnouncementXML	Submission_2026-04-13_07:19:05.505.xml
DigitalObjectIdentifier	https://doi.org/10.6019/PXD067389
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Sasha Singh
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: NEWT:9606;
ModificationList	adenosine diphosphoribosyl (ADP-ribosyl) modified residue; acetylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2025-08-15 10:51:20	ID requested
⏵ 1	2026-04-13 07:19:06	announced

10.6019/PXD067389;

10.1021/acs.jproteome.5c01012;

Kasai T, Weiss L, Wohlfahrt J, Matamalas JT, Shlayen G, Kirsch ZJ, Aikawa M, Aikawa E, Singh SA, Impact of Variations in Commercial Standard Operating Procedure on Plasma Proteome Recovery. J Proteome Res, 25(4):1941-1950(2026) [pubmed]

submitter keyword: Macrophage, proteomics, EThcD, HCD,ADP-ribosylation

Masanori Aikawa
contact affiliation	Center for Interdisciplinary Cardiovascular Sciences, Division of Cardiovascular Medicine, Department of Medicine, Brigham Women's Hospital, Harvard Medical School, Boston, MA, 02115, United States
contact email	maikawa@bwh.harvard.edu
lab head
Sasha Singh
contact affiliation	Brigham and Women's Hospital, Harvard Medical School
contact email	sasingh@bwh.harvard.edu
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/2026/04/PXD067389

PRIDE project URI

• PRIDE
  1. PXD067389
     1. Label: PRIDE project
     2. Name: An evaluation of high-field asymmetric-waveform ion mobility spectrometry coupled to electron-transfer/higher-energy collision dissociation for ADP-ribosylation proteomics