PXD068570

PXD068570 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 quadrupole-ion trap-Orbitrap (Fusion Lumos) also improves EThcD-dependent ADP-ribosyl peptide sequencing. Methods: ADP-ribosyl peptides derived from the human macrophage-like cell line THP-1 were analyzed on the Fusion Lumos fronted with a FAIMS Pro device. FAIMS plus/minus gas-phase segmentation (GPS) of the MS1 scan was applied to HCD and EThcD acquisitions. ADP-ribosyl peptide spectra were annotated using the SEQUEST-HT algorithm and RiboMaP, an annotation tool specific for ADP-ribosylated peptide spectra. Results: HCD-dependent ADP-ribosyl peptide identifications were enriched at higher compensation voltages as compared to EThcD. The net number of unique ADP-ribosyl and non-ADP-ribosyl (contaminant) peptides across compensation voltages 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 also confirmed that while multiple injections of peptides employing distinct compensation voltages maximized the number of EThcD-dependent ADP-ribosyl peptide identifications, their associated XCorr and p-series scores decreased. The most frequent ADP-ribosyl acceptor site was lysine, followed by serine, and that the proportion of ADP-ribosylated serine sites increased when THP-1 cells were activated with IFN-γ. Conclusions: Although FAIMS increases EThcD-dependent sequencing depth of ADP-ribosyl peptides, the gains are less than when using HCD. The ability to filter out doubly charged non-ADP-ribosylated peptides at increasingly higher negative compensation voltages benefits HCD, but not EThcD since this dissociation method works optimally with highly charged peptides, non-ADP-ribosyl and ADP-ribosyl alike.
HostingRepository	PRIDE
AnnounceDate	2025-12-26
AnnouncementXML	Submission_2025-12-26_10:15:55.569.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD068570
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	2025-09-19 06:10:13	ID requested
⏵ 1	2025-12-26 10:15:56	announced

10.6019/PXD068570;

10.3389/FCHBI.2025.1709253;

submitter keyword: None

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, 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

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PRIDE project URI

• PRIDE
  1. PXD068570
     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