Dataset Information

An evaluation of high-field asymmetric-waveform ion mobility spectrometry coupled to electron-transfer/higher-energy collision dissociation for ADP-ribosylation proteomics

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Macrophage

SUBMITTER: Sasha Singh

LAB HEAD: Masanori Aikawa

PROVIDER: PXD067389 | Pride | 2026-04-13

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	01_TK_THP-1_IFN-g_ADPr_EThcD_mz400-900_CV-50-60-70_1.raw	Raw
	01_TK_THP-1_IFN-g_ADPr_EThcD_mz400-900_CV-50-60-70_2.raw	Raw
	01_TK_THP-1_IFN-g_ADPr_EThcD_mz400-900_CV-50-60-70_3.raw	Raw
	01_TK_THP-1_IFN-g_ADPr_EThcD_mz400-900_CV-50-60-70_4.raw	Raw
	01_TK_THP-1_IFN-g_ADPr_EThcD_mz400-900_CV-50-60-70_5.raw	Raw

Items per page:

1 - 5 of 78

Publications

Impact of Variations in Commercial Standard Operating Procedure on Plasma Proteome Recovery.

Kasai Taku T Weiss Luisa L Wohlfahrt Jessica J Matamalas Joan T JT Shlayen Gabriel G Kirsch Zachary J ZJ Aikawa Masanori M Aikawa Elena E Singh Sasha A SA

Journal of proteome research 20260325 4

Advancements in mass spectrometry and complementary technologies now enable comprehensive, high-resolution plasma proteomics. Plasma is a key biofluid for clinical research, harboring potential disease-informative biomarkers. Commercial sources of nondiseased, healthy donor plasma samples are often used for proteomic workflow development and as controls for clinical studies. The overarching assumption is that standard operating procedures for plasma preparation are comparable across different so ...[more]

PMID: 41879802

Similar Datasets

Project 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.