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

Objective: ADP-ribosylation plays an important role in many cellular processes. Our previous work implemented field asymmetric-waveform ion mobility spectrometry (FAIMS) and in-source collision-induced dissociation (CID) on the quadrupole-Orbitrap to increase ADP-ribosyl peptide yield and acceptor site confidence of higher-energy collisional dissociation (HCD)-dependent ADP-ribosyl peptide identifications. In this study, we evaluated whether FAIMS on the quadrupole-ion trap-Orbitrap also improves electron-transfer/higher-energy collision dissociation (EThcD)-dependent ADP-ribosyl peptide sequencing.

Methods: ADP-ribosyl peptides derived from THP-1 cells were analyzed on the Fusion Lumos fronted with a FAIMS Pro device. ADP-ribosyl peptides were sequenced using either HCD or EThcD and 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 than those that used EThcD. The net number of unique ADP-ribosyl and non-ADP-ribosyl (contaminant) peptides across compensation voltages increased by 4.1- and 4.0-fold, respectively, for HCD, and 2.0- and 4.1-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. The proportion of ADP-ribosylated serine sites increased when THP-1 cells were activated with interferon γ (IFN-γ).

Conclusion: Although FAIMS increases the EThcD-dependent sequencing depth of ADP-ribosyl peptides, the gains are less than when using HCD. The ability to filter out doubly charged contaminant peptides at increasingly higher negative compensation voltages benefits HCD but not EThcD because this dissociation method works optimally with highly charged peptides, non-ADP-ribosyl and ADP-ribosyl alike.

Introduction

Adenosine diphosphate ribosylation (ADP-ribosylation) is a reversible post-translational modification involved in a variety of cellular processes, including DNA repair and replication, RNA processing, stress response, and inflammation (Fehr et al., 2020). The modification entails the transfer of the ADP-ribose moiety from NAD+ to specific amino acids, either as a monomeric ADP-ribosyl (MAR) or polymeric ADP-ribosyl (PAR) form. The reaction is catalyzed by the poly-ADP-ribosyltransferases/polymerases (PARPs). The most notable is PARP1, which is rapidly activated at sites of DNA damage where it modifies itself and other chromatin-associated proteins (Suskiewicz et al., 2023). Other PARPs (e.g., PARP9 and PARP14) are activated by interferon signaling and contribute to antiviral, pro-inflammatory, or anti-inflammatory responses (Fehr et al., 2020; Higashi et al., 2019; Iwata et al., 2016). We recently reported the novel finding that atherosclerotic lesions and fatty livers of Ldlr−/− mice contain ADP-ribosylated proteins of which several, including canonical high-density lipoprotein (HDL) proteins (e.g., APOA1, APOA2, and APOE), are commonly found in these two metabolically challenged organs. Moreover, we identified these proteins in their ADP-ribosylated forms in plasma, strongly suggesting that the lesional signals were derived from the liver via circulation (Santine et al., 2025). This study faced many challenges associated with enriching ADP-ribosylated proteins from aortae and plasma for subsequent mass spectrometric analysis. Nonetheless, our ongoing efforts on multiple fronts of mass spectrometry workflows for ADP-ribosylation enabled us to describe this new biology (Kasai et al., 2023; Kasai et al., 2025).

Protein ADP-ribosylation is complicated to study because of its dynamic nature—with rapid enzymatic turnover, numerous amino acid acceptor sites, and the highly labile nature of the modification (Suskiewicz et al., 2023). In recent years, advances in mass spectrometry-dependent workflows have significantly improved our ability to enrich the ADP-ribosylome. A major milestone was the development of the Af1521 macrodomain enrichment workflow, allowing for selective purification of ADP-ribosylated peptides (Buch-Larsen et al., 2020). The workflow also implements a poly-ADP-ribose-glycohydrolase (PARG) treatment step to reduce PARylated peptides to their MARylated form because only the monomer can be readily detected via mass spectrometry (Martel et al., 2016).

Mass spectrometric fragmentation strategies must be considered carefully for ADP-ribosylation proteomics because they influence both peptide identification and acceptor site localization, often excelling at one at the cost of the other. For instance, the commonly employed higher-energy collisional dissociation (HCD) method produces complex MS/MS fragments that are a result of complete and partial modification losses due to the labile nature of the ADP-ribose. HCD, therefore, complicates making confident acceptor site annotation, requiring specialized software to annotate partial modification losses for acceptor site localization (Kuraoka et al., 2022). Previous studies, including our own, demonstrated that electron-transfer dissociation (ETD)-based approaches, such as electron-transfer/higher-energy collision dissociation (EThcD), provide superior confidence in acceptor site localization by preserving the modification on the amino acid (Buch-Larsen et al., 2020; Kuraoka et al., 2022; Zee and Garcia, 2010; Rosenthal et al., 2011; Syka et al., 2004). Despite the benefit of acceptor site identification, EThcD is slower than HCD and is selective toward larger peptides because the electron transfer is more efficient with multiply charged peptides (Rosenthal et al., 2011; Syka et al., 2004).

In a previous study, we implemented in-source collision-induced dissociation (CID) to convert the ADP-ribosyl (ADPr) moiety to a simpler phosphoribosyl form that is less labile and more amenable to subsequent HCD-dependent acceptor site localization (Kasai et al., 2025). This strategy is an alternative for acceptor site localization if ETD is unavailable. In addition to in-source CID, we applied high-field asymmetric-waveform ion mobility spectrometry (FAIMS) to fractionate non-ADP-ribosyl peptide contaminants from ADP-ribosyl peptides. ADP-ribosyl peptides sequenced using HCD are conducive to enrichment by FAIMS due to their stability at highly negative compensation voltages (−60 V to −90 V) that filter out prototypical peptides (Kasai et al., 2023; Kasai et al., 2025). This stability is owed in part to the prevalence of 3+ charge states due to missed (tryptic) cleavages when the ADP-ribosylation is located on a lysine. We then questioned whether FAIMS in conjunction with EThcD would also improve efforts to increase ADP-ribosyl peptide sequencing depth. In this case, we turned to the quadrupole-ion trap-Orbitrap equipped with ETD capability to investigate the performance of EThcD when coupled to a FAIMS device.

Methods

Cell culture

THP-1 cells, a human monocytic cell line, were purchased from American Type Culture Collection (Cat# TIB-202) and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Cat# MT10040CV) in 10% fetal bovine serum (VWR International, Cat# 97068-085) with penicillin and streptomycin (Thermo Fisher Scientific, Cat# 15140163) at 37 °C in 5% CO₂. THP-1 cells were plated at a density of 3.0 × 10⁷ cells in 15 cm dishes and allowed to differentiate from their monocyte-like state into macrophage-like cells using RPMI supplemented with 100 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, Cat# P1585) for 2 days, followed by a media exchange back to RPMI alone for 1 day.

Interferon γ stimulation

THP-1-derived macrophages (2 × 15 cm dishes pooled per treatment and replicate) were treated with phosphate-buffered saline (PBS, VWR International, Cat# 12001-680) alone as a control or with 10 ng/mL interferon γ (IFN-γ, R&D Systems, Cat# 285-IF) in PBS, with n = 5 replicates per condition. After 6 h, the cells were washed twice with ice-cold PBS, and then the cells were lysed in modified RIPA buffer (50 mM Tris-HCl pH 7.4, 0.4 M NaCl [Sigma-Aldrich, Cat# S9888], 1.0 mM EDTA [Boston BioProducts, Cat# BM-150], 1.0% Nonidet P-40 [Sigma-Aldrich, Cat# 74385], 0.1% sodium deoxycholate [Sigma-Aldrich, Cat# D6750], 40 μM PJ34 [Abcam, Cat# ab120981], 1.0 μM ADP-HPD [MilliporeSigma, Cat# 118415], and protease inhibitor cocktail [Sigma-Aldrich, Cat# P8340]) as described previously (Higashi et al., 2019).

Proteolysis

THP-1 lysates were homogenized on ice using sonication. Homogenized lysates were precipitated in acetone (Thermo Fisher Scientific, Cat# A949-1). The cell pellets were resuspended in a denaturation buffer (6.0 M urea [Sigma-Aldrich, Cat# U4884], 2.0 M thiourea [Sigma-Aldrich, Cat# T7875], and 10 mM HEPES [Boston BioProducts, Cat# BBH-80]). The protein amount was determined by a Pierce 660 nm Protein Assay Reagent (Thermo Fisher Scientific, Cat# 22660). Proteins (5.0–10 mg) were reduced in 1.0 mM dithiothreitol (DTT, Thermo Fisher Scientific, Cat# 20290) and alkylated in 5.5 mM chloroacetamide (Sigma-Aldrich, Cat# C0267). Proteolysis was performed with trypsin (Thermo Fisher Scientific, Cat# 90058) in 20 mM ammonium bicarbonate (Sigma-Aldrich, Cat# 09830) overnight. The peptides were desalted using a Sep-Pak C18 Classic Cartridge (Waters, Cat# WAT051910) by following the manufacturer’s instructions. Using a Concentrator plus complete system (Eppendorf AG), the peptide sample was reduced to a final volume of 0.8 mL of affinity precipitation buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂ [Thermo Fisher Scientific, Cat# AM9530G], and 250 μM DTT, 50 mM NaCl). The peptide amount was determined by using a NanoDrop One Spectrophotometer at 205 nm (ε₂₀₅ = 31 methods, Thermo Fisher Scientific).

ADP-ribosyl peptide enrichment

The peptides (2–4 mg) were treated with PARG overnight (1.0 μg PARG per 1.0 mg peptide, Creative BioMart, Cat# PARG-31H) to obtain only MARylated peptides. ADP-ribosyl (MARylated) peptides were enriched using the Archaeoglobus fulgidus ribosylation binding protein eAf1521 macrodomain affinity pull-down, as described previously (Higashi et al., 2019; Kuraoka et al., 2022; Nowak et al., 2020). The peptides were then processed using the Microcon-30 kDa Centrifugal Filter Unit (MilliporeSigma, Cat# MRCF0R03) and desalted using an Oasis HLB cartridge (30 mg [Waters, 1 cc, Cat# WAT094225]) as per the manufacturer’s instructions. The peptides were suspended in loading buffer (5.0% acetonitrile [Thermo Fisher Scientific, Cat# A955-1] and 0.5% formic acid [Thermo Fisher Scientific, Cat# 28905] in water [Thermo Fisher Scientific, Cat# W6-1]) for mass spectrometric analysis.

Mass spectrometry

The Orbitrap Fusion Lumos was fronted with a FAIMS Pro and EASY-Spray Source, coupled to an Easy-nLC1200 HPLC pump (Thermo Fisher Scientific). The peptides were fractionated using a dual column set-up: an Acclaim™ PepMap™ 100 C18 HPLC column, 75 μm × 20 mm (Thermo Fisher Scientific, Cat# 164946), and an EASY-Spray™ PepMap™ Neo UHPLC Column, 75 μm × 150 mm (Thermo Fisher Scientific, Cat# ES75150PN). Binary mobile phases (A: water/0.1% formic acid; B: 95% acetonitrile/5% water/0.1% formic acid) were employed at a flow rate of 300 nL/min. The analytical gradient was from 5% to 21% B for 60 min, followed by 10 minutes of 21%–30% B, and a 15-min column wash alternating between 95% B and 5% B, held for 3 min each, in between a 2-min ramp up or down. Following the washes, the column was equilibrated at 95% B. The mass spectrometers were operated in positive mode.

HCD properties of ADP-ribosyl peptides

The MS1 resolution was set to 120,000 with a scan range of m/z 400–900. Ions with a charge state of 2–6 were selected for MS2 fragmentation, dynamic exclusion was 60 s, and the intensity threshold was set to 25,000. The total cycle time of the data-dependent mode was set to 3 s. The MS2 resolution was set to 120,000, the isolation window was 1.2 m/z, the maximum injection time was dynamic, and the collision energy was 28%.

EThcD properties of ADP-ribosyl peptides

The MS1 resolution was set to 120,000 with a scan range of m/z 400–900. Ions with a charge state of 2–6 were selected for MS2 fragmentation, dynamic exclusion was 60 s, and the intensity threshold was set to 25,000. The total cycle time of the data-dependent mode was set to 3 s, with a minimum number of six points across the chromatographic peak. The MS2 resolution was set to 120,000, the isolation window was 1.2 m/z, the maximum injection time was dynamic, and the supplemental activation collision energy was 22.5%.

Gas-phase segmentation (GPS) is defined as segmenting the MS1 scan range, analyzed as separate injections: m/z 400–655 and m/z 645–900. FAIMS was operated on the standard resolution set to static gas mode with a total carrier gas flow of 3.9 L/min. Single compensation voltages (CVs) of −40 V, −50 V, −60 V, −70 V, or −80 V or combined multiple CVs of −60 V, −70 V, and −80 V were applied for HCD properties (Kasai et al., 2023), while −40 V, −45 V, −50 V, −55 V, −60 V, −65 V, −70 V, −75 V, −80 V, −85 V, or −50 V, −60 V, and −70 V were applied for EThcD.

Spectral annotation

ADP-ribosyl peptide spectra were analyzed using Proteome Discoverer 2.4, in which the RiboMaP spectral annotation node was built (Kuraoka et al., 2022; Singh et al., 2022). The spectra were queried against the UniProt human fasta database (downloaded 18 January 2022; n = 100,730 entries) using the SEQUEST search engine algorithm. Trypsin was set as the digestion enzyme, allowing up to four missed cleavages and a minimum peptide length of six amino acids. ADP-ribosyl (+541.061 Da) of Asp, Glu, Lys, Arg, Ser, Thr, Tyr, and His; oxidation (+15.995 Da) of methionine; and acetylation (+42.011 Da) of the N-terminus were set as variable modifications. Carbamidomethylation (+57.021 Da) of cysteine was set as a static modification. Spectral mass tolerances were 10 ppm for the precursor and 20 mmu for the fragment ions. The peptide false discovery rate (FDR) was calculated using Percolator (target/decoy method), and peptide spectrum matches (PSMs) were filtered at 1.0% FDR (Kuraoka et al., 2022). The data acquired in this study are provided in Supplementary Table S1.

Data representation

The PSMs for each dataset were exported from Proteome Discoverer. Data plots were done using Microsoft Excel from Microsoft 365.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2025) partner repository with the dataset identifier PXD068570 and 10.6019/PXD068570.

Statistical analysis

All data are presented as the count number or mean ± SD with individual data dots. Student’s t-tests were performed for statistical analysis using Microsoft Excel.

Results

Study workflow

ADP-ribosyl peptides enriched from THP-1 cells treated with either PBS or IFN-γ were analyzed using FAIMS coupled to EThcD or HCD performed on a quadrupole-ion trap-Orbitrap instrument (Orbitrap Fusion Lumos) (Figure 1). Our main goal was to determine whether FAIMS could improve ADP-ribosyl peptide sequencing depth when using EThcD, as previously demonstrated by us when using HCD on the quadrupole-Orbitrap (Exploris 480) (Kasai et al., 2025). Our previous studies also demonstrated that non-ADP-ribosyl (contaminant) peptides are useful internal controls to compare/contrast the mass spectrometric properties of ADP-ribosyl peptides (Kasai et al., 2023; Kuraoka et al., 2022). Initial acquisition optimizations were carried out using ADP-ribosyl peptides pooled from the PBS and IFN-γ treatments.

Figure 1

Figure 1. Study workflow employing high-field asymmetric-waveform ion mobility spectrometry (FAIMS) versus gas-phase segmentation (GPS) of ADP-ribosyl (ADPr) peptides for subsequent EThcD- or HCD-dependent sequencing on the Orbitrap Fusion Lumos. THP-1 macrophage-like cells (THP-1) with or without IFN-γ were used as a source for ADP-ribosyl peptides.

ADP-ribosyl peptides exhibit distinct compensation voltage properties between EThcD and HCD

The ADP-ribose modification, when on a lysine, induces missed cleavages, shifting the charge state distribution toward 3+ and higher compared to prototypical or non-ADP-ribosyl peptides (Kasai et al., 2023; Kasai et al., 2025). When using FAIMS and HCD performed on the quadrupole-Orbitrap, this charge state shift renders ADP-ribosyl peptides stable at highly negative compensation voltages that filter out peptide contaminants that are predominantly 2+ charge states. This filtering increases the ADP-ribosyl peptides’ signal-to-noise and sequencing depth (Kasai et al., 2023; Kasai et al., 2025). We then investigated whether a similar phenomenon would benefit EThcD when coupled to FAIMS.

We determined that when performed on the Fusion Lumos, ADP-ribosyl (Supplementary Tables S2, S3) and non-ADP-ribosyl peptides (Supplementary Tables S4, S5) exhibited distinct compensation voltage properties when FAIMS was coupled to HCD (Figure 2A). ADP-ribosyl PSMs peaked at −60 V (448 PSMs), while non-ADP-ribosyl PSMs peaked at −50 V (1,683 PSMs). In addition, compensation voltages between 50 V and 80 V equaled or increased the number of ADP-ribosyl PSMs above and beyond HCD alone (i.e., without FAIMS) (Figure 2A). These findings are consistent with our previous results on the Exploris 480 (Kasai et al., 2023; Kasai et al., 2025). On the other hand, when using EThcD, ADP-ribosyl (Supplementary Tables S6, S7) and non-ADP-ribosyl PSMs (Supplementary Tables S8, S9) exhibited similar distributions across compensation voltages with identifications peaking between −55 V (ADPr, 206 PSMs; non-ADPr, 787 PSMs) and −60 V (ADPr, 202 PSMs; non-ADP-ribosyl 853 PSMs). Moreover, unlike HCD, no single compensation voltage increased the number of ADP-ribosyl PSMs above EThcD alone (Figure 2A). These data indicate that there is less of a benefit to filtering out 2+ charge states of peptide contaminants because they are not the preferred substrates for EThcD.

Figure 2

Figure 2. ADP-ribosyl peptide dissociation properties when using FAIMS with a scan range of m/z 400–900. (A) Distribution of peptide spectrum matches (PSMs) across compensation voltage for ADP-ribosyl and non-ADP-ribosyl peptides broken down by precursor charge (z). PBS and IFN-γ THP-1 ADP-ribosyl peptides were pooled for this analysis. (B) Plots depicting the cumulative number of ADP-ribosyl and non-ADP-ribosyl peptides identified with increasing negative compensation voltage.

To further illustrate the point, we plotted the cumulative number of unique ADP-ribosyl peptides with increasing compensation voltage and noted that the identifications plateau around −75 V with EThcD but continue to rise with HCD (Figure 2B). When compared to the acquisitions without FAIMS, we gained 4.1-fold more ADP-ribosyl peptide identifications with HCD (217–882 ADP-ribosyl peptides) and 2.0-fold more with EThcD (242–495 ADP-ribosyl peptides). On the other hand, for non-ADP-ribosyl peptides, the net gains were 4.0 and 4.1-fold, respectively, for HCD and EThcD (Figure 2B). These data underscore that while EThcD-dependent sequencing of ADP-ribosyl peptides does benefit from FAIMS, the gains are less than those for HCD.

Optimization of FAIMS coupled to EThcD for ADP-ribosyl peptide sequencing

Our earlier works used GPS as the sole gas-phase fractionation strategy to sequence deeper into the ADP-ribosylome (Higashi et al., 2019; Kuraoka et al., 2022), with the caveat that a given GPS scan range equally enriches ADP-ribosyl and non-ADP-ribosyl peptides (Kasai et al., 2023). To determine to what extent we could increase the number of ADP-ribosyl peptides using either GPS or FAIMS, we tested two standard GPS/MS1 scan ranges versus two FAIMS strategies, as guided by our previous study (Figure 3A) (Kasai et al., 2023).

Figure 3

Figure 3. Impact of FAIMS on EThcD-dependent ADP-ribosyl PSM yields. (A) Acquisition methods tested. (B) Number of ADP-ribosyl PSMs and their XCorr values and p-series scores for each method. Differences were evaluated using unpaired two-tailed Student’s t-test. (C) Venn diagram representing the number of ADP-ribosyl peptide sequences and the distributions of acceptor sites in THP-1 treated with PBS (control) or IFN-γ. (D) Example ADP-ribosyl peptide identified as a lysine-modified in control and as serine-modified in IFN-γ. RiboMaP annotations of p-ions are highlighted in yellow. The adenosine monophosphate [AMP]+ peak is the HCD-induced fragment ion from the ADP-ribose, and the P5-ion is the complementary precursor peptide ion. The P1-ion corresponds to a complete loss of the ADP-ribose from the precursor peptide.

A single MS1 scan range (Method 1: m/z 400–900) injection yielded 271 ADP-ribosyl PSMs, and a GPS strategy entailing two independent injections (Method 2: m/z 400–655, m/z 650–900 scan range) yielded 232 PSMs (Figure 3B). FAIMS using three distinct compensation voltages with a single MS1 scan range (Method 3) increased the number of ADP-ribosyl PSMs to 278, but the best result was achieved when the same MS1 scan range was acquired three times using distinct compensation voltages (Method 4) (Figure 3B). While Method 4 increased the number of ADP-ribosyl PSMs to 503, we also noted the decrease in the associated XCorr values and p-series scores (Figure 3B). The p-series score reflects the number of HCD (of the EThcD method)-induced p-ions (partial or complete ADP-ribosyl modification losses) in the spectra (Kuraoka et al., 2022; Singh et al., 2022). Lower XCorr values and p-series scores reflect the overall lower signals of the sequenced ADP-ribosyl peptides (and any peptide in general for XCorr). These data demonstrate that although we sequenced more ADP-ribosyl peptides with Method 4, the higher number came at the cost of fewer annotated peaks per spectrum. Nonetheless, as we demonstrated previously, ADP-ribosyl peptides of interest can be subjected to targeted MS/MS as a means to increase signal-to-noise and the number of annotated fragment peaks for increased confidence in identification (Higashi et al., 2019; Kuraoka et al., 2022).

We then analyzed our ADP-ribosyl peptides enriched from THP-1 cells treated with PBS (control) or IFN-γ. We enriched ADP-ribosyl peptides from a pool of two 15 cm culture dishes per replicate (n = 5 replicates). Using acquisition Method 3, we identified a total of 538 ADP-ribosyl peptide sequences, of which 242 and 74 contained unique acceptor sites for control and IFN-γ (Figure 3A). We also noted a shift toward the proportion of serine acceptor sites in IFN-γ (18%) compared to the control (7%) (Figure 3C). For example, a peptide from the mitochondrial protein peptidyl-prolyl cis-trans isomerase F was identified as modified at either a lysine (position 8) in both PBS and IFN-γ or at serine (position 7) but only in the IFN-γ condition (Figure 3D).

Discussion

In this study, we sought to enhance ADP-ribosyl peptide identification by coupling FAIMS to EThcD, performed on the quadrupole-ion trap-Orbitrap. FAIMS is an atmospheric pressure ion mobility technique that provides gas-phase separation of ions by their mobility under asymmetric electric fields, while still being a reliable method for label-free proteome quantification (Bekker-Jensen et al., 2020). FAIMS has been shown to improve proteome coverage by reducing chemical noise, which improves dynamic range and detection limits, increasing precursor selectivity (Swearingen and Moritz, 2012). EThcD-dependent ADP-ribosyl peptide sequencing retains the intact modification for acceptor site localization, with the caveat that highly charged peptides are the preferred substrates for the electron-transfer reaction.

Our study demonstrated that while EThcD-dependent sequencing of ADP-ribosyl peptides does benefit from FAIMS, the gain is less than that when using HCD. The distinguishing variable is the charge state prevalence of the background, non-ADP-ribosyl peptides that contaminate ADP-ribosyl peptide enrichment protocols. Contaminant peptides carry predominantly 2+ charge states that are filtered out at the high negative compensation voltages (Figure 2A). While removal of these background peptides suits HCD-dependent efforts, it does not impact EThcD in the same manner because 2+ charge state peptides are not preferred substrates for this dissociation method. Higher charge state contaminant peptides also benefit from FAIMS, effectively competing against the ADP-ribosyl peptides. The recent launch of the quadrupole-Orbitrap equipped with ETD (Kessler et al., 2025) provides researchers yet another instrument platform to further investigate the dissociation properties of modified peptides, including ADP-ribosylated peptides. Multiple dissociation options on a single instrument permit researchers to leverage the pros of each method for their modified peptide studies.

Conclusion

FAIMS coupled to EThcD improves ADP-ribosyl peptide sequencing depth but not necessarily at the higher negative compensation voltages that benefit HCD.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

Ethics statement

Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.

Author contributions

TK: Conceptualization, Formal Analysis, Data curation, Methodology, Writing – review and editing, Investigation, Writing – original draft. GS: Writing – original draft, Writing – review and editing. YN: Writing – review and editing, Investigation. MA: Supervision, Writing – review and editing. SS: Writing – review and editing, Conceptualization, Supervision, Writing – original draft.

Funding

The authors declare that financial support was received for the research and/or publication of this article. This study was in part supported by research grants from Kowa Company, the National Heart, Lung, and Blood Institute (R01HL126901 and R01HL149302 to MA; R01HL174066 to SS and MA), and the American Heart Association (2024A003947 to SS). Other than funding, the study did not involve Kowa.

Conflict of interest

TK and YN were employees of Kowa Company, Ltd., Nagoya, Japan.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author(s) declared that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process or the final decision.

Generative AI statement

The authors declare that no Generative AI was used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchbi.2025.1709253/full#supplementary-material

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