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 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.

研究目标：ADP-核糖基化（ADP-ribosylation）是一类重要的翻译后修饰，在细胞过程中发挥关键作用。本团队此前的工作在四极杆-轨道阱（quadrupole-Orbitrap，Exploris 480）质谱平台上，采用包括场非对称离子迁移谱（FAIMS）在内的多种气相分离策略及源内碰撞诱导解离（in-source CID）技术，提升了依赖高能量碰撞解离（HCD）的ADP-核糖基（ADPr）肽段的鉴定产率与受体位点置信度。本研究旨在评估四极杆-离子阱-轨道阱（quadrupole-ion trap-Orbitrap，Fusion Lumos）质谱平台联用FAIMS，是否同样可改善依赖电子转移诱导碰撞解离（EThcD）的ADP-核糖基肽段测序效果。
实验方法：从人巨噬细胞样细胞系THP-1中提取的ADP-核糖基肽段，在搭载FAIMS Pro装置的Fusion Lumos平台上进行质谱分析。针对MS1扫描的FAIMS正负气相分段（GPS）策略，被应用于HCD与EThcD两种数据采集模式。采用SEQUEST-HT算法及ADP-核糖基化肽段光谱专用注释工具RiboMaP，对ADP-核糖基肽段的质谱数据进行注释。
实验结果：相较于EThcD模式，依赖HCD的ADP-核糖基肽段鉴定在更高补偿电压（CV）下得到显著富集。与未使用FAIMS的对照组相比，不同补偿电压下获取的独特ADP-核糖基肽段及非ADP-核糖基（污染）肽段的净数量，在HCD模式下分别提升了3.2倍与3.8倍，在EThcD模式下则分别提升了2.0倍与3.6倍。本研究同时证实，尽管采用不同补偿电压进行多次肽段进样可最大化依赖EThcD的ADP-核糖基肽段鉴定数量，但对应的XCorr与p系列分值会出现下降。最常见的ADP-核糖基受体位点为赖氨酸，其次为丝氨酸；当THP-1细胞经干扰素-γ（IFN-γ）激活后，ADP-核糖基化丝氨酸位点的占比显著升高。
研究结论：尽管FAIMS可提升依赖EThcD的ADP-核糖基肽段测序深度，但该提升效果弱于HCD模式。通过更高幅值的负补偿电压过滤双电荷非ADP-核糖基肽段的能力，对HCD模式更为有利，而对EThcD模式则无明显益处，因为该解离方法对高电荷肽段（无论是否为ADP-核糖基化肽段）均能实现最优解离效果。