Kasai Taku, Nakamura Yuto, Aikawa Masanori, Singh Sasha A
Department of Medicine, Center for Interdisciplinary Cardiovascular Sciences, Division of Cardiovascular Medicine, Brigham Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Division of Cardiovascular Medicine, Center for Excellence in Vascular Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Rapid Commun Mass Spectrom. 2025 Feb 28;39(4):e9961. doi: 10.1002/rcm.9961.
ADP-ribosylation is a posttranslational modification whose higher-energy collisional dissociation (HCD) products are dominated by complete or partial modification losses, complicating peptide sequencing and acceptor site localization efforts. We tested whether in-source collision-induced dissociation (CID) performed on a quadrupole-Orbitrap could convert ADPr to the smaller phosphoribose-HO derivative to facilitate HCD-dependent peptide sequencing.
ADP-ribosyl (ADPr) peptides derived from the human macrophage-like cell line THP-1 were analyzed on a quadrupole-Orbitrap. We monitored the dissociation of ADPr (+ 541.061 Da) to phosphoribosyl-HO (+ 193.997 Da) peptides while varying the source and high-field asymmetric waveform ion mobility mass spectrometry (FAIMS) compensation voltages. Xcorr and ptmRS were used to evaluate peptide sequencing and acceptor site confidence, respectively. Phosphoribosyl-HO acceptor sites were compared with those determined by electron-transfer higher-energy collision dissociation (EThcD), performed on a quadrupole-ion trap-Orbitrap.
In-source CID of ADPr peptides to their phosphoribosyl-HO derivatives increased with increasing source voltage (up to 50 V), as judged by monitoring the corresponding modification loss ([adenosine monophosphate/AMP]) and the number of identified phosphoribosyl-HO peptide identifications. The average Xcorr increased from 1.36 (ADPr) to 2.26 (phosphoribosyl-HO), similar to that achieved with EThcD for ADPr peptides (2.29). The number of high-confidence acceptor sites (> 95%) also increased, from 31% (ADPr) to 70% (phosphoribosyl-HO), which was comparable to EThcD (70%).
In-source CID converts ADP-ribosyl to phosphoribosyl-HO peptides that are more amenable to HCD-dependent peptide sequencing, providing an alternative method for acceptor site determination when ETD-based methods are not available.
ADP-核糖基化是一种翻译后修饰，其高能碰撞解离（HCD）产物主要是完全或部分修饰损失，这使得肽测序和受体位点定位工作变得复杂。我们测试了在四极杆-轨道阱上进行的源内碰撞诱导解离（CID）是否能将ADPr转化为较小的磷酸核糖-HO衍生物，以促进基于HCD的肽测序。
对源自人巨噬细胞样细胞系THP-1的ADP-核糖基（ADPr）肽在四极杆-轨道阱上进行分析。我们在改变源电压和高场不对称波形离子迁移质谱（FAIMS）补偿电压的同时，监测ADPr（+541.061 Da）向磷酸核糖-HO（+193.997 Da）肽的解离。分别使用Xcorr和ptmRS评估肽测序和受体位点置信度。将磷酸核糖-HO受体位点与在四极杆-离子阱-轨道阱上进行的电子转移高能碰撞解离（EThcD）所确定的位点进行比较。
通过监测相应的修饰损失（[单磷酸腺苷/AMP]）和鉴定出的磷酸核糖-HO肽的数量判断，ADPr肽向其磷酸核糖-HO衍生物的源内CID随着源电压增加（高达50 V）而增加。平均Xcorr从1.36（ADPr）增加到2.26（磷酸核糖-HO），与ADPr肽的EThcD结果（2.29）相似。高置信度受体位点（>95%）的数量也从31%（ADPr）增加到70%（磷酸核糖-HO），与EThcD（70%）相当。
源内CID将ADP-核糖基转化为更适合基于HCD的肽测序的磷酸核糖-HO肽，当基于电子转移解离（ETD）的方法不可用时，为受体位点确定提供了一种替代方法。
