In-Source Collision-Induced Dissociation (CID) Improves Higher-Energy Collisional Dissociation (HCD)-Dependent Fragmentation of ADP-Ribosyl Peptides

IF 1.8 3区化学 Q4 BIOCHEMICAL RESEARCH METHODS Rapid Communications in Mass Spectrometry Pub Date : 2024-12-04 DOI:10.1002/rcm.9961

Taku Kasai, Yuto Nakamura, Masanori Aikawa, Sasha A. Singh

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Abstract

Rationale

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-H₂O derivative to facilitate HCD-dependent peptide sequencing.

Methods

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-H₂O (+ 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-H₂O acceptor sites were compared with those determined by electron-transfer higher-energy collision dissociation (EThcD), performed on a quadrupole-ion trap-Orbitrap.

Results

In-source CID of ADPr peptides to their phosphoribosyl-H₂O 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-H₂O peptide identifications. The average Xcorr increased from 1.36 (ADPr) to 2.26 (phosphoribosyl-H₂O), 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-H₂O), which was comparable to EThcD (70%).

Conclusions

In-source CID converts ADP-ribosyl to phosphoribosyl-H₂O 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.

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源内碰撞诱导解离（CID）改善了adp -核糖基肽的高能碰撞解离（HCD）依赖的碎片化。

原理：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，使肽测序和受体位点定位工作复杂化。我们测试了在四极轨道rap上进行的源内碰撞诱导解离（CID）是否可以将ADPr转化为更小的磷酸- h2o衍生物，以促进hcd依赖性肽的测序。方法：采用四极轨道rap对人巨噬细胞样细胞系THP-1提取的adp -核糖基（ADPr）肽进行分析。在不同的源电压和高场不对称波形离子迁移率质谱（FAIMS）补偿电压下，我们监测了ADPr （+ 541.061 Da）解离成磷酸波基- h2o （+ 193.997 Da）肽。Xcorr和ptmRS分别用于评估肽序列和受体位点置信度。将磷酸核糖基- h2o受体位点与在四极离子阱-轨道阱上通过电子转移高能碰撞解离（EThcD）测定的受体位点进行了比较。结果：通过监测相应的修饰损失（[腺苷单磷酸/AMP]+）和鉴定出的磷酸核糖基- h2o肽的数量，可以判断ADPr肽对其磷酸核糖基- h2o衍生物的源内CID随着源电压的增加而增加（最高可达50 V）。平均Xcorr从1.36 （ADPr）增加到2.26（磷酸核糖基- h2o），与EThcD获得的ADPr肽相似（2.29）。高置信度受体位点的数量（> 95%）也从31% （ADPr）增加到70%（磷酸核糖基- h2o），与EThcD（70%）相当。结论：In-source CID将adp -核糖基转化为磷酸核糖基- h2o肽，更适合于hcd依赖性肽测序，在没有基于etd的方法时，为受体位点的确定提供了一种替代方法。

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来源期刊

Rapid Communications in Mass Spectrometry 化学-分析化学

CiteScore

4.10

自引率

5.00%

发文量

219

审稿时长

2.6 months

期刊介绍： Rapid Communications in Mass Spectrometry is a journal whose aim is the rapid publication of original research results and ideas on all aspects of the science of gas-phase ions; it covers all the associated scientific disciplines. There is no formal limit on paper length ("rapid" is not synonymous with "brief"), but papers should be of a length that is commensurate with the importance and complexity of the results being reported. Contributions may be theoretical or practical in nature; they may deal with methods, techniques and applications, or with the interpretation of results; they may cover any area in science that depends directly on measurements made upon gaseous ions or that is associated with such measurements.