Carbamidomethylation Side Reactions May Lead to Glycan Misassignments in Glycopeptide Analysis,Analytical Chemistry

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Carbamidomethylation Side Reactions May Lead to Glycan Misassignments in Glycopeptide Analysis
Analytical Chemistry ( IF 6.7 ) Pub Date : 2015-05-22 00:00:00 , DOI: 10.1021/acs.analchem.5b01121
Zsuzsanna Darula ₁ , Katalin F. Medzihradszky _{1,

2}

Affiliation

Iodoacetamide is perhaps the most widely used reagent for the alkylation of free sulfhydryls in proteomic experiments. Here, we report that both incomplete derivatization of Cys side chains and overalkylation of the peptides may lead to the misassignment of glycoforms when LC–MS/MS with electron-transfer dissociation (ETD) alone is used for the structural characterization of glycopeptides. Accurate mass measurements do not help, because the elemental compositions of the misidentified and correct modifications are identical. Incorporation of “higher-energy C-trap dissociation” (HCD), i.e., beam-type collision-induced dissociation data into the database searches with ETD data may prove decisive in most cases. However, the carbamidomethylation of Met residues leads to sulfonium ether formation, and the resulting fixed positive charge triggers a characteristic fragmentation, that eliminates the normal Y₁ fragment from the HCD spectra of N-linked glycopeptides, producing an abundant Y₁-48 Da ion instead (the nominal mass difference is given relative to the unmodified amino acid sequence), that easily can be mistaken for the side chain loss from Met sulfoxide. In such cases, good quality ETD data may indicate the discrepancy, and will also display abundant fragments due to CH₃–S–CH₂CONH₂ elimination from the charge-reduced precursor ions. Our observations also draw attention to the underreported interference of different unanticipated covalent modifications.

中文翻译：

糖胺甲基化副反应可能导致糖肽分析中的糖基错配

碘乙酰胺也许是蛋白质组学实验中用于游离巯基烷基化的最广泛使用的试剂。在这里，我们报道当仅将LC-MS / MS与电子转移解离（ETD）一起用于糖肽的结构表征时，Cys侧链的不完全衍生化和肽的过度烷基化都可能导致糖型的错误分配。准确的质量测量无济于事，因为错误识别和正确修改的元素组成是相同的。在大多数情况下，将“高能C陷阱解离”（HCD）（即束型碰撞诱导的解离数据）与ETD数据结合到数据库搜索中可能证明是决定性的。但是，Met残基的氨基甲酰甲基化会导致形成ether醚，N-连接糖肽的HCD光谱中的_1个片段，产生大量的Y ₁ -48 Da离子（相对于未修饰的氨基酸序列，给出了标称质量差），很容易将其误认为是Met造成的侧链损失亚砜。在这种情况下，高质量的ETD数据可能表明存在差异，并且由于从电荷减少的前体离子中消除了CH ₃ -S-CH ₂ CONH ₂，因此还会显示大量碎片。我们的观察结果还引起人们对未预料到的不同意想不到的共价修饰干扰的关注。

更新日期：2015-05-22

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