Carbamidomethylation Side Reactions May Lead to Glycan Misassignments in Glycopeptide Analysis

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.

碘乙酰胺或许是蛋白质组学实验中应用最为广泛的游离巯基烷基化试剂。本研究发现，当仅采用结合电子转移解离（electron-transfer dissociation，ETD）的液相色谱-串联质谱（LC-MS/MS）进行糖肽结构表征时，半胱氨酸（Cys）侧链衍生化不完全以及肽段过度烷基化均可能导致糖型的错误归属。精确质量测定对此无法提供有效帮助，因为被错误归属的修饰与正确修饰的元素组成完全一致。在多数情况下，将束型碰撞诱导解离（即高能C阱解离（higher-energy C-trap dissociation，HCD））数据与ETD数据结合用于数据库检索，可有效解决该问题。然而，甲硫氨酸（Met）残基的氨基甲酰甲基化会引发锍醚形成，由此产生的固定正电荷会触发特征性碎裂：N连接糖肽的HCD谱图中会缺失正常的Y1离子，转而生成丰度较高的Y1-48 Da离子（标称质量差相对于未修饰氨基酸序列计算），该离子极易被误认为由甲硫氨酸亚砜的侧链丢失所产生。在此类情形下，高质量的ETD数据可揭示这一差异，同时还会呈现因电荷还原前体离子发生CH3-S-CH2CONH2消除而产生的丰度较高的碎片离子。本研究结果同时也提醒学界，不同未预期共价修饰的干扰尚未被充分报道。