化学
烷基化
碘乙酸
碘代乙酰胺
赖氨酸
试剂
质谱法
单同位素质量
半胱氨酸
翻译后修饰
色谱法
丙烯酰胺
串联质谱法
去酰胺
组合化学
生物化学
氨基酸
有机化学
聚合物
共聚物
酶
催化作用
作者
Wendong Ma,Irene Ang,Kate M K Lei,Melody Man Ting Lam,Pengwei Zhang,Terence Poon
标识
DOI:10.1021/acs.analchem.2c01261
摘要
Emerging evidence suggests that advanced glycation end-products (AGEs) such as Nε-(carboxymethyl)lysine (CML) and Nε-(carboxymethyl)lysine (CEL) may play important roles in certain human diseases. Reliable analytical methods are needed for their characterizations and measurements. Pitfalls have been reported for applications of LC–MS/MS to identify various types of post-translational modifications, but not yet for the case of AGEs. Here, we showed that in the absence of manual inspection, cysteine alkylation with 2-iodoacetamide (IAA) can result in false-positive/ambiguous identifications of CML >20%. They were attributed to offsite alkylation together with incorrect monoisotopic peak assignment (pitfall 1) or together with deamidation (pitfall 2). For pitfall 1, false-positive identifications can be alleviated using a peptide mass error tolerance ≤5 ppm during the database search. Pitfall 2 results in ambiguous modification assignments, which may be overcome by using other alkylation reagents. According to calculations of theoretical mass shifts, the use of other common alkylation reagents (iodoacetic acid, 2-chloroacetamide, and acrylamide) should face similar pitfalls. The use of acrylamide can result in false-positive identifications of CEL instead of CML. Subsequently, we showed that compared to IAA, the use of N-isopropylacrylamide (NIPAM) as an alkylation reagent achieved similar levels of proteome coverage, while reducing the offsite alkylation reactions at lysine by more than five times. Furthermore, false-positive/ambiguous identifications of CML due to the two types of pitfalls were absent when using NIPAM. NIPAM alkylation results in a unique mass shift that allows reliable identifications of CML and most likely other AGEs, such as CEL.
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