Analytical Biochemistry Pub Date : 2022-07-31 , DOI: 10.1016/j.ab.2022.114823
Elodie Logerot 1 , Guillaume Cazals 1 , Antony Memboeuf 2 , Christine Enjalbal 1
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α-amidation of peptide sequences is a common post-translational modification in the living world. Since the majority of these C-terminal amidated peptides are bioactive, there is hence a great interest to identify and characterize them from biological matrices and natural extracts. Regarding conventional separative methods dedicated to peptides (such as HPLC or CE), elution protocols must be carefully optimized hampering straightforward LC-MS analysis of complex samples. From a mass spectrometry point of view, they are difficult to pinpoint owing to the only 1 Da mass difference between the post-translational amidated and the corresponding native carboxylated forms producing overlapping isotopic contributions of both molecular ions. To circumvent this analytical difficulty, usage of energy-resolved tandem mass spectrometry experiments and of the survival yield technique was investigated. Pair of peptides were thus dissociated in positive and negative mode according to the survival yield technique, in MS2 and MS3 experiments, in order to separate them giving a reliable MS/MS methodology to detect such post-translationally modified sequence.
中文翻译:
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利用存活率技术揭示 C 末端肽酰胺化
肽序列的α-酰胺化是生物界常见的翻译后修饰。由于这些 C 末端酰胺化肽中的大多数具有生物活性,因此从生物基质和天然提取物中鉴定和表征它们引起了极大的兴趣。关于专用于肽的传统分离方法(如 HPLC 或 CE),必须仔细优化洗脱方案,从而阻碍复杂样品的直接 LC-MS 分析。从质谱的角度来看,由于翻译后酰胺化形式和相应的天然羧化形式之间仅有 1 Da 的质量差异,它们很难确定,这两种分子离子产生重叠的同位素贡献。为了规避这个分析困难,研究了能量分辨串联质谱实验和存活率技术的使用。因此,根据存活率技术,在 MS 中,一对肽以正向和负向模式解离2和 MS 3实验,以便将它们分开,提供可靠的 MS/MS 方法来检测这种翻译后修饰的序列。