Abstract Labeling of peptides and proteins using chelators binding metal ions has become a novel approach for quantitative proteomics in recent years. The aim of this work was the optimization of a new method for peptide derivatization with lanthanide labels (holmium, lutetium, and thulium) followed by nano high performance liquid chromatography (nano-HPLC) separation with UV detection. Matrix-assisted laser desorption ionization–mass spectrometry (MALDI MS) was used to confirm the derivatization and to identify the derivatized peptides. Peptides were labeled with the three different lanthanide metals using a bifunctional DOTA-based (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) reagent. The results demonstrate that the derivatization reaction using the chelating reagent DOTA-NHS-ester was effective for single peptides and peptide mixtures. Furthermore, an efficient pre-cleaning method was applied by nano-HPLC using a C-18 trap column for elimination of the excess of labeling reagent. The application of the optimized method to label peptides in a Cytochrome C digest delivered comparable results to those obtained with model peptides.

中文翻译：

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通过纳米 HPLC 研究用镧系元素-NHS-酯-DOTA 标记肽

摘要 近年来，使用结合金属离子的螯合剂标记肽和蛋白质已成为定量蛋白质组学的一种新方法。这项工作的目的是优化使用镧系元素标记（钬、镥和铥）进行肽衍生化的新方法，然后通过紫外检测进行纳米高效液相色谱 (nano-HPLC) 分离。基质辅助激光解吸电离质谱 (MALDI MS) 用于确认衍生化并鉴定衍生化肽。使用基于双功能 DOTA（1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸）的三种不同镧系元素金属标记肽。结果表明，使用螯合剂 DOTA-NHS-酯的衍生反应对单一肽和肽混合物有效。此外，通过使用 C-18 捕集柱的纳米 HPLC 应用了一种有效的预清洁方法，以消除过量的标记试剂。应用优化的方法来标记细胞色素 C 消化物中的肽，其结果与使用模型肽获得的结果相当。