Analytical Biochemistry and Mass Spectrometry Core Facility, Department of Pharmacy, University of Groningen, Groningen, The Netherlands.
Rapid Commun Mass Spectrom. 2013 Feb 28;27(4):546-52. doi: 10.1002/rcm.6479.
Cleavage of peptide bonds C-terminal to tyrosine and tryptophan after electrochemical oxidation may become a complementary approach to chemical and enzymatic cleavage. A chemical labeling approach specifically targeting reactive cleavage products is presented here and constitutes a promising first step towards the development of a new proteomics workflow.
Hexylamine was used to react with the spirolactone moieties generated after electrochemical oxidation and cleavage of tripeptides. The influence of pH and reaction time on the yield was determined and the excess of tagging reagent was optimized. Selective detection of the tagged cleavage products was achieved by precursor ion scanning in a triple quadrupole mass spectrometer.
Optimal labeling was reached under aqueous conditions when working at pH 10 with a reaction time of 0.5 min. The excess of hexylamine over spirolactone groups can be significantly decreased by working under non-aqueous conditions in pure acetonitrile to prevent spirolactone hydrolysis. The specific formation of hexylamine-containing y(1) reporter ions generated by collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) allows for selective detection by precursor ion scanning of the cleaved and labeled peptides.
This work presents a method for selective labeling and detection of electrochemically cleaved Tyr- and Trp-containing peptides for which reaction conditions have been optimized with hexylamine as labeling agent. This workflow offers new possibilities for electrochemical oxidation, cleavage and labeling of peptides and proteins.
在电化学氧化和色氨酸和酪氨酸 C 末端肽键的裂解后，裂解可能成为化学和酶裂解的补充方法。本文提出了一种专门针对反应性裂解产物的化学标记方法，这是朝着开发新蛋白质组学工作流程迈出的有希望的第一步。
己胺用于与电化学氧化和三肽裂解后生成的螺内酯部分反应。确定了 pH 值和反应时间对产率的影响，并优化了标记试剂的过量。通过三重四极杆质谱仪中的前体离子扫描实现了标记裂解产物的选择性检测。
在 pH 值为 10 时在水性条件下工作，反应时间为 0.5 分钟时达到最佳标记。通过在纯乙腈中在非水条件下工作，可以大大减少螺内酯基团上的己胺过量，以防止螺内酯水解。通过碰撞诱导解离（CID）串联质谱（MS/MS）产生的含有己胺的 y(1)报告离子的特异性形成允许通过前体离子扫描选择性检测裂解和标记的肽。
这项工作提出了一种用于选择性标记和检测含有 Tyr-和 Trp 的电化学裂解肽的方法，已优化了使用己胺作为标记剂的反应条件。该工作流程为肽和蛋白质的电化学氧化、裂解和标记提供了新的可能性。
