基质辅助激光解吸电离-飞行时间质谱法测定α/β-二羰基类化合物与标准瓜氨酸化肽段之间的衍生化反应活性  

Determination of the derivatization reactivity betweenα/β-dicarbonyl compounds and standard citrullinated peptides based on matrix-assisted laser desorption ionization-time-of-flight mass spectrometry

作　　者：李雁凤 周丹丹 陈旭飞 赵娟娟 高春丽 邱兴泰 唐紫超 邓楠 赵伟宁 边阳阳 LI Yanfeng;ZHOU Dandan;CHEN Xufei;ZHAO Juanjuan;GAO Chunli;QIU Xingtai;TANG Zichao;DENG Nan;ZHAO Weining;BIAN Yangyang(School of Life Sciences,Northwestern University,Xi’an 710069,China;Xiamen Jinnuohua Biotechnology Co.,Ltd.,Xiamen 361000,China;Analysis and Testing Shared Center of Xi’an Jiaotong University,Xi’an 710049,China;School of Pharmacy,Shenzhen Technology University,Shenzhen 518118,China)

机构地区：[1]西北大学生命科学学院,陕西西安710069 [2]厦门金诺花生物技术有限公司,福建厦门361000 [3]西安交通大学分析测试共享中心,陕西西安710049 [4]深圳技术大学药学院,广东深圳518118

出　　处：《色谱》2024年第7期711-720,共10页Chinese Journal of Chromatography

基　　金：国家自然科学基金项目(22274130,22107075);陕西省自然科学基金项目(22JHQ087,2023-BSF-322).

摘　　要：蛋白质瓜氨酸化是一种由生物酶调控的不可逆翻译后修饰,其与免疫相关疾病、癌症、神经系统性疾病和老年痴呆症等重大疾病的发生和发展密切相关。蛋白质的瓜氨酸化修饰丰度低、缺乏亲和标签、m/z变化小且易受同位素及脱酰胺化作用干扰,因此对蛋白质的瓜氨酸化修饰进行质谱分析面临着巨大挑战。通过对瓜氨酸侧链脲基进行化学衍生反应,可以提高瓜氨酸化肽段的m/z差值,引入亲和富集标签,可有效提高瓜氨酸化分析的灵敏度和精准度。本研究以标准瓜氨酸化肽段为研究对象,以基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF MS)为检测手段,对一系列二羰基类化合物与瓜氨酸化肽段之间的化学反应活性进行测定。在对α-二羰基类化合物的考察中发现,苯乙二醛、丙酮醛、1,2-环己二酮和1,10-邻二氮杂菲-5,6-二酮对瓜氨酸化肽段的衍生化效率很高,并且能够生成单一的衍生化产物;2,3-丁二酮、乙二醛与瓜氨酸化肽段之间的反应效率也很高,但会生成一系列副反应产物。在对β-二羰基类化合物的考察中发现,1,3-环己二酮、2,4-戊二酮在与瓜氨酸化肽段进行反应后,均可以产生单一的衍生化产物,但反应效率很低,不适用于瓜氨酸化肽段的化学衍生反应。实验结果表明,α-二羰基结构是实现高效、特异性瓜氨酸化肽段化学衍生反应的基础,α-二羰基类化合物中不同的侧链结构又决定了衍生化产物的结构、衍生化效率及副产物的生成。通过进一步合成含有亲和标签的α-二羰基类化合物,可以实现瓜氨酸化肽段的特异性富集和精准鉴定,有助于瓜氨酸化蛋白质及其位点鉴定的新方法开发。Protein citrullination is an irreversible post-translational modification process regulated by peptidylarginine deiminases(PADs)in the presence of Ca 2+.This process is closely related to the occurrence and development of autoimmune diseases,cancers,neurological disorders,cardiovascular and cerebrovascular diseases,and other major diseases.The analysis of protein citrullination by biomass spectrometry confronts great challenges owing to its low abundance,lack of affinity tags,small mass-to-charge ratio change,and susceptibility to isotopic and deamidation interferences.The methods commonly used to study the protein citrullination mainly involve the chemical derivatization of the urea group of the guanine side chain of the peptide to increase the mass-to-charge ratio difference of the citrullinated peptide.Affinity-enriched labels are then introduced to effectively improve the sensitivity and accuracy of protein citrullination by mass spectrometry.2,3-Butanedione or phenylglyoxal compounds are often used as derivatization reagents to increase the mass-to-charge ratio difference of the citrullinated peptide,and the resulting derivatives have been observed to containα-dicarbonyl structures.To date,however,no relevant studies on the reactivity of dicarbonyl compounds with citrullinated peptides have been reported.In this study,we determined whether sixα-dicarbonyl and twoβ-dicarbonyl compounds undergo derivatization reactions with standard citrullinated peptides using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry(MALDI-TOF MS).Among theα-dicarbonyl compounds,2,3-butanedione and glyoxal reacted efficiently with several standard citrullinated peptides,but yielded a series of by-products.Phenylglyoxal,methylglyoxal,1,2-cyclohexanedione,and 1,10-phenanthroline-5,6-dione also derivated efficiently with standard citrullinated peptides,generating a single derivative.Thus,a new derivatization method that could yield a single derivative was identified.Among theβ-dicarbonyl compounds,1,3-cyc

关 键 词：基质辅助激光解吸电离-飞行时间质谱 二羰基结构 瓜氨酸化肽段 化学衍生 

分 类 号：O658[理学—分析化学]