Protein Isoaspartate Methyltransferase-Mediated 18O-Labeling of Isoaspartic Acid for Mass Spectrometry Analysis
Protein Isoaspartate Methyltransferase-Mediated 18O-Labeling of Isoaspartic Acid for Mass Spectrometry Analysis
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DOI：
10.1021/ac202652z
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发表时间：
2012-01-17
影响因子：
7.4
通讯作者：
Zhou, Zhaohui Sunny
中科院分区：
文献类型：
--
作者：
Liu, Min;Cheetham, Janet;Zhou, Zhaohui Sunny
异天冬氨酸(isasp，isod)或β-天冬氨酸(beta-Asp)是一种普遍存在的蛋白质和多肽的非酶修饰，是天冬氨酸(Asp)异构化或天冬酰胺(ASN)脱酰胺反应的产物。由于异天冬氨酸和天冬氨酸之间没有质量差异，所以对异天冬氨酸的灵敏和特异的检测，特别是在复杂的样品中，仍然具有挑战性。在这里，我们报道了一种新的天冬氨酸异构化的O-18标记的新方法，通过两步法：首先通过蛋白质异门冬氨酸甲基转移酶(PIMT，EC 2.1.1.77)将isAsp多肽特异性甲基化成相应的甲酯，然后甲酯在O-18-水中水解以再生isAsp。在isAsp上，O-16被O-18特异性取代导致质量位移2 Da，这可以用标准质谱学，如碰撞诱导解离(CID)和数据分析算法来自动和明确地识别。在一种单抗和β-三角洲睡眠诱导肽(DSIP)中检测和定位了几种异天冬氨酸多肽。
Arising from spontaneous aspartic acid (Asp) isomerization or asparagine (Asn) deamidation, isoaspartic acid (isoAsp, isoD, or beta-Asp) is a ubiquitous nonenzymatic modification of proteins and peptides. Because there is no mass difference between isoaspartyl and aspartyl species, sensitive and specific detection of isoAsp, particularly in complex samples, remains challenging. Here we report a novel assay for Asp isomerization by isotopic labeling with O-18 via a two-step process: the isoAsp peptide is first specifically methylated by protein isoaspartate methyltransferase (PIMT, EC 2.1.1.77) to the corresponding methyl ester, which is subsequently hydrolyzed in O-18-water to regenerate isoAsp. The specific replacement of O-16 with O-18 at isoAsp leads to a mass shift of 2 Da, which can be automatically and unambiguously recognized using standard mass spectrometry, such as collision-induced dissociation (CID), and data analysis algorithms. Detection and site identification of several isoAsp peptides in a monoclonal antibody and the beta-delta sleep-inducing peptide (DSIP) are demonstrated.