Chemo-enzymatic detection of protein isoaspartate using protein Isoaspartate methyltransferase and hydrazine trapping
Chemo-enzymatic detection of protein isoaspartate using protein Isoaspartate methyltransferase and hydrazine trapping
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DOI：
10.1021/ac800251q
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发表时间：
2008-05-15
影响因子：
7.4
通讯作者：
Zhou, Zhaohui Sunny
中科院分区：
文献类型：
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作者：
Alfaro, Joshua F.;Gillies, Laura A.;Zhou, Zhaohui Sunny
异天冬氨酸形成是一种普遍存在的翻译后修饰，由自发的天冬氨酸脱酰胺或天冬氨酸异构化引起。异天冬氨酸的形成将亚甲基插入到蛋白质骨架中，产生“扭结”，并可能极大地改变蛋白质的结构和功能，从而在无数的生物过程、人类疾病和蛋白质药物开发中发挥关键作用。在此，我们报告了一种化学酶检测方法，用于异天冬氨酸蛋白，该方法特别允许含异天冬氨酸蛋白的亲和富集。在初始阶段，蛋白质异天冬氨酸甲基转移酶选择性地将异天冬氨酸转化为相应的甲酯。随后，不稳定的甲酯被强亲核试剂（如肼）捕获在水溶液中形成肼。稳定的肼产物可以通过标准的蛋白质组学技术进行分析，如基质辅助激光解吸电离和电喷雾电离质谱。此外，化学捕获步骤允许我们引入几种标记策略来进行产品鉴定和定量，例如通过丹酚衍生物进行紫外可见和荧光检测。最重要的是，肼产物可以通过醛树脂亲和层析富集，从而大大降低样品的复杂性。因此，我们的方法代表了第一个对异天冬氨酸蛋白进行亲和富集的技术，应该可以用于系统和全面的表征异天冬氨酸，特别是在复杂的系统中。
Isoaspartate formation is a ubiquitous post-translation modification arising from spontaneous asparagine deamidation or aspartate isomerization. The formation of isoaspartate inserts a methylene group into the protein backbone, generating a "kink", and may drastically alter protein structure and function, thereby playing critical roles in a myriad of biological processes, human diseases, and protein pharmaceutical development. Herein, we report a chemo-enzymatic detection method for the isoaspartate protein, which in particular allows the affinity enrichment of isoaspartate-containing proteins. In the initial step, protein isoaspartate methyltransferase selectively converts isoaspartates into the corresponding methyl esters. Subsequently, the labile methyl ester is trapped by strong nucleophiles in aqueous solutions, such as hydrazines to form hydrazides. The stable hydrazide products can be analyzed by standard proteomic techniques, such as matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry. Furthermore, the chemical trapping step allows us to introduce several tagging strategies for product identification and quantification, such as UV-vis and fluorescence detection through a dansyl derivative. Most significantly, the hydrazide product can be enriched by affinity chromatography using aldehyde resins, thus drastically reducing sample complexity. Our method hence represents the first technique for the affinity enrichment of isoaspartyl proteins and should be amendable to the systematic and comprehensive characterization of isoaspartate, particularly in complex systems.