Enrichment and analysis of nonenzymatically glycated peptides: Boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry
Enrichment and analysis of nonenzymatically glycated peptides: Boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry
复制标题
DOI：
10.1021/pr070112q
复制
发表时间：
2007-01-01
影响因子：
4.4
通讯作者：
Metz, Thomas O.
中科院分区：
文献类型：
--
作者：
Zhang, Qibin;Tang, Ning;Metz, Thomas O.
D-葡萄糖对肽和蛋白质的非酶糖化在糖尿病的发病机制中具有重要意义，特别是在糖尿病并发症的发展中。然而，在自下而上的蛋白质组学研究中，不存在有效的高通量方法来鉴定含有这种低丰度翻译后修饰的蛋白质。在本报告中，苯硼酸亲和层析用于两步富集方案，以在体外从人血清糖化中选择性地首先分离糖化蛋白质，然后分离糖化胰蛋白酶肽。随后通过交替电子转移解离 (ETD) 和碰撞诱导解离 (CID) 串联质谱分析富集的肽。当首先对蛋白质进行富集，然后对肽水平进行富集时，与 CID 模式（占所有已识别肽的 17.0%）相比，ETD 片段化模式可以识别显着更高数量的糖化肽（占所有已识别肽的 87.6%）。这项研究表明，苯硼酸亲和层析与 LC-MS/MS 联用并使用 ETD 作为裂解模式是分析糖化蛋白的有效方法，并且可能在糖尿病研究中具有广泛的应用。
Nonenzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low-abundance posttranslational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron-transfer dissociation (ETD) and collision induced dissociation ( CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.