Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease
Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease
复制标题
DOI：
10.1021/pr0340173
复制
发表时间：
2003-09-01
影响因子：
4.4
通讯作者：
Ames, JM
中科院分区：
文献类型：
--
作者：
Brock, JWC;Hinton, DJS;Ames, JM
利用电喷雾液相色谱-质谱（ESI-LC-MS）进行蛋白质组学分析，比较了RNase的糖基化（Amadori加合物形成）和羧甲基化位点，并评估了Amadori加合物在晚期糖基化终产物（AGE）形成中的作用，n是-（羧甲基）赖氨酸（CIVIL）的一个元素。将RNase （13.7 mg/mL, 1 mM）与葡萄糖（0.4 M）在37℃下空气下于磷酸盐缓冲液（0.2 M, pH 7.4）中孵育14天。通过色氨酸的ESI-LC-MS分析，RNase的糖基化位点依次为K41、K7、K1、K37。其中K41、K7和K37也是CIVIL形成的主要位点。在其他实验中，RNase在厌氧条件下（1 mM DTPA， N-2净化）孵育以形成amadori修饰蛋白，然后在好氧条件下孵育以形成AGE。糖基化的主要位点依次为K41、K7、K1、K37，羧甲基化的主要位点依次为K41、K7、K37。RNase也用1-5 mM乙二醛孵育，这比实验条件下葡萄糖自氧化形成的量要大得多，但赖氨酸残基只有微量修饰，主要是在K41处。我们得出以下结论：(1)CIVIL的主要形成途径是蛋白质上Amadori加合物的自氧化，而不是葡萄糖自氧化产生的乙二醛；(2)与糖基化一样，羧甲基化是一种受邻近氨基酸和结合配体（如磷酸盐或磷酸化化合物）影响的蛋白质位点特异性修饰。即使在蛋白质修饰的总体程度较低的情况下，在少数反应位点定位高比例的修饰可能对理解衰老和糖尿病中蛋白质功能的丧失以及AGE抑制剂的设计具有重要意义。
Proteomic analysis using electrospray liquid chromatography-mass spectrometry (ESI-LC-MS) has been used to compare the sites of glycation (Amadori adduct formation) and carboxymethylation of RNase and to assess the role of the Amadori adduct in the formation of the advanced glycation end-product (AGE), N-is an element of-(carboxymethyl)lysine (CIVIL). RNase (13.7 mg/mL, 1 mM) was incubated with glucose (0.4 M) at 37 degreesC for 14 days in phosphate buffer (0.2 M, pH 7.4) under air. On the basis of ESI-LC-MS of tryptic peptides, the major sites of glycation of RNase were, in order, K41, K7, K1, and K37. Three of these, in order, K41, K7, and K37 were also the major sites of CIVIL formation. In other experiments, RNase was incubated under anaerobic conditions (1 mM DTPA, N-2 purged) to form Amadori-modified protein, which was then incubated under aerobic conditions to allow AGE formation. Again, the major sites of glycation were, in order, K41, K7, K1, and K37 and the major sites of carboxymethylation were K41, K7, and K37. RNase was also incubated with 1-5 mM glyoxal, substantially more than is formed by autoxidation of glucose under experimental conditions, but there was only trace modification of lysine residues, primarily at K41. We conclude the following: (1) that the primary route to formation of CIVIL is by autoxidation of Amadori adducts on protein, rather than by glyoxal generated on autoxidation of glucose; and (2) that carboxymethylation, like glycation, is a site-specific modification of protein affected by neighboring amino acids and bound ligands, such as phosphate or phosphorylated compounds. Even when the overall extent of protein modification is low, localization of a high proportion of the modifications at a few reactive sites might have important implications for understanding losses in protein functionality in aging and diabetes and also for the design of AGE inhibitors.