"One-pot" methylation in glycomics application: Esterification of sialic acids and permanent charge construction
"One-pot" methylation in glycomics application: Esterification of sialic acids and permanent charge construction
复制标题
DOI：
10.1021/ac070091j
复制
发表时间：
2007-05-15
影响因子：
7.4
通讯作者：
Li, Jianjun
中科院分区：
文献类型：
--
作者：
Liu, Xin;Li, Xianyu;Li, Jianjun
建立了一种简单、快速的“一锅法”甲基化方法，结合完全非特异性蛋白水解消化和甲基化，用于N-连接聚糖的分析。将由链霉蛋白酶E消化糖蛋白制备的Asn-聚糖混合物通过阳离子交换柱，将羧酸转化为Na+形式，然后用碘甲烷甲基化。衍生物可以很容易地用亲水性亲和层析柱纯化。通过基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF）和MALDI-TOF/TOF进行质谱分析。质谱数据表明，除了在天冬酰胺残基的氨基中形成季铵之外，羧酸被甲基化。三个模型糖蛋白，包括核糖核酸酶B，卵清蛋白和转铁蛋白，被用来证明这种技术的优点。结果表明，除了形成永久电荷外，还实现了唾液酸的稳定化。与未衍生化N-聚糖的分析相比，检测灵敏度提高了约10倍。通过血清转铁蛋白的聚糖谱进一步评估了新技术，证明该技术是表征蛋白质糖基化的灵敏方法。
A simple and rapid "one-pot" methylation method to esterify sialic acids and construct a permanent charge was developed for N-linked glycan analysis, which combined complete nonspecific proteolytic digestion and methylation. A mixture of Asn-glycans prepared from Pronase E digestion of the glycoprotein was passed through a cation-exchange column to convert carboxylic acids to the Na+ form before being methylated with methyl iodide. Derivatives could be easily purified with a hydrophilic affinity chromatography cartridge. Mass spectrometry analysis was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and MALDI-TOF/TOF. The mass spectrometric data indicated that carboxylic acids were methylated in addition to the formation of a quaternary ammonium in the amino group of asparagine residues. Three model glycoproteins, including ribonuclease B, ovalbumin, and transferrin, were employed to demonstrate the merits of this technique. Results showed that the stabilization of sialic acid was achieved in addition to the formation of a permanent charge. Compared to the analysis of underivatized N-glycans, detection sensitivity improved similar to 10-fold. The new technique was further evaluated with glycan profiling of serum transferrin and proved to be a sensitive method for the characterizing protein glycosylation.