Soo Evelyn C, Aubry Annie J, Logan Susan M, Guerry Patricia, Kelly John F, Young N Martin, Thibault Pierre
Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada K1K 0H1.
Anal Chem. 2004 Feb 1;76(3):619-26. doi: 10.1021/ac034875i.
A novel method employing CE-ESMS and precursor ion scanning was developed for the selective detection of nucleotide-activated sugars. By using precursor ion scanning for fragment ions specific to the different nucleotide carriers, i.e., ions at m/z 322 for cytidine monophosphate, m/z 323, 385, and 403 for uridine diphosphate, m/z 362, 424, and 442, for guanosine diphosphate, and m/z 346, 408, and 426 for adenosine diphosphate, it was possible to selectively detect sugar nucleotides involved in the biosynthesis of glycoconjugates such as glycoproteins and lipopolysaccharides. Enhancement of sensitivity was achieved using N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) as a sample stacking buffer and provided detection limits between 0.2 and 3.8 pmol.mL(-)(1). The present CE-ESMS method provided linear dynamic ranges over the concentrations 0.2-164 nM (r(2) = 0.952-0.997) for different nucleotide sugar standards. The application of this method is demonstrated for the identification of intracellular pools of sugar nucleotides in wild type and isogenic mutants from the bacterial pathogen Campylobacter jejuni. By using product ion scanning (with and without front-end collision-induced dissociation), it was possible to determine the precise nature of unexpected sugar nucleotides involved in the biosynthesis of pseudaminic acid, a sialic acid-like sugar previously observed on the flagellin of some pathogenic bacteria.
开发了一种采用毛细管电泳-电喷雾质谱联用（CE-ESMS）和前体离子扫描的新方法，用于选择性检测核苷酸激活的糖类。通过对不同核苷酸载体特有的碎片离子进行前体离子扫描，即胞苷一磷酸的m/z 322离子、尿苷二磷酸的m/z 323、385和403离子、鸟苷二磷酸的m/z 362、424和442离子以及腺苷二磷酸的m/z 346、408和426离子，可以选择性地检测参与糖蛋白和脂多糖等糖缀合物生物合成的糖核苷酸。使用N-(2-羟乙基)哌嗪-N'-(2-乙磺酸)作为样品堆积缓冲液提高了灵敏度，检测限在0.2至3.8 pmol·mL⁻¹之间。目前的CE-ESMS方法对于不同的核苷酸糖标准品，在0.2 - 164 nM的浓度范围内提供了线性动态范围（r² = 0.952 - 0.997）。该方法的应用在鉴定空肠弯曲菌野生型和同基因突变体中糖核苷酸的细胞内池方面得到了证明。通过使用产物离子扫描（有无前端碰撞诱导解离），可以确定参与假氨基糖生物合成的意外糖核苷酸的确切性质，假氨基糖是一种先前在一些病原菌鞭毛蛋白上观察到的类似唾液酸的糖类。
