Affinity capture and elution/electrospray ionization mass spectrometry assay of phosphomannomutase and phosphomannose isomerase for the multiplex analysis of congenital disorders of glycosylation types Ia and Ib.
Affinity capture and elution/electrospray ionization mass spectrometry assay of phosphomannomutase and phosphomannose isomerase for the multiplex analysis of congenital disorders of glycosylation types Ia and Ib.
复制标题
磷酸甘露糖变位酶和磷酸甘露糖异构酶的亲和捕获和洗脱/电喷雾电离质谱测定，用于糖基化类型 Ia 和 Ib 先天性疾病的多重分析。
DOI：
10.1021/ac0205053
复制
发表时间：
2003
影响因子：
7.4
通讯作者：
Gelb,MichaelH
中科院分区：
文献类型：
--
作者：
Li,Yijun;Ogata,Yuko;Freeze,HudsonH;Scott,CRonald;Turecek,Frantisek;Gelb,MichaelH
我们报道了亲和捕获-洗脱电喷雾质谱法（ACESI-MS）的新应用，以测定磷酸甘蔗渣异异构酶（PMM）和磷酸甘蔗渣异构酶（PMI），这两种酶缺乏时分别导致先天性糖基化cdg - Ia型和Ib型疾病。这种以质谱为基础的分析的新特点是，它允许人们区分和量化酶的产物是同分异构体的底物，同时存在于复杂的混合物中，如培养的人类细胞匀浆。这是通过偶联分析实现的，其中PMM和PMI初级产品在体外与酵母转酮醇酶进行另一种酶促反应，改变要通过质谱检测的产品的质量。亲和纯化过程是全自动的，质谱分析以一种适合高通量应用的方式进行多路复用。
We report a new application of affinity capture-elution electrospray mass spectrometry (ACESI-MS) to assay the enzymes phosphomannomutase (PMM) and phosphomannose isomerase (PMI), which when deficient cause congenital disorders of glycosylation CDG-type Ia and type Ib, respectively. The novel feature of this mass-spectrometry-based assay is that it allows one to distinguish and quantify enzymatic products that are isomeric with their substrates that are present simultaneously in complex mixtures, such as cultured human cell homogenates. This is achieved by coupled assays in which the PMM and PMI primary products are in vitro subjected to another enzymatic reaction with yeast transketolase that changes the mass of the products to be detected by mass spectrometry. The affinity purification procedure is fully automated, and the mass spectrometric analysis is multiplexed in a fashion that is suitable for high-throughput applications.