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

被引:14
作者
Li, YJ
Ogata, Y
Freeze, HH
Scott, CR
Turecek, FE
Gelb, MH
机构
[1] Univ Washington, Dept Chem, Seattle, WA 98195 USA
[2] Burnham Inst, Glycobiol & Carbohydrate Chem Program, La Jolla, CA 92037 USA
关键词
D O I
10.1021/ac0205053
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
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.
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收藏
页码:42 / 48
页数:7
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