Affinity Capture and Elution/Electrospray Ionization Mass Spectrometry Assay of Phosphomannomutase and Phosphomannose Isomerase for the Multiplex Analysis of Congenital Disorders of Glycosylation Type
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- 作者：Yijun Li ; Yuko Ogata ; Hudson H. Freeze ; C. Ronald Scott ; Franti
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- 刊名：Analytical Chemistry
- 出版年：2003
- 出版时间：January 1, 2003
- 年：2003
- 卷：75
- 期：1
- 页码：42 - 48
- 全文大小：120K
- 年卷期：v.75,no.1(January 1, 2003)
- ISSN：1520-6882
文摘
We report a new application of affinity capture-elutionelectrospray mass spectrometry (ACESI-MS) to assay theenzymes phosphomannomutase (PMM) and phosphomannose isomerase (PMI), which when deficient causecongenital disorders of glycosylation CDG-type Ia and typeIb, respectively. The novel feature of this mass-spectrometry-based assay is that it allows one to distinguish andquantify enzymatic products that are isomeric with theirsubstrates that are present simultaneously in complexmixtures, such as cultured human cell homogenates. Thisis achieved by coupled assays in which the PMM and PMIprimary products are in vitro subjected to another enzymatic reaction with yeast transketolase that changes themass of the products to be detected by mass spectrometry.The affinity purification procedure is fully automated, andthe mass spectrometric analysis is multiplexed in afashion that is suitable for high-throughput applications.