A Tandem Mass Spectrometry Triplex Assay for the Detection of Fabry, Pompe, and Mucopolysaccharidosis-I (Hurler)
A Tandem Mass Spectrometry Triplex Assay for the Detection of Fabry, Pompe, and Mucopolysaccharidosis-I (Hurler)
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DOI：
10.1373/clinchem.2010.152009
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发表时间：
2010-12-01
影响因子：
9.3
通讯作者：
Scott, C. Ronald
中科院分区：
文献类型：
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作者：
Duffey, Trisha A.;Bellamy, Garland;Scott, C. Ronald
背景：我们试图建立一种串联质谱分析方法，其中3种溶酶体酶（α -葡萄糖苷酶、α -半乳糖苷酶a和α - l -伊杜糖醛酸酶）的酶活性可以通过使用单一测定缓冲液来定量测定干血斑。方法：用3种不同底物和内标在单一缓冲液中孵育3mm干血斑打孔。用乙酸乙酯对样品进行了简单的液-液萃取。提取液干燥后用溶剂重悬，进样于串联质谱仪中。采用多反应监测对产品及内标进行监测。结果：3种溶酶体酶成功测定，统计数据可接受。该分析可通过使用最少量的一次性耗材和设备进行。包括数据分析在内的整个过程以48小时为周期。5990个匿名新生儿干血点的数据显示酶活性近似钟形分布（平均值为19.0、11.5和3.5 mu mol）。h(1)。α -葡萄糖苷酶、α -半乳糖苷酶A和α -L-伊杜糖醛酸酶（L - blood）分别为（-1）。无血条件下的空白值分别为0.13、0.24和0.45 μ mol。h(1)。（L血）（-1）。通过一次分析3种酶，如果所有酶的酶活性值都很低（例如，如果在分析中存在血液不足），就会发现有问题的样品进行重新分析。结论：该方法表明，在单一缓冲液中，用最少的供应和人工进行三重检测，可以适应于高通量新生儿筛查实验室，用于Pompe， Fabry和粘多糖病- i （Hurler）病的分析。(C) 2010年美国临床化学学会
BACKGROUND: We sought to develop a tandem mass spectrometry assay in which the enzymatic activities of 3 lysosomal enzymes (alpha-glucosidase, alpha-galactosidase A, and alpha-L-iduronidase) could be quantified in dried blood spots by using a single assay buffer.METHODS: A 3-mm dried blood spot punch was incubated in a single assay buffer with 3 different substrates and internal standards. The sample was processed by a simple liquid-liquid extraction by using ethyl acetate. The extract was dried down and resuspended in solvent for injection into the tandem mass spectrometer. Products and internal standards were monitored by multiple reaction monitoring.RESULTS: Assay for the 3 lysosomal enzymes was successfully achieved with acceptable statistics. The assay can be performed by using a minimal quantity of disposable supplies and equipment. The entire procedure fits into a 48-h cycle including data analysis. Data from 5990 anonymous newborn dried blood spots showed an approximate bell-shaped distribution of enzymatic activities (mean values of 19.0, 11.5, and 3.5 mu mol . h(-1) . (L blood)(-1) for alpha-glucosidase, alpha-galactosidase A, and alpha-L-iduronidase, respectively. Blank values obtained in the absence of blood were 0.13, 0.24, and 0.45 mu mol . h(-1) . (L blood)(-1), respectively). By assaying 3 enzymes at once, problematic samples are spotted for reanalysis if enzyme activity values are low for all enzymes (for example, if insufficient blood is present in the assay).CONCLUSIONS: This method demonstrates that a triplex assay in a single buffer and with minimal supplies and labor can be adapted to a high-throughput newborn screening laboratory for the analysis of Pompe, Fabry, and mucopolysaccharidosis-I (Hurler) diseases. (C) 2010 American Association for Clinical Chemistry