Tryptic Peptide Analysis of Ceruloplasmin in Dried Blood Spots Using Liquid Chromatography-Tandem Mass Spectrometry: Application to Newborn Screening
Tryptic Peptide Analysis of Ceruloplasmin in Dried Blood Spots Using Liquid Chromatography-Tandem Mass Spectrometry: Application to Newborn Screening
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DOI：
10.1373/clinchem.2008.111989
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发表时间：
2008-12-01
影响因子：
9.3
通讯作者：
Hahn, Si Houn
中科院分区：
文献类型：
--
作者：
Dewilde, Amy;Sadilkova, Katerina;Hahn, Si Houn
背景：全世界都在进行新生儿筛查，以识别患有可治疗先天性疾病的婴儿。最近的串联质谱 (MS/MS) 应用显着扩展了使用单个干血点 (DBS) 筛查 > 50 种代谢疾病的能力。代谢紊乱特别适合筛查的特征是存在特定的小分子代谢物。然而，许多可治疗的疾病，如威尔逊病，其特点是血浆或循环血细胞内大蛋白缺失或减少，目前没有经济有效的筛查方法。方法：我们开发了一种定量 DBS 中铜蓝蛋白 (CP) 的方法，用于新生儿威尔逊病筛查。使用同位素标记肽内标和液相色谱三重四极杆质谱 (LC-MS/MS) 对胰蛋白酶消化的 DBS 样品中的 U 特异性肽进行定量。结果：校准曲线在 20 至 95 mg/dL (200-950 mg/L) 范围内呈线性。 Cl:1 浓度为 25、35 和 55 mg/dL（250,350 和 550 mg/L）时，测定内不精密度（平均 CV）分别为 9.2%、10.7% 和 10.2%。 19 个不同批次的测定间不精密度分别为 8.9%、5.8% 和 6.9%。对先前测试的 CP 患者样本进行的方法比较研究给出了可比较的结果，定量下限约为 0.7 mg/dL (7 mg/L)。结论：我们的研究支持，在胰蛋白酶消化后，使用 LC-MS/MS 测定 DBS 中的 CP 定量来进行新生儿威尔逊病筛查是可行的。这种方法应该适用于新生儿筛查其他可治疗的遗传性疾病，例如以大蛋白作为生物标志物的原发性免疫缺陷。 (C) 2008 美国临床化学协会
BACKGROUND: Newborn screening to identify infants with treatable congenital disorders is carried out worldwide. Recent tandem mass spectrometry (MS/MS) applications have markedly expanded the ability to screen for >50 metabolic diseases with a single dried blood spot (DBS). The feature that makes metabolic disorders particularly amenable to screening is the presence of specific small-molecule metabolites. Many treatable disorders such as Wilson disease, however, are characterized by absent or diminished large proteins in plasma or within circulating blood cells, for which there are currently no cost-effective screening methods.METHODS: We developed an assay for quantifying ceruloplasmin (CP) in DBS for newborn screening of Wilson disease. U-specific peptides from DBS samples digested by trypsin were quantified using isotopically labeled peptide internal standards and liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS).RESULTS: The calibration curve was linear from 20 to 95 mg/dL (200-950 mg/L). Intraassay imprecision (mean CV) for Cl:1 concentrations of 25, 35, and 55 mg/dL (250,350, and 550 mg/L) was 9.2%,10.7%, and 10.2%, respectively. Interassay imprecision for 19 different batches was 8.9%, 5.8%, and 6.9%. A method comparison study on previously tested patient samples for CP gave comparable results with lower limit of quantification, around 0.7 mg/dL (7 mg/L).CONCLUSIONS: Our study supports that newborn screening for Wilson disease is feasible using LC-MS/MS assay for CP quantification in DBS after tryptic digestion. This approach should be applicable to newborn screening for other treatable genetic conditions, such as primary immunodeficiencies, that have large proteins as biomarkers. (C) 2008 American Association for Clinical Chemistry