Yu Chaowen, Huang Shuodan, Wang Ming, Zhang Juan, Liu Hao, Yuan Zhaojian, Wang Xingbin, He Xiaoyan, Wang Jie, Zou Lin
Center for Clinical Molecular Medicine, Children's Hospital of Chongqing Medical University, Chongqing 400014, China; Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing 400014, China; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing 400014, China; Key Laboratory of Pediatrics in Chongqing, Chongqing 400014, China.
Center for Neonatal Screening, Meizhou Maternal and Child Health Care Hospital, Guangdong 514000, China.
J Proteomics. 2017 Feb 10;154:78-84. doi: 10.1016/j.jprot.2016.12.008. Epub 2016 Dec 20.
Traditional methods for thalassemia screening are time-consuming and easily affected by cell hemolysis or hemoglobin degradation in stored blood samples. Tandem mass spectrometry (MS/MS) proved to be an effective technology for sickle cell disorders (SCD) screening. Here, we developed a novel MS/MS method for β-thalassemia screening from dried blood spots (DBS). Stable isotopic-labeled peptides were used as internal standards for quantification and calculation of the α:β-globin ratios. We used the α:β-globin ratio cutoffs to differentiate between normal individuals and patients with thalassemia. About 781 patients and 300 normal individuals were analyzed. The α:β-globin ratios showed significant difference between normal and β-thalassemia patients (P<0.01), particularly when the disease was homozygous or double heterozygous with another α- or β-thalassemia mutation. In the parallel study, all cases screened for suspected thalassemia from six hundred DBS samples by using this MS/MS method were successfully confirmed by genotyping. The intra-assay and inter-assay CVs of the ratios ranged from 2.4% to 3.9% and 4.7% to 7.1%, and there was no significant sample carryover or matrix effect for this MS/MS method. Combined with SCD screening, this MS/MS method could be used as a first-line screening assay for both structural and expression abnormalities of human hemoglobin.
Traditional methods for thalassemia screening were depending on the structural integrity of tetramers and could be affected by hemolysis and degradation of whole blood samples, especially when stored. We used proteospecific peptides produced by the tryptic digestion of each globin to evaluate the production ratio between α- and β-globin chains, which turned out to be quite stable even when stored for more than two months. Though most of the peptides were specific to α-globin or β-globin, we only chose four most informative peptides and its stable isotopic-labeled peptides as internal standards for analysis, which could obtain a high accuracy. Currently, we are the first to address the application of MS/MS for thalassemia screening, when combined with SCD screening, this MS/MS method could be used as a first-line screening assay for both structural and expression abnormalities of human hemoglobin.
传统的地中海贫血筛查方法耗时且容易受到储存血样中细胞溶血或血红蛋白降解的影响。串联质谱（MS/MS）被证明是一种用于镰状细胞病（SCD）筛查的有效技术。在此，我们开发了一种从干血斑（DBS）中筛查β地中海贫血的新型MS/MS方法。使用稳定同位素标记的肽作为内标来定量和计算α:β珠蛋白比率。我们使用α:β珠蛋白比率临界值来区分正常个体和地中海贫血患者。分析了约781例患者和300例正常个体。正常人和β地中海贫血患者之间的α:β珠蛋白比率显示出显著差异（P<0.01），特别是当疾病为纯合子或与另一种α或β地中海贫血突变的双重杂合子时。在平行研究中，使用这种MS/MS方法从600份DBS样本中筛查出的所有疑似地中海贫血病例均通过基因分型成功确诊。该比率的批内和批间变异系数分别为2.4%至3.9%和4.7%至7.1%，并且这种MS/MS方法没有明显的样品残留或基质效应。结合SCD筛查，这种MS/MS方法可作为人类血红蛋白结构和表达异常的一线筛查检测方法。
传统的地中海贫血筛查方法依赖于四聚体的结构完整性，并且可能受到全血样本溶血和降解的影响，尤其是在储存时。我们使用每种珠蛋白经胰蛋白酶消化产生的蛋白特异性肽来评估α和β珠蛋白链之间的产生比率，结果发现即使储存超过两个月该比率也相当稳定。尽管大多数肽对α珠蛋白或β珠蛋白具有特异性，但我们仅选择了四个信息量最大的肽及其稳定同位素标记的肽作为分析内标，这可以获得较高的准确性。目前，我们是首个探讨MS/MS在地中海贫血筛查中的应用的，当与SCD筛查结合时，这种MS/MS方法可作为人类血红蛋白结构和表达异常的一线筛查检测方法。
