Division of Molecular Medicine, Clinical Proteomics Unit, St. John's Research Institute, St. John's National Academy of Health Sciences, Bangalore 560034, India.
J Proteome Res. 2013 Jul 5;12(7):3215-22. doi: 10.1021/pr4000625. Epub 2013 Jun 6.
Unambiguous analysis of hemoglobin variants is critical in the diagnosis of hemoglobinopathies. In diagnostic laboratories, alkaline gel electrophoresis and automated HPLC are used in identifying variants. In specific instances, comigration of hemoglobin variant bands in gel and coelution of different variants or elution of variants with unmatched library information in HPLC can result in ambiguities in interpretation. Hemoglobin variants mostly arise from point mutations leading to very high sequence homology between normal and variant hemoglobin. In addition, unavailability of a variant database compatible with proteomics data analysis software makes mass spectrometry based variant analysis very challenging. In the present study, we standardized a nanoLC-MS based method for variant analysis to achieve substantially high sequence coverage. We developed three hemoglobin variant databases, specific to three different proteolytic enzymes, compatible with proteomics search engine software. The above nanoLC-MS method and the compatibility of the customized databases were validated by analysis of a sickle hemoglobin variant. Six other hemoglobin variants were characterized wherein diagnosis reports based on conventional tools were ambiguous. The novelty of our method lies in its simplicity and accuracy of the analysis with minimal manual intervention. The presently described method may be used in the future for the routine hemoglobin variant diagnosis.
血红蛋白变异体的明确分析在血红蛋白病的诊断中至关重要。在诊断实验室中，碱性凝胶电泳和自动化 HPLC 用于鉴定变异体。在特定情况下，凝胶中血红蛋白变异体带的共迁移以及不同变异体的共洗脱或 HPLC 中与库信息不匹配的变异体的洗脱可能导致解释上的歧义。血红蛋白变异体主要由导致正常和变异血红蛋白之间非常高的序列同源性的点突变引起。此外，由于缺乏与蛋白质组学数据分析软件兼容的变异体数据库，基于质谱的变异体分析极具挑战性。在本研究中，我们标准化了一种基于纳升液相色谱-质谱（nanoLC-MS）的变异体分析方法，以实现高序列覆盖率。我们开发了三个血红蛋白变异体数据库，分别针对三种不同的蛋白水解酶，与蛋白质组学搜索引擎软件兼容。上述 nanoLC-MS 方法和定制数据库的兼容性通过对镰状血红蛋白变异体的分析进行了验证。还对另外六个血红蛋白变异体进行了特征描述，其中基于常规工具的诊断报告存在歧义。我们方法的新颖之处在于其简单性和准确性，分析过程中干预最小。目前描述的方法将来可能用于常规血红蛋白变异体诊断。
