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Capillary Electrophoresis with Mass Spectrometry for Top-Down Identification of Hemoglobin Variants

Updated: Feb 5, 2023





Author: Ruben Yiqi Luo, PhD, DABCC, Assistant Professor, Department of Pathology, School of Medicine, Stanford University



Hemoglobin variants are the hemoglobin molecules with modified amino acid sequences as a result of genetic mutations, and they constitute a major type of hemoglobinopathy. So far, over 1400 hemoglobin variants have been discovered and reported. Identification of hemoglobin variants, particularly the variant forms of subunits α, β, γ, and δ, are of significant value in the clinical diagnosis of hemoglobinopathy. In clinical laboratories, the conventional methods to identify hemoglobin variants include gel electrophoresis, capillary electrophoresis (CE), and high-performance liquid chromatography (HPLC). These methods differentiate hemoglobin variants by their size and charge properties, measured by electrophoretic migration in a medium or retention to an ion-exchange resin. However, the resolving power of the conventional methods can be inadequate due to the lack of structural characterization, resulting in ambiguity in the identification of hemoglobin variants. In Dr. Ruben Yiqi Luo’s recent publication in Clinical Chemistry, he demonstrated a novel approach using CE coupled with a high-resolution mass spectrometry (HR-MS) through a specifically designed neutral-coating nanoelectrospray ion source. By this means, Dr. Luo’s team achieved detailed structural characterization of hemoglobin variants in two steps: (1) intact-protein analysis that preliminarily identifies hemoglobin subunits by precursor ions, and (2) top-down analysis that characterizes the primary structures of hemoglobin subunits by fragment ions produced from particular precursor ions. They have constructed a database of hemoglobin variants, to which the CE-HR-MS analytical results can be matched to identify what hemoglobin variants are present in clinical specimens. It should be noted that the neutral-coating CE plays an essential role in this application. Its excellent separation efficiency not only allows for a simple dilute-and-shoot sample preparation procedure but also facilitates the analysis of heterozygote samples that contain a normal hemoglobin subunit as well as a variant with a very small mass change.


In clinical laboratory practice, it is known that conventional electrophoretic methods for Hb analysis have limited resolving power to identify Hb variants due to the lack of structural characterization. While gene sequencing can be used as a confirmatory test for Hb variants, the high cost limits its availability in clinical laboratories, and moreover, the clinical gene sequencing service usually does not cover all Hb subunit genes (especially γ and δ genes). With the ability to characterize the primary structure of hemoglobin, the CE-HR-MS method complements conventional methods for the identification of hemoglobin variants. It can be positioned as a reflex test method, which means a preliminary screening method is first implemented and the CE-HR-MS test will be triggered if hemoglobin variants are suspected in the primary screening. In addition to the amino acid sequence, the CE-HR-MS method can figure out the post-translational modifications of a hemoglobin subunit. So far, acetylation of a lysine residue and glutathionylation of a cysteine residue in hemoglobin subunits have been accurately identified. When the CE-HR-MS method is employed for hemoglobin analysis, the information of the post-translational modifications can be obtained and may potentially assist with the diagnosis and treatment of hematological disorders.


中性涂层毛细管电泳与高分辨率质谱联用用于自顶向下蛋白质分析鉴定血红蛋白变体

血红蛋白变体是由于基因突变导致氨基酸序列发生改变的血红蛋白分子,这类变体组成了血红蛋白病的一个主要类型。 迄今为止,已发现并报道了 1400 多种血红蛋白变体。 血红蛋白变体的鉴定,特别是亚基α、β、γ和δ的变异形式,对血红蛋白病的临床诊断具有重要价值。 在临床实验室中,识别血红蛋白变体的常规方法包括凝胶电泳、毛细管电泳(CE),以及高效液相色谱( HPLC)。 这些方法通过大小和电荷特性来区分血红蛋白变体,通过介质中的电泳迁移或在离子交换树脂中的保留来测量。 然而,由于缺乏结构特征,传统方法的分辨能力可能不足,导致血红蛋白变体的鉴定模糊不清。 在 Ruben Yiqi Luo 博士最近发表在《临床化学》上的文章中,他展示了一种通过专门设计的纳升电喷雾离子源将 CE 与高分辨率质谱 (HR-MS) 结合使用的新方法。 通过这种方式,Luo博士的团队分两步实现了对血红蛋白变体的详细结构特征的解析:(1)完整蛋白分析,通过前体离子初步识别血红蛋白亚基;(2)自顶向下分析,通过识别特定前体离子产生的碎片离子解析血红蛋白亚基的一级结构。 他们构建了一个血红蛋白变异体数据库,CE-HR-MS 分析结果可以与之匹配,以确定临床样本中存在哪些血红蛋白变异体。 应该注意的是,中性涂层CE 在此应用中起着至关重要的作用。 其出色的分离效率不仅允许简单的稀释-注射样本制备程序,而且还有助于分析包含正常血红蛋白亚基以及质量变化非常小的变体的杂合子样品。

在临床检验实践中,由于缺乏结构解析,传统的基于电泳的血红蛋白分析方法对于血红蛋白变体的鉴定能力是有限的。尽管基因测序可以作为血红蛋白变体的验证性方法,但高昂的成本限制了其在临床实验室的可用性,而且通常临床基因测序服务并不涵盖所有血红蛋白亚基基因,尤其是γ和δ基因。 由于能够鉴定血红蛋白的一级结构,CE-HR-MS 方法是对血红蛋白变体鉴定的传统方法的有效补充。在实践中,可以将其作为升级检测方法:如果样本在初步筛选中怀疑含有血红蛋白变体,则进一步用CE-HR-MS方法进行测试。 除了氨基酸序列外,CE-HR-MS 方法还可以检测血红蛋白亚基的翻译后修饰。 迄今为止,已经准确鉴定了血红蛋白亚基中赖氨酸残基的乙酰化和半胱氨酸残基的谷胱甘肽化。 当采用 CE-HR-MS 方法进行血红蛋白分析时,可以获得翻译后修饰的信息,并可能有助于血液疾病的诊断和治疗。


Reference

Ruben Yiqi Luo, Carolyn Wong, James Qiangwei Xia, Bertil E. Glader, Run-Zhang Shi, James L. Zehnder, “Neutral-Coating Capillary Electrophoresis Coupled with High-Resolution Mass Spectrometry for Top-Down Identification of Hemoglobin Variants”, Clinical Chemistry, 69, 56-67, 2022.

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