Capillary Zone Electrophoresis-Top-Down Tandem Mass Spectrometry for In-Depth Characterization of Hemoglobin Proteoforms in Clinical and Veterinary Samples.,Analytical Chemistry

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Capillary Zone Electrophoresis-Top-Down Tandem Mass Spectrometry for In-Depth Characterization of Hemoglobin Proteoforms in Clinical and Veterinary Samples.
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-07-06 , DOI: 10.1021/acs.analchem.0c01350
Alexander Stolz _{1,

2} , Ylva Hedeland ₃ , Liesa Salzer ₁ , Jennifer Römer _{1,

4} , Reidun Heiene ₅ , Laurent Leclercq ₆ , Hervé Cottet ₆ , Jonas Bergquist ₇ , Christian Neusüß ₁

Affiliation

Hemoglobin (Hb) constitutes an important protein in clinical diagnostics—both in humans and animals. Among the high number of sequence variants, some can cause severe diseases. Moreover, chemical modifications such as glycation and carbamylation serve as important biomarkers for conditions such as diabetes and kidney diseases. In clinical routine analysis of glycated Hb, sequence variants or other Hb proteoforms can cause interference, resulting in wrong quantification results. We present a versatile and flexible capillary zone electrophoresis-mass spectrometry screening method for Hb proteoforms including sequence variants and modified species extracted from dried blood spot (DBS) samples with virtually no sample preparation. High separation power was achieved by application of a 5-layers successive multiple ionic polymer layers-coated capillary, enabling separation of positional isomers of glycated α- and β-chains on the intact level. Quantification of glycated Hb was in good correlation with the results obtained in a clinical routine method. Identification and characterization of known and unknown proteoforms was performed by fragmentation of intact precursor ions. N-Terminal and lysine glycation could be identified on the α- and β-chain, respectively. The versatility of the method was demonstrated by application to dog and cat DBS samples. We discovered a putative new sequence variant of the β-chain in dog (T38 → A). The presented method enables separation, characterization, and quantification of intact proteoforms, including positional isomers of glycated species in a single run. Combined with the simple sample preparation, our method represents a valuable tool to be used for deeper characterization of clinical and veterinary samples.

中文翻译：

毛细管区带电泳-自顶向下串联质谱法在临床和兽医样品中对血红蛋白蛋白形式的深度表征

血红蛋白（Hb）构成人类和动物临床诊断中的重要蛋白质。在大量的序列变异中，有些会引起严重的疾病。而且，诸如糖基化和氨基甲酰化的化学修饰作为诸如糖尿病和肾脏疾病之类的疾病的重要生物标志物。在糖化Hb的临床常规分析中，序列变异或其他Hb蛋白形式可能引起干扰，导致错误的定量结果。我们提出了一种用于血红蛋白形式的通用且灵活的毛细管区带电泳-质谱筛选方法，包括从干血斑（DBS）样品中提取的序列变体和修饰物种，几乎不需要样品制备。通过施加5层连续的多层离子聚合物层涂覆的毛细管可实现高分离力能够在完整水平上分离糖化α链和β链的位置异构体。糖化血红蛋白的定量与临床常规方法获得的结果高度相关。已知和未知蛋白形式的鉴定和表征是通过完整的前体离子的碎片进行的。N末端和赖氨酸糖基化可以分别在α链和β链上鉴定。该方法的多功能性通过应用于狗和猫的DBS样品得到了证明。我们发现了狗（T38→A）中β链的一个推定的新序列变异。提出的方法能够在一次运行中分离，表征和定量完整的蛋白形式，包括糖基化物种的位置异构体。结合简单的样品制备，

更新日期：2020-08-04

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