Double alkylation with maleimide-PEG-biotin: An enrichment method for cysteine redox states

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry Pub Date : 2025-05-06 DOI:10.1016/j.ab.2025.115892

Jung Mi Lim , Rodney L. Levine

引用次数: 0

Abstract

Cysteine alkylation is widely used in mass-spectrometric based proteomic studies. The oxidation state of each cysteine can be determined by labeling free thiols with one alkylating agent and disulfides with a second alkylating agent that differs in mass from the first. We have developed an improved method utilizing biotin-conjugated maleimides to specifically label cysteine residues in the thiol state and in disulfide linkage. The biotin tag effectuates very efficient enrichment of cysteine containing peptides, greatly increasing sensitivity for those peptides. We also achieve very high recovery of the biotinylated peptides from an avidin column by elution with hexafluoro-2-propanol (HFIP). The method offers improved mapping of the cysteine proteome and its oxidation state.

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马来酰亚胺-聚乙二醇-生物素双烷基化：半胱氨酸氧化还原态的富集方法

半胱氨酸烷基化被广泛应用于基于质谱的蛋白质组学研究。每种半胱氨酸的氧化态可以通过用一种烷基化剂标记游离硫醇和用质量不同于第一种烷基化剂标记二硫化物来确定。我们开发了一种改进的方法，利用生物素偶联马来酰亚胺来特异性标记巯基和二硫键中的半胱氨酸残基。生物素标签可以非常有效地富集含有半胱氨酸的肽，大大提高了对这些肽的敏感性。我们还通过六氟-2-丙醇（HFIP）洗脱，从亲和素柱中获得了非常高的生物素化肽回收率。该方法提供了改进的半胱氨酸蛋白质组及其氧化状态的映射。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Analytical biochemistry 生物-分析化学

CiteScore

5.70

自引率

0.00%

发文量

283

审稿时长

44 days

期刊介绍： The journal''s title Analytical Biochemistry: Methods in the Biological Sciences declares its broad scope: methods for the basic biological sciences that include biochemistry, molecular genetics, cell biology, proteomics, immunology, bioinformatics and wherever the frontiers of research take the field. The emphasis is on methods from the strictly analytical to the more preparative that would include novel approaches to protein purification as well as improvements in cell and organ culture. The actual techniques are equally inclusive ranging from aptamers to zymology. The journal has been particularly active in: -Analytical techniques for biological molecules- Aptamer selection and utilization- Biosensors- Chromatography- Cloning, sequencing and mutagenesis- Electrochemical methods- Electrophoresis- Enzyme characterization methods- Immunological approaches- Mass spectrometry of proteins and nucleic acids- Metabolomics- Nano level techniques- Optical spectroscopy in all its forms. The journal is reluctant to include most drug and strictly clinical studies as there are more suitable publication platforms for these types of papers.