米 薇, 王 晶, 应万涛, 贾 伟, 蔡 耘, 钱小红＊

(1.中国计量科学研究院生物能源环境所,北京100013;2.蛋白质组学国家重点实验室,北京蛋白质组研究中心,北京放射与辐射医学研究所,北京102206)

基于共价色谱分离的含半胱氨酸肽段富集策略

米 薇1, 王 晶1, 应万涛2, 贾 伟2, 蔡 耘2, 钱小红2＊

(1.中国计量科学研究院生物能源环境所,北京100013;2.蛋白质组学国家重点实验室,北京蛋白质组研究中心,北京放射与辐射医学研究所,北京102206)

多维色谱分离、串联质谱分析技术已在蛋白质组研究中得到广泛应用。然而生物样品的蛋白质以及全酶切肽段具有高度的复杂性,这严重干扰了蛋白质高通量、规模化的分析。通过标签肽段富集进行样品预分离可以降低体系的复杂程度。本文建立了一种基于共价色谱技术选择性分离富集含半胱氨酸肽的方法,从而降低了样品体系的复杂程度。首先以牛血清白蛋白(BSA)的酶切肽段为模型,对富集条件进行了优化和考察,并在此基础上通过5种蛋白质酶切肽段混合物的富集对该方法进行了验证。结果证明此方法的重现性好,富集效率高,富集特异性好,能有效地富集鉴定含半胱氨酸肽段。所建立的方法在复杂体系的蛋白质组研究中具有广泛的应用前景,为复杂样品的蛋白质高通量、自动化、规模化鉴定和定量研究提供了实用技术。

共价色谱;液相色谱-串联质谱;含半胱氨酸肽;富集;蛋白质组

Abstract:Automated multidimensional liquid Chromatography-tandem mass spectrometry(LCMS/MS)is routinely applied in large scale proteome profiling.How ever global proteome analysis remains a technical challenge due to the issues associated with sample complexity by tryptic digestion.The application of tag containing pep tide enrichment approach for sample pre-separation could reduce the complexity of protein digest.Here,we demonstrated a simple and highly efficient cysteine-containing pep tide enrichment method using a thiol specific covalent resin.The cysteine-containing pep tides from the tryptic digests of the complex proteinmixtures were selected by covalent Chromatography based on thiol-disulfide exchange,identified by mass spectrometry.The strategy was firstly optimized and evaluated by using the tryptic pep tides of bovine serum album in(BSA).Then the method was applied with a relatively complicated sample from a five standard protein mixture.The results of these studies show that the enrichment method of cysteine-containing pep tides is highly specific,efficient and reproducible.The effectiveness of this method in reducing the sample complexity and improving the identification of peptides by mass spectrometry has enabled high-throughput,automatic and large-scale qualitative and quantitative proteome analyses.

Key words:covalent Chromatography;liquid Chromatography-tandem mass spectrometry;cysteine-containing pep tides;enrichment;proteome

蛋白质组学的基本目标是尽可能地鉴定到生物体所表达的全套蛋白质。……