基于共價(jià)色譜分離的含半胱氨酸肽段富集策略

米 薇, 王 晶, 應(yīng)萬濤, 賈 偉, 蔡 耘, 錢小紅＊

(1.中國計(jì)量科學(xué)研究院生物能源環(huán)境所,北京100013;2.蛋白質(zhì)組學(xué)國家重點(diǎn)實(shí)驗(yàn)室,北京蛋白質(zhì)組研究中心,北京放射與輻射醫(yī)學(xué)研究所,北京102206)

基于共價(jià)色譜分離的含半胱氨酸肽段富集策略

米 薇1, 王 晶1, 應(yīng)萬濤2, 賈 偉2, 蔡 耘2, 錢小紅2＊

(1.中國計(jì)量科學(xué)研究院生物能源環(huán)境所,北京100013;2.蛋白質(zhì)組學(xué)國家重點(diǎn)實(shí)驗(yàn)室,北京蛋白質(zhì)組研究中心,北京放射與輻射醫(yī)學(xué)研究所,北京102206)

多維色譜分離、串聯(lián)質(zhì)譜分析技術(shù)已在蛋白質(zhì)組研究中得到廣泛應(yīng)用。然而生物樣品的蛋白質(zhì)以及全酶切肽段具有高度的復(fù)雜性,這嚴(yán)重干擾了蛋白質(zhì)高通量、規(guī)模化的分析。通過標(biāo)簽肽段富集進(jìn)行樣品預(yù)分離可以降低體系的復(fù)雜程度。本文建立了一種基于共價(jià)色譜技術(shù)選擇性分離富集含半胱氨酸肽的方法,從而降低了樣品體系的復(fù)雜程度。首先以牛血清白蛋白(BSA)的酶切肽段為模型,對(duì)富集條件進(jìn)行了優(yōu)化和考察,并在此基礎(chǔ)上通過5種蛋白質(zhì)酶切肽段混合物的富集對(duì)該方法進(jìn)行了驗(yàn)證。結(jié)果證明此方法的重現(xiàn)性好,富集效率高,富集特異性好,能有效地富集鑒定含半胱氨酸肽段。所建立的方法在復(fù)雜體系的蛋白質(zhì)組研究中具有廣泛的應(yīng)用前景,為復(fù)雜樣品的蛋白質(zhì)高通量、自動(dòng)化、規(guī)模化鑒定和定量研究提供了實(shí)用技術(shù)。

共價(jià)色譜;液相色譜-串聯(lián)質(zhì)譜;含半胱氨酸肽;富集;蛋白質(zhì)組

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

蛋白質(zhì)組學(xué)的基本目標(biāo)是盡可能地鑒定到生物體所表達(dá)的全套蛋白質(zhì)。……