蛋白質高效分離兩維色譜柱的選擇優化

洪廣峰, 高明霞, 晏國全, 關 霞, 陶 芊, 張祥民,2＊

(1.復旦大學化學系,上海200433;2.復旦大學生物醫學研究院,上海200433)

蛋白質高效分離兩維色譜柱的選擇優化

洪廣峰1, 高明霞1, 晏國全1, 關 霞1, 陶 芊1, 張祥民1,2＊

(1.復旦大學化學系,上海200433;2.復旦大學生物醫學研究院,上海200433)

為了構建高效的離子交換/反相二維液相色譜(IEC/RPLC)分離平臺系統,提高復雜蛋白質樣品的分離效率,對色譜柱進行了評價與篩選。通過對實際人肝蛋白質樣品的分離效果的比較,選擇確定了TSKgel D EAE-5PW弱陰離子交換色譜柱(WAX)作為第一維色譜分離柱;考察了同一規格的10支代表性反相色譜柱(250mm×4.6 mm,5μm,30nm,C4、C8或C18),通過評價其對尿嘧啶、硝基苯、萘和芴的分離性能以及對3種標準蛋白質樣品的非特異性吸附、對人肝蛋白質樣品的WAX餾分的分離效果,最終確定以Jupiter300C4反相色譜柱作為第二維色譜分離柱。對兩維色譜柱的選擇優化為蛋白質高效分離二維液相色譜平臺的搭建提供了可靠基礎。

蛋白質組學;多維液相色譜;蛋白質;Top-dow n技術

Abstract:In order to optimize two-dimensional liquid Chromatographic(2D-LC)columns for highly efficient separation of proteins,several liquid Chromatographic columns were investigated and evaluated.Weak anion-exchange(WAX)column was chosen as the first dimension because of its extensive protein separation power.By comparison of different WAX Chromatographic columns for hum an liver protein separation,TSKgelD EAE-5PW column was selected as the first dimension of a2D-LC system.For the second dimension,ten typical reversed-phase(RP)LC columns(250mm×4.6mm,5μm,30nm)were investigated and evaluated.Their silica based RP stationary phases were butyl(C4),octyl(C8)or octadecyl(C18).To evaluate the retention behavior and non-specific protein adsorption ability of these ten columns,four neutral com pounds(uracil,nitrobenzene,naphthalene and fluorene)and three standard proteins(cytochrome C,myoglobin and album in from chicken egg white)were adopted and separated by RPLC.Meantime,WAX fractions were used to investigate the separation ability of different alkyl-bonded silica stationary phase columns for complex protein samples.B y comparison of column separation efficiency,adsorption of intact proteins and sample analysis,Jupiter 300C4column was finally employed for its excellent separation ability.Optimization of WAX and RPLC columns offers reliable foundation for the construction of2D-LC protein separation system s.

Key words:proteomics;multidimensional liquid Chromatography;intact protein;Top-down

隨著人類基因組計劃的完成,蛋白質組學已成為后基因組研究的重點。相對于基因組學而言,由于蛋白質表達的多樣性和翻譯后修飾的復雜性,其研究更加依賴于新技術新方法的發展。生物色譜與質譜技術的進步推動了蛋白質組學研究的迅猛發展,形成了基于完整蛋白質分離的Top-dow n路線和基于肽段串級質譜鑒定的B ottom-up路線[1-4]。雙向凝膠電泳技術盡管在蛋白質組學研究中的重要性眾所周知,然而其本身仍存在……