毛細管整體柱在線二維分離系統應用于人體軟骨的蛋白質組分析

謝沙潔, 王方軍, 晏 丹, 周廣東, 葉明亮, 鄒漢法＊

(1.中國科學院大連化學物理研究所,中國科學院分離分析化學重點實驗室,國家色譜研究分析中心,遼寧大連116023;2.組織工程國家工程研究中心,上海交通大學醫學院附屬第九人民醫院整形外科,上海200011)

毛細管整體柱在線二維分離系統應用于人體軟骨的蛋白質組分析

謝沙潔1, 王方軍1, 晏 丹2, 周廣東2, 葉明亮1, 鄒漢法1＊

(1.中國科學院大連化學物理研究所,中國科學院分離分析化學重點實驗室,國家色譜研究分析中心,遼寧大連116023;2.組織工程國家工程研究中心,上海交通大學醫學院附屬第九人民醫院整形外科,上海200011)

將已建立的7cm柱長的磷酸基團強陽離子交換富集整體柱與85cm柱長的C12烷基反相整體柱結合的在線二維分離平臺應用于軟骨提取蛋白的蛋白質組分析。對20μg軟骨提取蛋白的酶解產物進行14個鹽梯度的分級,然后對14個餾分進行反相色譜梯度分離及串聯質譜鑒定,成功地鑒定得到了7 434個獨立肽段對應的1 901個非冗余蛋白質。對所鑒定到的蛋白質進行定位分類,結果表明鑒定到的大部分蛋白質是來自于軟骨細胞內部的低豐度蛋白質,這對于許多關節類疾病的研究有重要意義。

整體柱;在線二維分離;蛋白質組學;軟骨組織

Abstract:In Shotgun proteome analysis,where nano-flow is adopted to increase the sensitivity as w ell as extrem ely com p licated samples such as proteolytic digest are inevitably confronted,monolithic capillary columns are widely used to improve the liquid Chromatography separation performance.It is known that cartilage contains extensive amounts of extracellular matrix(ECM),in which collagens and aggrecans being the most abundant macromolecules.It is obvious that the high content of ECM components causes a challenge in the comprehensive proteome analysis of cartilage.In this study,a7cm×150μmi.d.phosphate strong cation exchange(SCX)monolithic capillary column was coup led with an85cm×75μmi.d.C12reversed-phase monolithic capillary column for online two-dim ensional separation of20μg tryp tic digest of proteins extracted from hum an cartilage.After14salt steps fractionation and following gradient separation coup led with tandem mass spectrometry detection,finally7 434unique pep tides,corresponding to1 901distinct proteins were positively identified.Then,the identified proteins were analyzed by Gene Ontology(GO),and it was found that most of the identified proteins were com e from articular chondrocytes with low abundance,which is important for the researches of articular diseases.

Key words:monolithic column;online two-dimensional separation;proteomics;cartilage tissue

“鳥槍法”(Shotgun)蛋白質組學是將蛋白質混合物先進行酶解,然后通過鑒定酶解后的肽段來鑒定蛋白質的一種“從下至上”(bottom up)的蛋白質組學研究策略[1-3]。毛細管納升液相色譜與串聯質譜聯用(μHPLC-MS/MS)是“鳥槍法”蛋白質組學的技術核心,通過毛細管液相色譜對微量蛋白質酶解樣品的高效分離,極大地提高了質譜對復雜樣品進行蛋白質鑒定分析的能力[4]。……