本文選題：福氏志賀菌 + 基質(zhì)輔助激光解吸電離��； 參考：《中國病原生物學(xué)雜志》2016年08期

【摘要】：目的建立多維液相色譜質(zhì)譜組合體系,用以獲得福氏志賀菌更完整的蛋白質(zhì)組信息。方法對福氏2a志賀菌301株(Sf2a301)全菌蛋白進(jìn)行預(yù)分離,順序抽提胞漿蛋白和膜蛋白。應(yīng)用二維液相色譜基質(zhì)輔助激光解吸附飛行時(shí)間串聯(lián)質(zhì)譜(2DLC-MALDI-TOF/TOF)和二維液相色譜電噴霧串聯(lián)質(zhì)譜(2DLC-ESI-MS/MS)構(gòu)成的多維液相色譜質(zhì)譜組合體系進(jìn)行蛋白分離和鑒定,分別應(yīng)用MASCOT和SEQUEST軟件搜索NCBI上Sf2a301的蛋白質(zhì)數(shù)據(jù)庫,并對兩種串聯(lián)質(zhì)譜的互補(bǔ)性及組合后優(yōu)勢進(jìn)行分析。結(jié)果 MALDI-TOF/TOF和ESI-MS/MS分別鑒定到Sf2a301株的960個(gè)蛋白和729個(gè)蛋白,總共鑒定1 231個(gè)蛋白;兩種技術(shù)方法鑒定蛋白質(zhì)的平均氨基酸序列覆蓋率分別為14.3%和13.9%,組合鑒定后覆蓋率升高為15.7%。MALDI和ESI兩種離子化方式具有互補(bǔ)性,表現(xiàn)為MALDI更傾向于離子化分子質(zhì)量偏小、堿性、胰酶消化后羧基末端為精氨酸的肽段;ESI更傾向于離子化分子質(zhì)量偏大、疏水、胰酶消化后羧基末端為賴氨酸的肽段。結(jié)論應(yīng)用2DLC-MALDI-TOF/TOF和2DLC-ESI-MS/MS鑒定Sf2a301全蛋白質(zhì)組,既互相確認(rèn)又相互補(bǔ)充。多維液相色譜質(zhì)譜組合體系無論在蛋白質(zhì)鑒定數(shù)量還是可信程度上都優(yōu)于單一串聯(lián)質(zhì)譜,可作為復(fù)雜樣品的蛋白質(zhì)組學(xué)研究技術(shù)平臺。
[Abstract]:Objective to establish a multi-dimensional liquid chromatography-mass spectrometry system to obtain more complete proteome information of Shigella flexneri.Methods the whole bacterial protein of Shigella flexneri 301 strain Sf2a301) was preisolated and cytosolic protein and membrane protein were extracted sequentially.Two dimensional liquid chromatographic matrix assisted laser desorption time of flight mass spectrometry (2DLC-MALDI-TOF / TOF / TOF) and two-dimensional liquid chromatography / electrospray tandem mass spectrometry (2DLC-ESI-MS / MS) were used to isolate and identify proteins.The protein database of Sf2a301 on NCBI was searched by MASCOT and SEQUEST software, and the complementarities and advantages of the two tandem mass spectrometry were analyzed.Results 960 proteins and 729 proteins of Sf2a301 strain were identified by MALDI-TOF/TOF and ESI-MS/MS respectively, and 1 231 proteins were identified.The average amino acid sequence coverage of the two methods for protein identification was 14.3% and 13.9%, respectively. After combination identification, the average amino acid sequence coverage increased to 15.7%.MALDI and ESI, which were complementary to each other. The results showed that MALDI was more inclined to ionization molecular weight and alkalinity.The peptide segment with arginine end after trypsin digestion is more inclined to ionization molecular mass and hydrophobicity, and the carboxyl end after trypsin digestion is lysine peptide segment.Conclusion 2DLC-MALDI-TOF/TOF and 2DLC-ESI-MS/MS were used to identify the whole Sf2a301 proteome, which were mutually confirmed and complementary.Multidimensional liquid chromatography-mass spectrometry combination system is superior to single tandem mass spectrometry in terms of protein identification quantity and reliability. It can be used as a proteomics research platform for complex samples.
【作者單位】： 華北理工大學(xué)生命科學(xué)學(xué)院;
【基金】：國家自然科學(xué)基金項(xiàng)目(No.81302323) 河北省高等學(xué)�？茖W(xué)技術(shù)研究項(xiàng)目(No.QN20131059) 河北省自然科學(xué)基金項(xiàng)目(No.2013209194) 華北理工大學(xué)培育基金項(xiàng)目(No.GP201518);華北理工大學(xué)博士科研啟動項(xiàng)目
【分類號】：R446.5
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本文編號：1755324