長(zhǎng)雙歧桿菌NCC2705葡萄糖與乳糖代謝的比較蛋白質(zhì)組學(xué)
何湘1 **,，劉大偉1 **,，孫忠科1，王芳1,，姜錚1,，趙紅慶1，陳宣男2,，黃留玉1,，袁靜1*
（1軍事醫(yī)學(xué)科學(xué)院疾病預(yù)防控制研究所，北京100071）
（2吉林大學(xué)公共衛(wèi)生學(xué)院,，長(zhǎng)春 130021）
摘要：【目的】以本實(shí)驗(yàn)室前期構(gòu)建的長(zhǎng)雙歧桿菌NCC2705菌株蛋白質(zhì)參考圖譜為基礎(chǔ),，研究長(zhǎng)雙歧桿菌發(fā)酵乳糖和葡萄糖的比較蛋白質(zhì)組學(xué)?！痉椒ā坎捎肐mageMaster 2D Elite Platnum Version 5.0 比較分析3倍以上蛋白差異點(diǎn),；利用MALDI-TOF進(jìn)行差異蛋白鑒定, 每個(gè)蛋白質(zhì)點(diǎn)的肽指紋圖譜在長(zhǎng)雙歧桿菌NCC2705菌株的蛋白質(zhì)數(shù)據(jù)庫(kù)用Mascot進(jìn)行檢索；采用Pro-Q磷酸化試劑進(jìn)行磷酸化蛋白的染色,?！窘Y(jié)果】鑒定到31個(gè)蛋白表達(dá)發(fā)生顯著變化，在乳糖發(fā)酵中14 個(gè)蛋白上調(diào)17個(gè)蛋白下調(diào),。這些蛋白為親水性酸性蛋白，它們基因的CAI值均在0.5以上,，主要包括糖代謝相關(guān)蛋白,、應(yīng)激蛋白、轉(zhuǎn)錄和翻譯相關(guān)蛋白,，還有一些未知功能的蛋白,。此外，有兩個(gè)蛋白：轉(zhuǎn)醛縮酶（BL0715,，transaldolase,，tal）L3蛋白點(diǎn)和丙酮酸激酶（BL0988, pyruvate kinase, pyk）G9蛋白點(diǎn)發(fā)生了磷酸化作用?！窘Y(jié)論】長(zhǎng)雙歧桿菌NCC2705在乳糖中生長(zhǎng)快于葡萄糖,，它們的降解途徑是相同的；轉(zhuǎn)醛縮酶和丙酮酸激酶發(fā)生了翻譯后修飾作用,，推測(cè)轉(zhuǎn)醛縮酶在43T和47S發(fā)生了磷酸化,，而丙酮酸激酶在65S發(fā)生了磷酸化。
關(guān)鍵詞：比較蛋白質(zhì)組,；乳糖代謝,；雙向電泳；Pro-Q Diamond 染色,；MALDI-TOF,；磷酸化蛋白
中圖分類號(hào)：Q935 文獻(xiàn)標(biāo)識(shí)碼：A 文章編號(hào)：0001-6209 (2008) 11-1451-08
Proteomic analysis of Bifidobacteria longum strain NCC2705 grown on lactose and glucose
Xiang He 1**, Dawei Liu 1**, Zhongke Sun1, Fang Wang 1, Zheng Jiang 1,
Hongqing Zhao 1, Xuannan Chen 2, Liuyu Huang1, Jing Yuan 1*
(1 Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China)
(2 School of Public Health, Jilin University, Changchun 130021, China)
Abstract: [Objective] Based on a proteomic reference map of the important probiotic organism Bifidobacteria longum NCC2705 constructed by our previous research, we compared the proteomic profiles of Bifidobacteria longum strain NCC2705 grown on lactose or glucose to identify the catabolic route allowing lactose fermentation. [Methods] We considered the proteins differentially expressed if their relative volume deviated more than 3-fold with ImageMaster 2D Elite version 5.0 software. Interesting spots were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, and phosphorylation analysis of proteins with mobility changes by Pro-Q Diamond Stain. [Results] The identified spots represent 31 protein entries, 14 up-regulated proteins, 17 down-regulated proteins. These identified proteins, which were hydrophilic proteins and their genes with CAI value above 0.5 represented the most abundant proteins, included key stress proteins, metabolism-related proteins, and proteins related to translation. Two proteins including Tal (BL0715, transaldolase, L3) and Pyk (BL0988, pyruvate kinase, G9) exhibited clear post-translational modification. [Conclusion] Proteomic comparison of glucose- and lactose-grown cells revealed that lactose and glucose were catabolized via the same degradation pathway, and the rate of glucose assimilation was higher than that of lactose. Spot and protein analysis revealed that post-translational modifications might be common in these proteins. Pro-Q Diamond staining analysis revealed that lactose trigger Tal phosphorylation at 43 T /47 S, and inhibited Pyk phosphorylation at 65 S. These proteins were identified for the first time as bifidobacterial phosphoproteins.
Keywords: Comparative proteome; the catabolism of lactose; 2D-PAGE; Pro-Q Diamond staining; phosphoproteins
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