He Xiang, Liu Dawei, Sun Zhongke, Wang Fang, Jiang Zheng, Zhao Hongqing, Chen Xuannan, Huang Liuyu, Yuan Jing
Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China.
Wei Sheng Wu Xue Bao. 2008 Nov;48(11):1451-8.
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.
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.
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.
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.
基于我们之前构建的重要益生菌长双歧杆菌NCC2705的蛋白质组参考图谱，比较长双歧杆菌菌株NCC2705在乳糖或葡萄糖上生长时的蛋白质组图谱，以确定允许乳糖发酵的分解代谢途径。
如果使用ImageMaster 2D Elite 5.0软件其相对体积偏差超过3倍，我们认为这些蛋白质存在差异表达。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）分析鉴定感兴趣的斑点，并通过Pro-Q Diamond染色对迁移率发生变化的蛋白质进行磷酸化分析。
鉴定出的斑点代表31个蛋白质条目，14个上调蛋白质，17个下调蛋白质。这些鉴定出的蛋白质都是亲水性蛋白质，其CAI值高于0.5的基因代表了最丰富的蛋白质，包括关键应激蛋白、代谢相关蛋白和翻译相关蛋白。包括Tal（BL0715，转醛醇酶，L3）和Pyk（BL0988，丙酮酸激酶，G9）在内的两种蛋白质表现出明显的翻译后修饰。
对在葡萄糖和乳糖上生长的细胞进行蛋白质组比较显示，乳糖和葡萄糖通过相同的降解途径进行分解代谢，且葡萄糖同化率高于乳糖。斑点和蛋白质分析表明，翻译后修饰在这些蛋白质中可能很常见。Pro-Q Diamond染色分析显示，乳糖在43T/47S处触发Tal磷酸化，并在65S处抑制Pyk磷酸化。这些蛋白质首次被鉴定为双歧杆菌磷酸化蛋白。
