铜绿假单胞菌SJTD-1的磷酸化蛋白质组学

doi:10.16183/j.cnki.jsjtu.2017.12.007

摘要/Abstract

摘要： 制备了铜绿假单胞菌SJTD-1菌株的蛋白质组酶解产物，并利用二氧化钛磁珠从酶解产物中富集磷酸化肽.首次利用纳升液相色谱-质谱技术鉴定磷酸化肽的磷酸化位点，最终共鉴定获得13条磷酸化肽，分别来自12个磷酸化蛋白，其中有7个磷酸化蛋白在假单胞菌中是首次发现.分析鉴定到的磷酸化蛋白，发现大多与DNA复制、物质转运、能量代谢等密切相关.这是首次对铜绿假单胞菌SJTD-1菌株进行磷酸化蛋白质组学分析，为SJTD-1在DNA复制、物质和能量代谢以及细胞膜转运等方面的研究奠定基础.

关键词: 铜绿假单胞菌, 磷酸化蛋白, 液相色谱质谱

Abstract: Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai 200240, China) Abstract： SJTD-1, a novel kind of Pseudomonas aeruginosa, is isolated from oil-contaminated soil. It has been demonstrated that this bacterium can metabolize n-alkanes (C12 to C30) in our laboratory. Pseudomonas aeruginosa SJTD-1 may have wide applications in oil pollution. In our study, phosphopeptides of Pseudomonas aeruginosa SJTD-1 were enriched by TiO2 and 13 phosphopeptides from 12 phosphoproteins were first characterized by nano-liquid chromatography-mass spectrometry (nano LC-MS). Among these identified phosphoproteins, 7 phosphoproteins were first discovered in Pseudomonas aeruginosa. We found most of these identified phosphoproteins were related to DNA replication, material transport and energy metabolism. This is the first analysis of phosphorylation proteomic in Pseudomonas aeruginosa SJTD-1, and our results provide evidence for further research on DNA replication, material or energy metabolism and membrane transport in Pseudomonas aeruginosa.

Key words: Pseudomonas aeruginosa, phosphoproteins, liquid chromatography-mass spectrometry (LC-MS)

中图分类号:

O 657.7

邢雪娇a，李文婷b，侯敬丽c，刘建华b. 铜绿假单胞菌SJTD-1的磷酸化蛋白质组学[J]. 上海交通大学学报（自然版）, 2017, 51(12): 1448-1455.

XING Xuejiaoa，LI Wentingb，HOU Jinglic，LIU Jianhuab. Phosphoproteomics of Pseudomonas aeruginosa SJTD-1[J]. Journal of Shanghai Jiaotong University, 2017, 51(12): 1448-1455.

参考文献

1］DUNN J D, REID G E, BRUENING M L. Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry［J］. Mass Spectrometry Reviews, 2010, 29(1): 29-54. ［2］LARSEN M R, THINGHOLM T E, JENSEN O N, et al. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns［J］. Molecular & Cellular Proteomics, 2005, 4(7): 873-886. ［3］ODA Y, NAGASU T, CHAIT B T. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome［J］. Nature Biotechnology, 2001, 19(4): 379-382. ［4］RUSH J, MORITZ A, LEE K A, et al. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells［J］. Nature Biotechnology, 2005, 23(1): 94-101. ［5］PINKSE M W H, MOHAMMED S, GOUW J W, et al. Highly robust, automated, and sensitive online TiO2-based phosphoproteomics applied to study endogenous phosphorylation in Drosophila melanogaster［J］. Journal of Proteome Research, 2007, 7(2): 687-697. ［6］李娟，蒋武辉，徐晓颖.二氧化钛结合超滤膜富集和分离肿瘤患者唾液中的磷酸化肽和唾液酸化糖肽［J］. 高等学校化学学报，2014, 35(10): 2073-2077. LI Juan, JIANG Wuhui，XU Xiaoying. Novel method coupled TiO2 with ultrafiltration membrane to enrich and separate phosphopetides and sialic acid-containing glycopeptides from saliva of cancer patient［J］. Chemical Journal of Chinese University, 2014, 35(10): 2073-2077. ［7］LIU H, XU J, LIANG R, et al. Characterization of the medium- and long-chain n-alkanes degrading pseudomonas aeruginosa strain SJTD-1 and its alkane hydroxylase genes［J］. PloS One, 2014, 9(8): e105506. ［8］LIU H, LIANG R, TAO F, et al. Genome sequence of pseudomonas aeruginosa strain SJTD-1, a bacterium capable of degrading long-chain alkanes and crude oil［J］. Journal of Bacteriology, 2012, 194(17): 4783-4784. ［9］孙文兵，侯敬丽. 假单胞菌SJTD-1全蛋白质组学研究［J］. 质谱学报，2015, 36(3): 193-198. SUN Wenbing, HOU Jingli. Proteome analysis of pseudomonas aeruginosa SJTD-1［J］. Journal of Chinese Mass Spectrometry Society, 2015, 36(3): 193-198. ［10］LIU H, SUN W B, LIANG R B, et al. iTRAQ-based quantitative proteomic analysis of Pseudomonas aeruginosa SJTD-1: A global response to n-octadecane induced stress［J］. Journal of Proteomics, 2015, 123: 14-28. ［11］OUIDIR T, JARNIER F, COSETTE P, et al. Potential of liquid-isoelectric-focusing protein fractionation to improve phosphoprotein characterization of Pseudomonas aeruginosa PA14［J］. Analytical and Bioanalytical Chemistry, 2014, 406(25): 6297-6309. ［12］OUIDIR T, JARNIER F, COSETTE P, et al. Extracellular Ser/Thr/Tyr phosphorylated proteins of Pseudomonas aeruginosa PA14 strain［J］. Proteomics, 2014, 14(17/18): 2017-2030. ［13］RAVICHANDRAN A, SUGIYAMA N, TOMITA M, et al. Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non-pathogenic Pseudomonas species［J］. Proteomics, 2009, 9(10): 2764-2775. ［14］OUIDIR T, JOUENNE T, HARDOUIN J. Post-translational modifications in Pseudomonas aeruginosa revolutionized by proteomic analysis［J］. Biochimie, 2016, 125: 66-74. ［15］GARNAK M, REEVES H C. Phosphorylation of isocitrate dehydrogenase of Escherichia coli［J］. Science, 1979, 203(4385): 1111-1112. ［16］STOVER C K, PHAM X Q, ERWIN A L, et al. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen［J］. Nature, 2000, 406(6799): 959-964. ［17］TAVARES-CARREN F, PATEL K B, VALVANO M A. Burkholderia cenocepacia and salmonella enterica ArnT proteins that transfer 4-amino-4-deoxy-l-arabinose to lipopolysaccharide share membrane topology and functional amino acids［J］. Scientific Reports, 2015, 5: 10773. ［18］RYAN A, WANG C J, LAURIERI N, et al. Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases［J］. Protein Cell, 2010, 1(8): 780-790 ［19］SUN Y Y, CHI H, SUN L. Pseudomonas fluorescens filamentous hemagglutinin, an iron-regulated protein, Is an important virulence factor that modulates bacterial pathogenicity［J］. Frontiers in Microbiology, 2016, 7: 1320. ［20］KOCHETOV G A, SOLOVJEVA O N. Structure and functioning mechanism of transketolase［J］. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 2014, 1844(9): 1608-1618. ［21］VIMALA A, HARINARAYANAN R. Transketolase activity modulates glycerol-3-phosphate levels in Escherichia coli［J］. Molecular Microbiology, 2016, 100(2): 263-277. ［22］SAHA A, CONNELLY S, JIANG J, et al. Akt phosphorylation and regulation of transketolase is a nodal point for amino acid control of purine synthesis［J］. Molecular Cell, 2014, 55(2): 264-276.