Comparison of proteins expressed by Pseudomonas aeruginosa strains representing initial and chronic isolates from a cystic fibrosis patient:: an analysis by 2-D gel electrophoresis and capillary column liquid chromatography-tandem mass spectrometry
Comparison of proteins expressed by Pseudomonas aeruginosa strains representing initial and chronic isolates from a cystic fibrosis patient:: an analysis by 2-D gel electrophoresis and capillary column liquid chromatography-tandem mass spectrometry
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DOI：
10.1099/00221287-146-10-2495
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发表时间：
2000-10-01
期刊：
影响因子：
--
通讯作者：
Goldberg, JB
中科院分区：
文献类型：
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作者：
Hanna, SL;Sherman, NE;Goldberg, JB
囊性纤维化（CF）患者慢性肺部感染中分离的铜绿假单胞菌具有不同于最初感染CF患者的表型，以及不同于其他临床或环境分离株的表型。蛋白表达采用二维（2-D）凝胶电泳，蛋白鉴定采用微毛细管柱液相色谱-串联质谱（mu LC/MS/MS）测序。本分析选择的分离株来自CF患者的痰液：菌株383具有环境分离株典型的非黏液样表型，菌株2192具有慢性CF肺部感染分离株典型的黏液样表型，通过限制性内切酶分析、随机扩增多态性DNA-PCR和脉冲场凝胶电泳证实菌株383和2192基因相同。对蛋白提取条件进行优化，从临床分离的数百种蛋白中分离出一致的高分辨率，通过二维凝胶考马斯氏染色检测，选择14种蛋白进行分析；这些蛋白通过凝胶内胰蛋白酶消化产生的肽的LC/MS/MS鉴定，并与假单胞菌基因组计划的翻译数据进行比较；该技术的优化已允许比较菌株383和2192表达的蛋白质。
Isolates of Pseudomonas aeruginosa from chronic lung infections in cystic fibrosis (CF) patients have phenotypes distinct from those initially infecting CF patients, as well as from other clinical or environmental isolates, To gain a better understanding of the differences in these isolates, protein expression was followed using two-dimensional (2-D) gel electrophoresis and protein identification by peptide sequencing using micro-capillary column liquid chromatography-tandem mass spectrometry (mu LC/MS/MS). The isolates selected for this analysis were from the sputum of a CF patient: strain 383 had a nonmucoid phenotype typical of isolates from the environment, and strain 2192, obtained from the same patient, had a mucoid phenotype typical of isolates from chronic CF lung infections, Strains 383 and 2192 were confirmed to be genetically identical by restriction endonuclease analysis, random amplified polymorphic DNA-PCR, and pulsed-field gel electrophoresis, Conditions of protein extraction were optimized for consistent high-resolution separation of several hundred proteins from these clinical isolates as detected by Coomassie staining of 2-D gels, Fourteen proteins were selected for analysis; this group included those whose expression was common between both strains as well as unique for each strain, The proteins were identified by mu LC/MS/MS of the peptides produced by an in-gel tryptic digestion and compared to translated data from the Pseudomonas Genome Project; optimization of this technique has allowed for the comparison of proteins expressed by strains 383 and 2192.