10kDa以上幽门螺杆菌感染相关尿液差异表达蛋白的高效液相色谱-质谱分析

High Performance Liquid Chromatogram-Mass Spectrometry for Helicobacter pylori Infection-associated Differentially Expressed Proteins in Urine with Relative Molecular Mass More than 10 kDa

背景:除消化性溃疡和胃癌外,幽门螺杆菌(Hp)感染尚与多种胃肠外疾病密切相关。目前临床上尚无Hp感染的体液检测方法。目的:鉴定相对分子质量在10 k Da以上的Hp感染相关尿液差异表达蛋白,为Hp感染的体液诊断提供潜在生物学标记物。方法:志愿者留取晨起中段尿,并行13C-尿素呼气试验以明确Hp感染情况。Hp阴性和阳性尿液样本各取15例,分别混合后提取蛋白,以高效液相色谱-质谱系统分离蛋白、采集数据,行label-free定量分析。另取Hp阴性尿液样本15例和阳性尿液样本11例,对差异表达分析结果进行全扫描验证。应用IPA软件对鉴定出的尿液差异表达蛋白进行生物信息学分析。结果:Label-free定量分析共检测到475个尿液蛋白,并确定其中42个为差异表达蛋白。经外部扫描验证,最终鉴定出11个显著上调的差异表达蛋白。生物信息学分析揭示了这些差异表达蛋白的分子功能、参与的生物学通路、相关疾病等信息。结论:鉴定出的11个10 k Da以上尿液差异表达蛋白有望成为诊断Hp感染的生物学标记物,并可为Hp感染与胃肠外疾病的相关性研究提供分子水平的证据。

Background:Helicobacter pylori( Hp) is an important pathogen for peptic ulcer and gastric cancer,and is reportedly associated with a variety of extragastrointestinal diseases.However,there is no body fluid detection technique for Hp infection in clinical practice.Aims:To identify Hp infection-associated differentially expressed proteins in urine with relative molecular mass more than 10 kDa and provide potential biomarkers for diagnosis of Hp infection through body fluid detection.Methods:Midstream urine was collected from volunteers in the morning,and13C-urea breath test was performed to determine Hp infection.Each of 15 Hp-negative and 15 Hp-positive urine samples were mixed respectively for protein extraction.Spectra data were acquired by high performance liquid chromatogram-mass spectrometry,and label-free technology was used for relative quantitative analysis.The other 26 urine samples( 15 Hp-negative and 11 Hp-positive)were used for validation by full scan.IPA software was employed for bioinformatics analysis.Results:A total of 475 urinary proteins were detected by label-free quantitative analysis and 42 differentially expressed proteins were identified.Finally,11 significantly up-regulated differentially expressed proteins were confirmed by external scanning validation.Bioinformatics analysis revealed the molecular functions,biological pathways,and related diseases of these differentially expressed proteins.Conclusions:These 11 differentially expressed proteins more than 10 kDa identified in urine might bepotential biomarkers for diagnosis of Hp infection and provide molecular evidence for the correlation of Hp infection with extragastrointestinal diseases.