Coupling Pressurized Capillary Electrochromatography with Mass Spectrometry and Cell Metabonomics Study
|School||Shanghai Jiaotong University|
|Keywords||pressurized capillary electrochromatography (pCEC) massspectrometry (MS) lung cancer cell metabonomics|
With the advent of the post-genome era, systems biology includingmetabolomics, proteomics, and lipidomics has become an internationalfocusing hotspot in bio-research. The reduction of sample amount and theincrease of sample complexity lead to a growing desire to developminiaturized analytical instruments with superior performance in separationefficiency, resolution, peak capacity and speed.Pressurized capillary electrochromatography (pCEC) is a micro scaleseparation technology which has its retention mechanism based on bothchromatography and electrophoresis. Mass spectrometry (MS) is a widelyused detector which offers good selectivity and sensitivity. Thedevelopment of new ion source such as electrospray ionization (ESI) makesit possible for the online coupling of MS with a variety of separationtechniques.In this study, pCEC was coupled with electrospray ionizationquadruple time-of-flight MS for the first time using a zero dead volume union interface. The effect factors were investigated, and the conditionswere optimized. It was proved that the pCEC-MS system possess superiorseparation capability in the analysis of biological samples. The study alsoproposed a cell metabolomics studies by using HPLC-MS. The results weresupportive and supplementary to the traditional metabolomics studies,provides a new platform for the study of disease mechanisms, screening ortreatment.This thesis is presented in five chapters. The main contents are asfollows.The first chapter describes the background and significance of the topic.The separation mechanism, instrumental scheme, detector module andapplications of pCEC were reviewed. The epidemiology, factors, pathologicstages and clinical examination of lung cancer as well as the origin anddevelopment, methods and applications of metabolomics research wereintroduced.The second chapter describes the designing and performanceinvestigation of pCEC-MS interface. Three interfaces were designed andcompared. For the first time, the pCEC was coupled with MS with a zerodead volume union interface. The effects of electrolytes in mobile phasesand flow rate on MS signal as well as the limit of detection and reproducibility of the system were investigated. Experimental resultsshowed that the pCEC-MS system was stable and sensitive, with a sampledetection limit of ng/mL level.The third chapter describes the application of pCEC-MS system inmetabolomics study of lung cancer. The conditions of applied voltage,mobile phase, and gradient elution were optimized. A significant distinctionwas showed between lung cancer patients and healthy controls.49candidates of biomarkers were found. Compared with the control group, thelung cancer patient occur disorder in carnitine, uric acid, and amino acidmetabolism pathway.The fourth chapter describes metabonomics study of lung cancer cellsbased on high performance liquid chromatography and mass spectrometry.The metabolic profiles of the polar metabolites and the non-polarmetabolites in7lung tumor cell lines and a normal cell line were analyzedseparately. The results showed that the metabolites of the lung cancer celllines and normal cell line have significant differences. Further,10polarmetabolites and21non-polar metabolites which had a significantcontribution to classification were selected and preliminary identified due tothe accurate mass. Comparing to the normal cell line, the lung tumor celllines present an abnormal metabolism in protein, fatty acid, and phospholipids.The fifth chapter summarizes the whole thesis and looked into theprospects. The coupling of pCEC with MS combines both the feature ofpCEC’s fast speed separation, high column efficiency, and less consumptionof reagent with MS’s accuracy, specificity, and reliably, thus providing apowerful tool for the qualitative and quantitative analysis of complexbiochemical samples. The established cancer cell metabonomics reseachmethod possesses the advantage of simple and accurate, which can beexpected to become a new method for metabolomics studies.