| This study mainly addresses the application of rapid resolution liquid chromatography mass spectrometry/tandem mass spectrometry (RRLC-MS/MS) combined with electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI) for investigating the differences of these three ionization methods in acquiring small molecular metabolome of plasma, and integrated RRLC-MS/MS combined with ESI and APCI for plasma metabolomics has been established. Meanwhile, the combination of adavantage of RRLC-MS/MS in fast speed, high sensitivity, high selectivity, high-throughput with the non-deviation detecting and robust identification of NMR has been employed for the identification of lung cancer plasma metabolomics in order to provide biomarkers of small molecule for clinically evaluating therapeutic effects and prognosis analysis of lung cancer.Through analyzing metabolome differences between healthy and lung cancer plasma,66(detected by LC-MS) and12(detected by1H NMR) potential biomarkers closely related diagnosing lung cancer has been obtained, respectively. In the mean time,33metabolites responded to therapy has been acquired by analyzing the differences among lung cancer plasma samples over the periods of pre and post treatments, and these responded metabolites has further been sifted into potential biomarkers related to evaluating therapeutic effects by observing the dynamic change trends throught the pre, middle, and post treatment periods combined with clinical evaluation of therapeutic effects, and consequently10potential biomarkers related to therapeutic effects’evalution has been achieved. In addition,1H NMR discovered10biomarkers responded to treatments, and8of them are closely related to therapeutic effects. This study proved the integrated ionization methods could better make up the disadvantages of ionization discrimination and efficiency differences encountered by MS with only a single ionization source. Also, the integrated ionization methods combined with1H NMR furtherly consummated the analytical methods for plasma metabolomics. 1. Study on the RRLC-MS methods suited for plasma metabolomicsRRLC-MS in combination with ESI, APCI and APPI in both positive and negative ion modes has been conducted to detedc healthy plasma for investigating the ability of different ionization methods in gaining plasma metaboleme, and the results has proved that integrated ionization RRLC-MS/MS is able to get more global metaboleme information. Further analysis on the applicability of three ionization methods for detecting different types of plasma metabolites indicates that the integration of ESI and APPI without APCI could better satisfy the requirement of metabolomics for obtaining comprehensive metabolites’information and high-throughput detection.2. The application of integrated ionization RRLC-MS/MS in lung cancer plasma metabolomicsThe integrated ESI and APPI with RRLC-MS/MS in combination of multivariate statistical analysis have been implemented in lung cancer plasma metabolomics. Through comparing the differences of plasma motabolime between healthy and cancer patients,66potential biomarkers related to diagnosing lung cancer has been produced, and27metabolited has been identified. Furtherly, differences of plasma metabolome between pre and post treatments of lung cancer patients has been assessed, and33metabolites responded to therapy has been obtained, and danyic change trends of these33metabolites has been analyzed among healthy, pre, middle, and post treatment plasma samples, and10potential biomarkers closely related to lung cancer genesis, development, treatment, and prognosis has been achieved, including carnitine10:0, sn-1lysoPC (16:1), sn-2lysoPC (18:2), sn-1lysoPC(16:0), glutamine, DHEAs, androsterone sulfate, oleic acid, linoleic acid, and glucose. The study results proved that integrated ionization methods could, to some degree, conquer the disadvantage of ionization discrimination and efficiency differences encountered by MS with only a single ionization source. The integrated ionization methods not only provide complementarity for each other in detecting metabolites with different polarity, but also validate the realiability of potential biomarkers discovered by the other side. The study results are very promising to convey the extremely important bases for clinically therapeutic effects evaluation and prognosis. 3.1H NMR-based lung cancer plasma metabolomicsThe1H NMR with multivariate statistical analysis has been introduced into lung cancer plasma metabolomics. Through comparing the differences of plasma metabolome between healthy and cancer plasma samples,12potential biomarkers related to diagnosing lung cancer has been achieved, and identified. Furtherly, differences of plasma metabolome between pre and post treatments of lung cancer patients has been evaluated, and10metabolites responded to therapy has been obtained, and danyic change trends of these10metabolites has been analyzed among healthy, pre, middle, and post treatment plasma samples, and8potential biomarkers closely related to lung cancer genesis, development, treatment, and prognosis has been achieved, including citrate, acetate, glucose, glutamine, puruvate, phospholipids choline, valine, cytidine, among which glucose, glutamine, and phospholipids choline linked to therapeutic effects evaluation were simultaneously discovered by1H NMR and LC-MS that validated the detection realiability. At the same time,1H NMR and LC-MS methods offered5and4unique potential biomarkers related to therapy evaluation, respectively, which suggested the combination of two spectrometry technologies could not only validate detection of each others, but also broaden the discovered ranges of potential biomarkers. The above potential biomarkers discovered by integrated LC-MS/MS and1H NMR metabolomics are likely to provide valuable bases for clinically diagnosing, assessing therapy, and prognosis analysis. |
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