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Development And Application Of Phospholipidomics Platform Based On LC-MS

Posted on:2012-09-17Degree:DoctorType:Dissertation
Country:ChinaCandidate:C ZhuFull Text:PDF
GTID:1110330368975318Subject:Pharmaceutical Engineering and Technology
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With the integrated development of metabolomics, the comprehensive analysis of all metabolites in bio-samples is processing systematically. To develop a robust and reliable method is crucial important for metabolomics study. Due to the different physicochemical properties of different compounds, the target platforms should be established and then combined for identification and quantification (IQ) of all metabolites.Phospholipids, a major component of all cell membranes, play a very important role in bioactivities. Phospholipids and its metabolism are closely associated with human health and diseases. Therefore, it is essential to develop an efficient method for phospholipids measurement for intensive study of diseases. In this dissertation, the development and application of a phospholipidomics platform were successfully achieved with using liquid chromatography-mass spectrometry (LC-MS) and multivariate statistical analysis (MVA).1, Bases on liquid chromatography coupled with time of flight mass spectrometry (LC-TOF/MS) and ion trap mass spectrometry (Ion trap-MS), a phospholipidomics platform was initially established and applied to the study of diabetes mellitus (DM) and diabetic nephropathy (DN). With the use of a diol column, isopropanol (IPA)/hexane/H2O based mobile phase and an isocratic elution,65 phospholipids species belonging to 7 classes were simultaneously identified and quantified in negative ionization mode in a single run of 45 min. The first application of this initial platform was to study the effect of a TCM formula called "Tangshenfang" (TSF) on phospholipids metabolism of OLETL rats. The results indicated that TSF was able to significantly regulate phospholipids metabolism of OLETF rats. According to quantitative results, a total of 8 phospholipids compounds which have significant concentration variation between control group and model group were considered as potential biomarkers of type II diabetes. The second application of this initial platform focuses on the biomarker discovery of diabetes mellitus (DM) and diabetic nephropathy (DN). The score plot of partial least squares discriminant analysis (PLS-DA) indicated the differences of phospholipids metabolism between healthy controls and patients. Employing PLS-DA and analysis of variance (ANOVA), we found 3 DM-specific biomarkers,8 DN-specific biomarkers and 2 novel biomarkers for distinguishing DM, DN and healthy individuals. Ultimately, the plasma concentrations of the two novel biomarkers in each group were determined by an external standard method.2, Due to the disadvantages of the initial phospholipidomics platform, such as, high sample volume needed, relatively long run time, retention time shifting and no internal standard calibration, we improved the initial platform using hydrophilic interaction liquid chromatography coupled with a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (HILIC-Ion trap-FT/MS). A novel and efficient method was developed for phospholipids separation. With the use of IPA extraction method, sample preparation becomes a simple and time-saving procedure and only needs 10μL of plasma. With the use of a diol column, acetornitrile (ACN)/water based mobile phase and a binary gradient, in a single run of 15 min,186 phospholipids species belonging to 9 classes were identified and quantified in negative ionization mode. The method was fully validated with different bio-samples following a standard validation procedure of metabolomics and successfully used to separate phospholipids extracts from different bio-samples. The improved phospholipidomics approach is characterized as a simple, efficient, high throughput, stable, robust and standard platform.3, This improved platform was applied to investigate abnormal phospholipids metabolism induced by central nervous system (CNS) disorder. An Experimental Autoimmune Encephalitis (EAE) rat model of multiple sclerosis showed significant difference of plasma phospholipids metabolism compared with the controls indicating the close relationship between phospholipids metabolism and multiple sclerosis. Some phospholipids were identified as biomarkers of the early stage of multiple sclerosis. Additionally, we did the integrated analysis of plasma phospholipidomics data and cerebrospinal fluid (CSF) metabolomics data and found the consistence to a certain extent between plasma phospholipids metabolism and CSF metabolism. This study provides a referable basis for plasma metabolomics study of CNS disorder.The developed phospholipidomics platform is a rapid, high-throughput and robust tool for integrated metabolomics study.
Keywords/Search Tags:phospholipidomics, HILIC-Ion trap-FT/MS, separation, identification, quantification, biomarker
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