Ultrasensitive CITP-MS based targeted proteomics technologies for protein identification and quantification

dc.contributor.advisorLee, Cheng Sen_US
dc.contributor.authorWang, Chenchenen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.description.abstractMass Spectrometry (MS) based technologies have enabled efficient and comprehensive proteomic profiling for biomarker discovery. However, due to sample complexity and large concentration variation, the obtained data is usually biased to endogeneous high abundance proteins while the important disease-related information went missing. Targeted proteomics enables the delivery of precise and sensitive qualitative/quantitative data of interest to researchers by focusing analysis on a preselected population of cells or proteins. This project aims to develop targeted proteomic technologies through capillary isotachophoresis (CITP)-based technique which is capable of selectively enriching trace compounds for a further improved sensitivity in both discovery and validation studies. By employing tissue microdissection and a CITP-based multidimensional separation platform, homogeneous glioma cells were isolated from unwanted cells and analyzed in search of glioblastoma biomarker. Comparative proteomic profiling of pure tumor cells from different grades of infiltrative astrocytomas revealed disease specific protein expression variation among grades. Further validation using immunohistochemistry demonstrated consistent results. This targeted tissue analyzing platform provided a sensitive and confident methodology for biomarker discovery within minute amount of samples. With the demonstrated outstanding analyzing capacity on targeted biomarker discovery, we moved on to developing ultrasensitive targeted quantitation techniques. We demonstrated online coupling of transient-CITP/CZE (capillary zone electrophoresis) with selective reaction monitoring (SRM) MS for the first time via a sheathliquid interface for improved sensitivity and selectivity. Ultrasensitive targeted quantitation was achieved through the incorporation of the selective enrichment capability of CITP/CZE with SRM MS, giving a limit of quantitation (LOQ) of 50 pM with a total sample loading of 50 attomoles. In order to further improve the sensitivity, we developed a novel sheathless interface which enables increased loading capacity and nanoflow operation by assembling a large size separation capillary and a small size porous emitter. LOQ was improved 5 times comparing to using the first sheathliquid interface, giving a LOQ of 10 pM with a total sample loading of 25 attomoles. This novel interface optimally preserved the high resolution and efficiency of CITP/CZE while improving the limited sample loading capacity, demonstrating a powerful analytic platform for targeted proteomic quantitation and validation.en_US
dc.subject.pquncontrolledanalytical chemistryen_US
dc.subject.pquncontrolledmass spectrometryen_US
dc.subject.pquncontrolledSRM quantificationen_US
dc.titleUltrasensitive CITP-MS based targeted proteomics technologies for protein identification and quantificationen_US


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