Development of Multidimensional Separations Using Microfluidic Devices for Proteomics Applications Public Deposited

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  • March 20, 2019
  • Chambers, Andrew George
    • Affiliation: College of Arts and Sciences, Department of Chemistry
  • This work describes the development of novel microfluidic tools for the analysis of complex peptide mixtures. Gradient elution electrochromatography utilizing a reversed-phase monolith stationary phase is demonstrated on a microchip device. Porous polymer monoliths were patterned within glass microchips by photopolymerization of acrylate monomers. Mobile phase gradients were readily produced on-chip by computer-controlled mixing of solvents. Isocratic and gradient separations of protein digests were preformed to evaluate this device. Several two-dimensional (2D) separation systems were constructed for online coupling of liquid chromatography (LC) and capillary electrophoresis (CE). First, a hybrid 2D LC-CE system was constructed to perform LC in a capillary column and transfer the effluent to a microchip for CE with laser-induced fluorescence (LIF) detection. This basic hybrid 2D LC-CE system was then directly interfaced with mass spectrometry (MS) detection by microfabricating an electrospray ionization (ESI) emitter on the microchip device. This system new was used for rapid peptide mass fingerprinting of monoclonal antibody digests to confirm antibody identity and modification. In a third 2D system, the LC dimension was also integrated to produce a microchip device capable of performing LC-CE-ESI. This microchip incorporated a sample trapping region and an LC channel packed with reversed-phase particles. In addition to LC-CE-ESI, this device was used for LC-ESI without any instrumental modifications. Finally, a dual microfluidic ESI source will be discussed for increasing the mass accuracy of microchip ESI measurements. This device featured two independent ESI emitters that were used to sequentially introduce ions from two solutions into a mass spectrometer. Using the second emitter to introduce a reference compound for internal calibration, accurate mass measurements (< 3 ppm mass error) were obtained with the microchip dual ESI device and time-of-flight MS.
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  • In Copyright
  • "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry."
  • Ramsey, J. Michael
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  • Chapel Hill, NC
  • Open access

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