Date of Award


Degree Name

Doctor of Philosophy



First Advisor

Kinsel, Gary


Proteomics is an increasingly important area of biological research and has gathered much attention over recent years. Major challenges that make a proteomic analysis difficult are sample complexity, diversity and dynamic range. Progress in the area of proteomics relies heavily on new analytical tools for the sensitive, selective, and high-throughput studies of target analytes. It is estimated that there are several hundred thousand proteins in a human cell. In order to be able to analyze such a complex sample, an analytical method must be capable of separating and detecting many different sample peaks. The complexity of such samples indicates that a single separation method will not be able to provide the needed resolution. If two methods that are orthogonal are combined, then the peak capacity of the combined system is the product of the two individual peak capacities. Development of such systems would cater to the current demands of proteomics studies. Matrix assisted laser desorption/ionization (MALDI) mass spectrometry has evolved into a primary analytical tool for proteomics research. MALDI is fast and efficient and has a high tolerance to non-volatile buffers and impurities. The samples for MALDI are typically applied to solid supports after having been subjected to off-line liquid or gel separations. Several methods have been reported involving various chromatographic or electrophoretic separation methods. However, the current methods often require highly sophisticated sample handling systems, which are often expensive and in need of skilled human resources. The current demands of proteomic analyses require fast, efficient and inexpensive methods for separation to fully harness the capability of MALDI mass spectrometry. In this work a microfluidic device has been designed to perform dynamic isoelectric focusing (DIEF) based protein separation with digital sample deposition directly on a MALDI target for offline analysis. DIEF is related to capillary isoelectric focusing which and can facilitate the interface without the loss of the separation resolution. Compared to traditional capillary isoelectric focusing (cIEF) DIEF uses additional high-voltage power supplies to control the pH gradient by manipulating the electric field. The proteins can be focused at a desired sampling position according to their isoelectric point, to be collected for further analysis by MALDI mass spectrometry. DIEF has a peak capacity of over a thousand and offers an ease of interfacing to other techniques making it a preferred separation method for the interface with mass spectrometric techniques such as MALDI. The design of the microfluidic device is based on a digital droplet fractionation. Multiple fractions of the sample solution from DIEF are generated to retain the resolution and to act as an additional separation mode. The microfluidic device is controlled by actuating pneumatic valves built into the device. The DIEF operational parameters were optimized according to the surface functionality and the design of the microfluidic device. A suitable MALDI sample preparation method was found by studying different existing methods. The methods were studied using test proteins prepared in solutions having the additives used in the experiment. A simple mixture of three proteins was used to demonstrate the application of the developed method. The separation between the proteins insulin, hemoglobin and the myoglobin was demonstrated by varying the separation resolution in three experiments.




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