Integrated protein analysis on microchips for capillary electrophoresis electrospray mass spectrometry

We have developed an integrated system to combine protein processing steps such as protein digestion, preconcentration and peptide separation together with electrospray mass spectrometry detection. This thesis will present the development of the microchip-ESMS interface and its application to protein preparation and analysis on-chip.; Our first effort was to build a reliable interface to couple the microchip and mass spectrometer together. Different designs for spraying directly from the edge of the chip at the channel outlet were tested. Based on a low dead volume microchip-capillary coupling technique, a gold coated capillary tip was coupled to the end of the microchip channel and served as the electrospray tip. This design gave stable electrospray and was used for subsequent studies. Separation of protein and peptides was achieved. A large volume sample stacking method used to preconcentrate samples in the microchip system was developed. Standard peptides were tested and a 50-fold improvement on detection limit with bradykinin was achieved. This preconcentration method is a useful way to enrich analyte ions with high electrophoretic mobilities in the direction opposite to EOF.; Instead of traditional protein solution phase digestion in microcentrifuge tubes, proteins were digested directly in a microchip with immobilized trypsin beads. Two kinds of on-chip digestion methods were used: on-chip reservoir digestion and integrated packed bed digestion. The integrated packed bed appeared to be the fastest digestion method, compared to on-chip reservoir digestion and traditional solution phase digestion. A flow rate of 0.5–1 μl/min was found adequate for complete consumption of cytochrome c or BSA, corresponding to a digestion time of 3–6 min at room temperature. The digests were separated in the microchip channel before mass spectrometry detection. A solid phase extraction step was then added. On-chip solid phase extraction of peptides was investigated, and it provided an efficient preconcentration method. A two-bed system for protein digestion and subsequent peptide SPE concentration was constructed on-chip. Submicromolar cytochrome c can be successfully digested and preconcentrated. This is the first time so many protein processing steps were integrated together on a microchip device with MS detection, which bump us closer to the final goal of a proteomic processing chip.