Microfluidic electrocapture technology in protein and peptide analysis

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Medical Biochemistry and Biophysics

Sammanfattning: After sequencing the genomes of several organisms, science in the postgenomic era now aims at a thorough study of the proteins present in a given tissue or organism. Since this task requires an enormous analytical effort, integrated microfluidic systems are envisioned as the solution to automated high throughput analysis of biomolecules. This thesis is focused on a microfluidic methodology and device that present several advantages over present technologies. The microfluidic device utilizes an electric field to capture molecules traveling in a flow stream. After capture, another medium is injected into the system that is of a desirable chemical composition or carries reagents, which are brought into contact with the captured molecules. The microfluidic device was employed as a concentrator for capillary electrophoresis (CE). Samples containing a mixture of proteins were concentrated and injected into a CE instrument. Detection limits were thereby improved from µM to nM protein levels. The device was further applied to desalting and removal of contaminants before MALDI-MS analysis. Polypeptides were captured followed by the injection of a solvent suitable for NIS analysis. Significant desalting and removal of CHAPS detergent was obtained for efficient analysis of peptides and proteins by MALDI-MS. In further study, the utilization of the electrocapture device to carry out microreactions is described. After the capture of a target protein, another medium containing enzymes and/or reagents is injected. Reduction, alkylation, and trypsin digestion, as well as sample cleanup, were carried out for peptide mass mapping by MALDI-MS. The use of the electrocapture device as a separation tool is also described. The separation process involves the capture and subsequent sequential release of peptides according to their electrophoretic mobility. Tryptic peptides from digestion of a mixture of proteins were separated and analyzed by MALDI-MS. A final study concerns the capture mechanism. It was found that negatively charged molecules are in fact immobilized in the flow stream due to a steady-state phenomenon created by the generation of areas with different electric field strengths along the fluidic channel. Herein we describe a flexible microfluidic device capable of processing polypeptides to resolve key analytical problems in protein and peptide analysis.

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