Microfluidic electrocapture technology in protein and peptide analysis

<p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p><p>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.</p><h3>List of scientific papers</h3><p>I. Astorga-Wells J, Swerdlow H (2003). Fluidic preconcentrator device for capillary electrophoresis of proteins. Anal Chem. 75(19): 5207-12. <br><a href="https://pubmed.ncbi.nlm.nih.gov/14708796">https://pubmed.ncbi.nlm.nih.gov/14708796</a><br><br></p><p>II. Astorga-Wells J, Jornvall H, Bergman T (2003). A microfluidic electrocapture device in sample preparation for protein analysis by MALDI mass spectrometry. Anal Chem. 75(19): 5213-9. <br><a href="https://pubmed.ncbi.nlm.nih.gov/14708797">https://pubmed.ncbi.nlm.nih.gov/14708797</a><br><br></p><p>III. Astorga-Wells J, Bergman T, Jornvall H (2004). Multistep microreactions with proteins using electrocapture technology. Anal Chem. 76(9): 2425-9. <br><a href="https://pubmed.ncbi.nlm.nih.gov/15117179">https://pubmed.ncbi.nlm.nih.gov/15117179</a><br><br></p><p>IV. Astorga-Wells J, Bergman T, Jornvall H (2004). Separation of peptides using electrocapture technology. [Manuscript]</p><p>V. Astorga-Wells J, Jornvall H, Bergman T (2004). Principle for immobilization of charged molecules in a microflow stream. [Manuscript]</p>