On-chip solid phase extraction and enzyme digestion using cationic PolyE-323 coatings and porous polymer monoliths...

We evaluate the compatibility and performance of polymer monolith solid phase extraction beds that incorporate cationic charge, with a polycationic surface coating, PolyE-323, fabricated within microfluidic glass chips. The PolyE-323 is used to reduce protein and peptide adsorption on capillary walls during electrophoresis, and to create anodal flow for electrokinetically driven nano-electrospray ionization mass spectrometry. A hydrophobic butyl methacrylate-based monolithic porous polymer was copolymerized with an ionizable monomer, [2-(methacryloyloxy)ethyl] trimethylammonium chloride to form a polymer monolith for solid phase extraction that also sustains anodal electroosmotic flow. Exposure of the PolyE-323 coating to the monolith forming mixture affected the performance of the chip by a minor amount; electrokinetic migration times increased by ∼5%, and plate numbers were reduced by an average of 5% for proteins and peptides. 1-mm long on-chip monolithic solid phase extraction columns showed reproducible, linear calibration curves (R(2)=0.9978) between 0.1 and 5 nM BODIPY at fixed preconcentration times, with a capacity of 2.4 pmol or 0.92 mmol/L of monolithic column for cytochrome c. Solution phase on-bed trypsin digestion was conducted by capturing model protein samples onto the monolithic polymer bed. Complete digestion of the proteins was recorded for a 30 min stop flow digestion, with high sequence coverage (88% for cytochrome c and 56% for BSA) and minimal trypsin autodigestion product. The polycationic coating and the polymer monolith materials proved to be compatible with each other, providing a high quality solid phase extraction bed and a robust coating to reduce protein adsorption and generate anodal flow, which is advantageous for electrospray.