Miniaturized membrane-based reversed-phase chromatography and enzyme reactor for protein digestion, peptide separation, and protein identification using electrospray ionization mass spectrometry.

Abstract

A commonly used capillary fitting is employed for housing miniaturized membrane chromatography for performing reversed-phase peptide separations. By placing a hydrophobic and porous polyvinylidene fluoride membrane around the end of a polymer sleeve, the assembly of capillary fitting not only provides the stationary phase, but also establishes the necessary flow paths using capillary connections. The miniaturized membrane chromatography system is coupled with a micro-enzyme reactor containing immobilized trypsins for performing rapid protein digestion, peptide separation, and protein identification using electrospray ionization mass spectrometry. Separation performance of cytochrome c digest in miniaturized membrane chromatography is compared with the results obtained from micro-LC and capillary LC. The efficacy and the potentials of miniaturized membrane chromatography in tryptic mapping are reported. The use of miniaturized membrane chromatography allows significant reduction in sample consumption together with enhanced detection sensitivity. By minimizing the void volume in miniaturized membrane chromatography, the elution times of cytochrome c peptides are significantly shortened in this study in comparison with our previous results, and are comparable with those in micro-LC and capillary LC using considerably higher mobile phase flow-rates.