A microscale electrospray interface incorporating a monolithic, poly(styrene-divinylbenzene) support for on-line liquid chromatography tandem mass spectrometry analysis of peptides and proteins

A methodology is described for creating a monolithic chromatography support within a pulled fused-silica electrospray needle, The monolith was formed from a mixture of styrene, divinylbenzene, 1-dodecanol, and toluene using 2 ,2'-azobis(isobutyronitrile) as the catalyst. The mixture was loaded into 1 50-mu m-i.d. fused-silica capillary tubing with a pulled 5-10-mu m needle t ip at one end. Polymerization at 65 degrees C followed by removal of the po rogen material yielded a stable, porous, monolithic support which had excel lent properties for the separation and on-line, electrospray, mass spectrom etry analysis of peptides and proteins. The performance of the monolith-fil led electrospray needles was compared with similar needles filled with comm ercial C18 silica and polymeric particulate supports. Separation efficienci es for both protein and peptide mixtures were generally equal to or better than the particulate supports at comparable pressures and now rates. The io n chromatograms derived from the on-line MS analysis were remarkably free f rom chemical background signals that often complicate the LC/MS analysis of femtomole amounts of sample, Good sequence coverage was obtained by LC/MS/ MS analysis of the peptide mixture obtained from a protein isolated by silv er-stained gel electrophoresis. The capability of the monolith to do peak p arking experiments was demonstrated by the characterization of an immunorea ctive HPLC fraction. The simple fabrication method, chromatographic perform ance, and robust nature of these microscale integrated column electrospray sources make them ideally suited for high-sensitivity tandem LC/MS analyses .