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 150-micron-i.d. fused-silica capillary tubing with a pulled 5-10-micron needle tip at one end. Polymerization at 65 degrees C followed by removal of the porogen material yielded a stable, porous, monolithic support which had excellent properties for the separation and on-line, electrospray, mass spectrometry analysis of peptides and proteins. The performance of the monolith-filled electrospray needles was compared with similar needles filled with commercial C18 silica and polymeric particulate supports. Separation efficiencies for both protein and peptide mixtures were generally equal to or better than the particulate supports at comparable pressures and flow rates. The ion chromatograms derived from the on-line MS analysis were remarkably free from 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 silver-stained gel electrophoresis. The capability of the monolith to do peak parking experiments was demonstrated by the characterization of an immunoreactive HPLC fraction. The simple fabrication method, chromatographic performance, and robust nature of these microscale integrated column electrospray sources make them ideally suited for high-sensitivity tandem LC/MS analyses.
Moore
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Division of Immunology, Beckman Research Institute of The City Of Hope, Duarte, California 91010, USA.
Licklider
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Schumann
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Lee
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D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D016208:Databases, Factual; D007475:Ion Exchange Resins; D013058:Mass Spectrometry / Q000379:methods*; D008832:Microchemistry / Q000379:methods; D008969:Molecular Sequence Data; D009339:Needles; D010455:Peptides / Q000737:chemistry*; D011137:Polystyrenes; D011506:Proteins / Q000737:chemistry*; D012117:Resins, Synthetic
D007475:Ion Exchange Resins; D010455:Peptides; D011137:Polystyrenes; D011506:Proteins; D012117:Resins, Synthetic; C003771:Amberlite XAD-2 resin