A MICROSCALE ELECTROSPRAY INTERFACE FOR ONLINE, CAPILLARY LIQUID-CHROMATOGRAPHY TANDEM MASS-SPECTROMETRY OF COMPLEX PEPTIDE MIXTURES

A microcapillary liquid chromatography (HPLC) system designed for the gradient elution of peptide and protein samples at now rates < 1 mu L/ min has been coupled to a triple-sector quadrupole mass spectrometer v ia a simple sheathless electrospray interface (microspray). The micros pray interface used a name-drawn, uncoated, fused silica needle with t ip outer diameters in the range of 15-20 mu m and an opening less than 5 mu m in diameter. Online sample filtration to prevent clogging of t he drawn needle was accomplished by using a hydrophilic PVDF membrane filter integrated into the needle assembly. The spray potential (0.5-1 kV) was applied directly to the sample stream through the capillary u nion, Stable electrospray conditions were obtained over the full range of the gradient (0-90% acetonitrile in water) and was generally indep endent of flow rate. Both off-line and online analyses of proteins and peptide digest mixtures were performed at sample levels less than 10 fmol. HPLC parameters could be optimized for either rapid LC/MS analys is or enhanced performance in LC/MS/MS experiments by modulation of th e eluting peak widths. Additionally, flow could be greatly reduced as selected components pass through the interface to prolong the time ava ilable to collect mass spectral data. The reduced spectral background and peak width manipulation facilitated the acquisition of peptide pro duct ion spectra (MS/MS) using real-time, automated instrument control procedures.