FABRICATION OF STABLE PACKED CAPILLARY REVERSED-PHASE COLUMNS FOR PROTEIN STRUCTURAL-ANALYSIS

Capillary column (less than or equal to 320-mu m ID) liquid chromatogr aphy is an essential tool for the separation and concentration of low- picomole amounts of proteins and peptides for mass-spectrometric based structural analysis. We describe a detailed procedure for the fabrica tion of stable and efficient 50- to 180-mu m ID polyimide fused-silica columns. Columns were packed by conventional slurry packing with reve rsed-phase silica-based supports followed by column bed consolidation with acetonitrile and sonication. PVDF membrane or internal fused-sili ca particles were employed for column end-frit construction. The abili ty of these columns to withstand high back pressures (300-400 bar) ena bled their use for rapid chromatography (>3400 cm/hr; i.e., similar to 40 mu l/min for 200-mu m ID columns) and the loading of large sample volumes (up to 500 mu l) The accurate low flow rates (0.4-4.0 mu l/min ) and precise gradient formation necessary to operate these columns we re achieved by a simple modification of conventional HPLC systems [Mor itz et al. (1992), J. Chromatogr. 599, 119-130]. Column performance wa s evaluated for ability to resolve low-fmol amounts of all components of a mixture of PTH-amino acids and to separate peptides for on-line L C/MS analysis of peptide mixtures derived from in situ digestion of 2- DE resolved protein spots.