Fabrication of stable packed capillary reversed-phase columns for protein structural analysis.
Capillary column (< or = 320-micron ID) liquid chromatography 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 fabrication of stable and efficient 50- to 180-micron ID polyimide fused-silica columns. Columns were packed by conventional slurry packing with reversed-phase silica-based supports followed by column bed consolidation with acetonitrile and sonication. PVDF membrane or internal fused-silica particles were employed for column end-frit construction. The ability of these columns to withstand high back pressures (300-400 bar) enabled their use for rapid chromatography (> 3400 cm/hr; i.e., approximately 40 microliters/min for 200-micron ID columns) and the loading of large sample volumes (up to 500 microliters). The accurate low flow rates (0.4-4.0 microliters/min) and precise gradient formation necessary to operate these columns were achieved by a simple modification of conventional HPLC systems [Moritz et al. (1992), J. Chromatogr. 599, 119-130]. Column performance was 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 LC/MS analysis of peptide mixtures derived from in situ digestion of 2-DE resolved protein spots.
Tong
D
D
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch), Parkville, Victoria, Australia.
Moritz
R L
RL
Eddes
J S
JS
Reid
G E
GE
Rasmussen
R K
RK
Dorow
D S
DS
Simpson
R J
RJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010669:Phenylthiohydantoin / Q000737:chemistry; D011145:Polyvinyls; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry*; D005465:Fluorocarbon Polymers
D000596:Amino Acids; D008567:Membranes, Artificial; D010669:Phenylthiohydantoin; D011145:Polyvinyls; D011506:Proteins; D012822:Silicon Dioxide; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers