Application of capillary reversed-phase high-performance liquid chromatography to high-sensitivity protein sequence analysis.
A continuous gradient elution method for capillary column (less than 0.32 mm I.D.) liquid chromatography was developed. Gradient eluent from a microbore liquid chromatograph was split ahead of the injector so that an accurate percentage (2-3%) of the mobile phase delivered by the pump flowed through the capillary column. The outlet of the column was connected to a length of 0.075 mm I.D. fused-silica capillary tubing which, in turn, was connected to a 6-mm optical path length longitudinal capillary flow cell. Fused-silica capillary columns of 0.32 mm I.D. were slurry-packed efficiently with 7-microns spherical, 300 A pore size, C8 bonded-phase particles, and evaluated in terms of their ability to resolve mixtures of proteins, peptides or phenylthiohydantoin (PTH)-amino acid derivatives. The gradient elution profiles agreed with those obtained using microbore (less than 2.1 mm I.D.) and larger bore columns. The minimum detectable amounts for proteins and PTH-amino acids on 0.32 mm I.D. capillary columns were 50 pg and 25 fmol, respectively. At a flow-rate of 3.6 microliters/min, proteins and peptides were recovered from the capillary columns in volumes of about 2-8 microliters. The use of a multiple-wavelength, forward-optics detector for identifying tryptophan- and tyrosine-containing peptides is discussed.
Moritz
R L
RL
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch), Parkville, Victoria, Australia.
Simpson
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D015850:Interleukin-6 / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet
D015850:Interleukin-6; D011506:Proteins; D011994:Recombinant Proteins