title: Reversed-phase isolation of peptides.
abstract: Reversed-phase high-performance liquid chromatography (HPLC) is a fundamental tool for the isolation and analysis of peptides. Peptides are separated on a hydrophobic stationary phase and eluted with a gradient of increasing organic solvent concentration. This unit presents protocol for separation of 5- to 500-pmol of peptides on a narrow-bore (2-mm-i.d.) or microbore (1-mm-i.d.) column. Smaller quantities can be separated capillary HPLC columns, as described. Capillary HPLC columns, however, require a gradient flow rate of 3 to 5 ml/min, which most current HPLC pumps cannot attain without modifications. A procedure is therefore provided for constructing a capillary HPLC system using readily available components. HPLC peaks that appear to be symmetrical may actually contain coeluting peptides. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and capillary electrophoresis are described for analysis of a small portion of an HPLC fraction to determine the number of components present in a small sample. These methods can be utilized to screen fractions prior to automated sequencing.
authors.affiliation: Genentech, Inc, South San Francisco, California.
authors.forename: William J
authors.initials: WJ
authors.lastname: Henzel
authors.affiliation: Genentech, Inc, South San Francisco, California.
authors.forename: John T
authors.initials: JT
authors.lastname: Stults
mesh_terms: D001671:Biochemistry / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014835:Volatilization
chemical_list: D010455:Peptides