UNIT 11.6 Reversed-Phase Isolation of Peptides

  1. William J. Henzel,
  2. John T. Stults

Published Online: 1 AUG 2001

DOI: 10.1002/0471140864.ps1106s24

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Henzel, W. J. and Stults, J. T. 2001. Reversed-Phase Isolation of Peptides. Current Protocols in Protein Science. 24:11.6:11.6.1–11.6.16.

Author Information

  1. Genentech, Inc., South San Francisco, California

Publication History

  1. Published Online: 1 AUG 2001
  2. Published Print: JUN 2001


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.