UNIT 11.6 Reversed-Phase Isolation of Peptides
Published Online: 1 AUG 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
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.
- Published Online: 1 AUG 2001
- 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.