Reversed-phase isolation of peptides.

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• Reversed-phase high-performance liquid chromatography (HPLC) is essential for peptide isolation and analysis.
• Peptides are separated using hydrophobic stationary phases and organic solvent gradients.

Purpose of the Study:

• To present protocols for separating 5- to 500-pmol of peptides using narrow-bore and microbore HPLC columns.
• To describe the construction of a capillary HPLC system for analyzing smaller peptide quantities.
• To introduce methods for assessing the purity of HPLC fractions.

Main Methods:

• Protocol for peptide separation on 2-mm-i.d. and 1-mm-i.d. HPLC columns.
• Procedure for constructing a capillary HPLC system with readily available components.
• Analysis of HPLC fractions using Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry and capillary electrophoresis.

Main Results:

• Successful separation protocols for various peptide quantities are presented.
• A method for building a capillary HPLC system is provided, addressing flow rate limitations.
• MALDI mass spectrometry and capillary electrophoresis effectively identify coeluting peptides in HPLC fractions.

Conclusions:

• The described HPLC methods and complementary analyses enhance peptide isolation and characterization.
• The capillary HPLC system construction enables analysis of smaller peptide samples.
• These techniques are valuable for screening HPLC fractions before automated sequencing.