Adaptation of reverse-phase high-performance liquid chromatography for the isolation and sequence analysis of peptides from plasma amyloid P-component

Abstract

Gel permeation and reverse-phase high-performance liquid chromatography were used to isolate chemical and enzymatic cleavage fragments of plasma amyloid P-component for amino acid sequence analysis. Optimal conditions for resolution of peptide mixtures were predetermined using analytical amounts (0.04–0.1 nmol) and volatile trifluoroacetic acid-acetonitrile or ammonium acetate-acetonitrile buffer systems. Thereafter, 100–200 nmol of each hydrolyzate was chromatographed on preparative columns. Size-exclusion chromatography using an acetic acid solvent containing n-propanol was found to be most useful for large-molecular-weight cyanogen bromide peptides while reverse-phase chromatography was best suited for the smaller enzymatically derived peptides. The high resolution and sensitivity of high-performance liquid chromatography using this dual approach has enabled the completion of greater than 95% of the sequence of P-component ( M r 23,500) using less than 10 mg.