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

✍ Scribed by Jacqueline K. Anderson; John E. Mole

• Publisher: Elsevier Science
• Year: 1982
• Tongue: English
• Weight: 740 KB
• Volume: 123
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(82)90466-3

✦ Synopsis

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-o. 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-molecularweight 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, 23,500) using less than 10 mg.