Preparation of Peptides and Proteins for Sequence Analysis at the Low Nanomole to Subnanomole Level by Reverse-Phase High-Performance Liquid Chromatography: Results for Cytochromes P450 and Fibronectin

Abstract

A number of key developments have led to the routine microsequence analysis (low nanomole to subnanomole range) of peptides and proteins in a number of laboratories. In spinning cup microsequence analysis, the developments were improved instrument design and performance (1–4), better methods for solvent and reagent purification (2, 5), automated conversion of amino acid anilinothiozolinone to phenylthiohydantoin (PTH) derivatives (6), high sensitivity separation and quantitation of PTH derivatives by reverse-phase HPLC (2, 7–9), and retention of µg amounts of proteins and peptides in the spinning cup with polybrene (2, 10). The ability to perform routine microsequence analysis on µg amounts of peptides and proteins has led to structural studies on relative rare substances which possess important biological properties. In our laboratory we have used this methodology to obtain structural information on human leukocyte and fibroblast interferons (11–15) and on bovine adrenal opioid peptides (16–18). Over the past two years these studies have led us to consider another critical aspect of microsequence analysis, namely the compatibility of sample preparation with microsequence analysis.