REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY FOR FRACTIONATION OF ENZYMATIC DIGESTS AND CHEMICAL CLEAVAGE PRODUCTS OF PROTEINS

This chapter discusses reversed-phase high-performance liquid chromatography for fractionation of enzymatic digests and chemical cleavage products of proteins. The extremely high resolving power and speed of reversed-phase high-performance liquid chromatography (HPLC) make it the current method of choice for fractionating complex mixtures of peptides derived from the enzymatic and chemical cleavage of proteins. With its high peak capacity, reversed-phase HPLC can readily bring about a 100- to 125-fold purification of a typical tryptic peptide with a gradient time of only about 90 min. Although reversed-phase HPLC provides an invaluable tool for the protein chemist, its utility is enhanced even further by coupling it with mass spectrometry. A reversed-phase HPLC tryptic peptide/ fast atom bombardment (FAB) mass spectrometric approach provides an elegant and rapid means to accurately determine the molecular weights of the resulting peptides. This information can, in turn, be used to rapidly verify the primary structures of proteins that have been deduced from their DNA sequences. © 1990, Elsevier Inc. All rights reserved.