Microsequence analysis: I. Peptide isolation using high-performance liquid chromatography.

Peptide mixtures, arising from such diverse sources as tissue extracts, urine or proteolysis, are usually fractionated and characterized by a combination of ion-exchange chromatography, gel-exclusion chromatography and/or electrophoresis. Appropriate combinations of these methods will normally yield a homogeneous product, but their costs, detection limits, analyses times and yields vary. The recently introduced technique of high-performance liquid chromatography is now being adapted to peptideprotein fractionation by numerous laboratories. The great versatility of this technique rests in the various column materials available for normal-phase adsorption (silica), reverse-phase adsorption (hydrocarbon bonded to silica) or ion exchange (ionic groups bonded to silica or polystyrene). To date proteins and large peptide fragments have been separated using both ion exchange (CM-glycophase/CPG [ 1 ] and DEAE-glycophase/CPG [2]) and molecular or steric exclusion supports (glycophase G/CPG [ 1,2]). Smaller peptides resulting from enzymatic hydrolyses [ 1,3-71, solid-phase syntheses [8] and tissue extractions [5,6,9,10] have been chromatographed and/or isolated using reverse phase Cl 8 columns and either isocratic or gradient elution systems. The detection systems, in most cases absorbance, limit both the buffer systems which can be employed and also the sensitivity. These restrictions