Use of Short Microbore HPLC Columns for the Concentration, Separation and Recovery of Subnanomole Amounts of Protein and Polypeptides for Microsequence Analysis

Recent advances in protein sequencing technology which have led to the development of the gas phase sequenator (1) now permit protein sequence information to be obtained from as little as 10–50 picomoles of material(2–4). Although a number of high resolution techniques permit the isolation of subnanomole amounts of protein or polypeptide in a pure form (e.g. HPLC(5,6), affinity chromatography(7,8) and polyacrylamide gel electrophoresis(8–10)), there are serious problems in recovering the sample in a form suitable for gas phase sequence analysis, i.e. in a small volume and free of interfering compounds. The ability to purify and to manipulate minute quantities of protein (e.g. concentrate, reduce and alkylate, desalt, generate fragments, etc.) is crucial to the success in obtaining accurate sequence at subnanomole levels.