Miniaturized protein microsequencer with PTH amino acid identification by capillary electrophoresis I. An argon pressurized delivery system for adsorptive and covalent sequencing

摘要

The design of a simple, highly miniaturized instrument for manual microsequence analysis of proteins and peptides is described. The reaction chamber is made of fused silica" capillary tubing with all reagents and solvents necessary for coupling and cleavage delivered via two valves and a syringe-based dispenser. Only two pressure regulators are required. A section of the flow-through reaction chamber is heated by thermoelectric modules to control the sequencing reaction temperature. Conversion of theextracted amino acid product to the more stable phenyithiohy-dantoin (PTH) form is performed off-line so that it may be dissolved in 1 μ1 buffer for identification. Approximately 0.1% of this PTH product is analyzed by micellar electrokinetic capillarychromatography (MECC) with thermo-op-tical absorbance detection (TOAD), providing femtomole detection of the phenylthiohydantoin ammo acids. Preliminary results at the 50 pmol level for both adsorptive and covalent sequencing methods are presented.