Microsequence analysis of peptides and proteins: V. Design and performance of a novel gas-liquid-solid phase instrument

Abstract

We describe the construction and performance of a novel, automated, Edman chemistry-based microsequencer. The reagent and solvent delivery system, the reaction cartridge for coupling and cleavage, and the conversion flask are all constructed from chemically inert perfluoroelastomers. The delivery valves are of a new design incorporating the use of electromagnetically actuated solenoids and zero-dead-volume construction, and may be connected in a modular fashion resulting in multiple inputs with a single output line which can be flushed with inert gas. The bottle closures are of a new design based on an all-Teflon compression fitting. The reaction cartridge and conversion flask are thermostated by solid-state heaters in an aluminum block. The overall size of the instrument is 25 × 34 × 14 in. The chemistry utilizes 2% aqueous triethylamine as the coupling base which is delivered to the reaction cartridge via a stream of nitrogen. The “gas-phase” delivery of the coupling base and the cleavage acid (trifluoroacetic acid) is modeled after the method described by R. M. Hewick et al ( J. Biol. Chem. 256, 7990–7997, 1981). The instrument has performed well over a period of 3 years in terms of low background peaks, sensitivity in the picomole range, and reliability of operation. The use of economical components, ease of construction and operation, and sensitive analytical capability make this instrument a useful tool for microsequence analysis of peptides and proteins.