Compact Protein Sequencer for the C-Terminal Sequence Analysis of Peptides and Proteins

Abstract

We describe the construction of a compact protein sequencer designed specifically for the C-terminal sequence analysis of peptides and proteins. This sequencer has a vertical flow path and is equipped with a continuous flow reactor (CFR). The flow paths for the various reagents and solvents have been minimized. A unique feature of this instrument is the design of a quadrate valve (quad valve) which permits the delivery of four solvents or reagents to the conversion flask (CF). Combination of two of these quad valves in series permits the delivery of eight solvents and reagents to the CFR. The CF contains three inputs from the top, one for transfer of the contents of the CFR, one which is used as a vent, and one for input of solvents or reagents from the CF quad valve. The CF drains from the bottom, connecting to a switching valve which allows delivery either to a waste bottle or to an on-line HPLC. Another unique feature of this instrument is the design of an optical flow detector which permits injection of approximately 90% of the contents of the CF for HPLC analysis. The overall size of the instrument (11 w × 16.5 h × 23.5 d in.) is smaller than commercially available instruments for protein sequencing and represents the first time an instrument has been constructed specifically for C-terminal sequence analysis. The utility of this instrument is demonstrated with the C-terminal sequence analysis of protein samples noncovalently applied to Zitex strips and with a peptide covalently attached to carboxylic acid-modified polyethylene film.