Use of o-phthalaldehyde to reduce background during automated Edman degradation

Background generated during the cleavage step of the Edman degradation is a major obstacle to extended automated amino acid sequencing. It was demonstrated recently by A. S. Bhown, J. C. Bennett, P. H. Morgan, and J. E. Mole (1981, Anal. Biochem. 112, 158-162) that introduction of fluorescamine into the spinning cup reduced background by blocking primary amino groups at cycles where a proline residue is at the exposed amino terminus. A convenient blocking reaction program using o-phthalaldehyde which can be intercalated into the sequencing format of an automated Beckman liquid-phase sequencer is presented. The advantages of o-phthalaldehyde in blocking of primary amines when proline is amino terminal arise from its stability in aqueous solution and the ease of programmed metering of delivery to the sequencer spinning cup. The blocking reaction proved successful not only in extending sequence analyses but also in the elimination of unwanted sequences in selected peptide mixtures without the necessity of purification of the target peptide.