Synthesis and Use of an Internal Amino Acid Sequencing Standard Peptide

Abstract

A sequenceable internal standard has been made that can be added to samples destined for automated amino acid sequencing and that provides a means for continuously monitoring instrument performance. The 41-residue standard peptide is composed of two unnatural amino acids, norleucine and succinyl-lysine, and has the structure [norleucine-(succinyl-lysine) 4] 8-norleucine. The phenyithiohydantoin derivatives of the latter two amino acids are well separated via reversed-phase HPLC from the phenyithiohydantoin derivatives of the 20 naturally occurring amino acids. In addition, the standard peptide can be readily synthesized and it has excellent solubility, stability, and sequencing characteristics. The placement of norleucine at every fifth residue in this peptide permits accurate repetitive yield and carryover calculations following runs that are as short as six cycles. Use of this internal standard permits the early detection of suboptimal instrument performance and is especially helpful in differentiating between two common reasons (blocked NH 2-terminus versus an instrument problem) for the failure of an automated protein sequencer to provide an NH 2-terminal sequence.