Design of peptide standards with the same composition and minimal sequence variation to monitor performance/selectivity of reversed-phase matrices.

The present manuscript extends our de novo peptide design approach to the synthesis and evaluation of a new generation of reversed-phase HPLC peptide standards with the same composition and minimal sequence variation (SCMSV). Thus, we have designed and synthesized four series of peptide standards with the sequences Gly-X-Leu-Gly-Leu-Ala-Leu-Gly-Gly-Leu-Lys-Lys-amide, where the N-terminal is either N(α)-acetylated (Series 1) or contains a free α-amino group (Series 3); and Gly-Gly-Leu-Gly-Gly-Ala-Leu-Gly-X-Leu-Lys-Lys-amide, where the N-terminal is either N(α)-acetylated (Series 2) or contains a free α-amino group (Series 4). In this initial study, the single substitution position, X, was substituted with alkyl side-chains (Ala<Val<Ile, in order of increasing hydrophobicity) or aromatic side-chains (Phe, Tyr). Peptide series pairs 1/2 and 3/4 thus represent SCMSV peptides, with the substitution site, X, being towards the N- or C-terminal, albeit with identical adjacent residues (Gly-X-Leu) to maintain the same environment around position X. In addition, peptide series pairs 1/3 and 2/4 enable an examination of the effect of a free, positively charged α-amino group on peptide retention behaviour relative to a blocked N-terminus. Peptide mixtures were run at pH 2 on columns with a variety of stationary phase selectivity (C(8), C(18), polar endcapped, polar embedded, ether-linked phenyl and Phenyl-Hexyl) under linear gradient conditions with acetonitrile or methanol as organic modifier. It was interesting to note that the addition of the hydroxyl group to the aromatic ring in a 12-residue Tyr SCMSV peptide pair had a dramatic effect on resolution compared to the Phe peptide pair. In addition, SCMSV peptide pairs with the β-branched Val and Ile side-chains at position X were the most difficult to separate compared to SCMSV peptides containing the aromatic side-chains Tyr and Phe. In this initial study, SCMSV peptide pairs proved to be a potent test of the selectivity of reversed-phase packing materials. In addition, mixtures of SCMSV peptide standards to assess overall capabilities of stationary phases to resolve complex peptide mixtures underlined the useful complementarity of combinations of different columns and elution conditions to maximize flexibility in peptide applications. Finally, our controlled, de novo designed peptide approach should spur the development of more quantitative selectivity parameters for peptide separations, such as those already available for small molecules, enhancing further the universal value of utilizing peptide standards to compare column performances in the separation of peptide mixtures.