High-throughput peptide synthesis and peptide purification strategy at the low micromol-scale using the 96-well format

The increasing demand for short- and medium-sized peptides in many fields of biological, medical and pharmaceutical research requires optimized and universally applicable high-throughput synthesis and purification techniques at the low-mumol scale. Here, we describe a continuous peptide synthesis/purification approach using the 96-well format. First, a mumol scale peptide synthesis on resin beads was optimized on a novel miniaturized 96-reaction vessel block employing standard Fmoc/tBu-chemistry. Almost 90% of the synthesized peptides contained the target sequence as the main component, as judged from matrix-assisted laser desorption/ionization (MALDI) mass spectra. Impurities were mostly related to partially protected peptides. Second, we tested the applicability of ion pair reversed-phase solid-phase extraction (IP-RP-SPE) to purify individual peptides. Depending on the length and predicted hydrophobicity of the peptides, elution was performed with 25 or 35% aqueous acetonitrile in the presence of 0.1% trifluoroacetic acid (TFA). Thus, scavengers used during TFA cleavage and partially protected peptides carrying very hydrophobic protecting groups were effectively removed. Using a narrow step gradient, the target peptides were even separated from deleted sequences and protected peptides with similar hydrophobicities. Third, we combined the mumol-scale synthesis in the 96-well format with purification by IP-RP-SPE on a 96-well micro-extraction plate format. This simple, fast and parallel approach was tested on 12-mer and 13-mer peptides to map epitopes of T- and B-cell clones, respectively. Approximately 80% of all peptides were obtained at purities >90% without purification by RP-HPLC. In summary, this novel approach has several advantages: (i) the mumol-scale reduced the cost of peptide synthesis, (ii) large numbers of peptides were purified faster, (iii) the volumes of eluents and waste were significantly reduced, and (iv) the RP-HPLC column was not contaminated with hydrophobic impurities.