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-micromol scale. Here, we describe a continuous peptide synthesis/purification approach using the 96-well format. First, a micromol 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 micromol-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 15-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 micromol-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.
Pipkorn
R
R
German Cancer Research Center, DKFZ, Heidelberg.
Boenke
C
C
Gehrke
M
M
Hoffmann
R
R
D000097:Acetonitriles; D000818:Animals; D000953:Antigens, Protozoan / Q000276:immunology; D002625:Chemistry, Organic / Q000295:instrumentation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D000939:Epitopes / Q000276:immunology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000276:immunology; D008863:Microspheres; D010455:Peptides / Q000138:chemical synthesis* / Q000302:isolation & purification; D015203:Reproducibility of Results; D012997:Solvents; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016176:T-Lymphocyte Subsets / Q000276:immunology; D014122:Toxoplasma / Q000276:immunology; D014269:Trifluoroacetic Acid
D000097:Acetonitriles; D000953:Antigens, Protozoan; D000939:Epitopes; D008565:Membrane Proteins; D010455:Peptides; C449007:ROP9 protein, Toxoplasma gondii; D012997:Solvents; D014269:Trifluoroacetic Acid; C032159:acetonitrile