12 channel multiplex peptide synthesizer

Peptides were synthesized using the stepwise solid-phase method by 9-fluorenylmethoxycarbonyl (Fmoc) chemistry on Wang resin (AnaSpec, Fremont, CA, USA) with a 12-channel multiplex peptide synthesizer (Protein Technologies, Tucson, AZ, USA) according to the manufacturer’s procedures. Detachment of peptide from the resin and removal of the side chain protection groups were done by incubating the resin with a mixture of trifloroacetic acid (TFA):Thioanisole:Water:Phenol:1,2-ethanedithio (82.5:5:5:5:2.5 v/v) for 2 hours.
The crude peptide was purified on a preparative Kinetex reversed-phase C18 column, 150 × 21.1 mm (Phenomenex, Torrance, CA, USA) using a BioCad Sprint (Applied Biosystems, Foster City, CA, USA). A flow rate of 30 mL/min with solvent A (0.1% TFA in deionized water) and solvent B (0.1% TFA in acetonitrile) was used. The column is equilibrated with 5% solvent B before sample injection. Elution is performed with a linear gradient from 5% solvent B to 100% solvent B in 60 min. The absorbance of the column effluent is monitored at 214 nm, and peak fractions are pooled and lyophilized.
The pure peptide fraction is identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) or electrospray ionization mass spectrometry (ESI-MS) and lyophilized.

Peptide was synthesized using a stepwise solid-phase method using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry on a Wang resin (AnaSpec, Fremont, CA) with a 12 channel multiplex peptide synthesizer (Protein Technologies, Tucson, AZ) as described previously (26 ). Briefly, peptide synthesis started from the C-terminus. The Fmoc group of the resin was removed with 20% piperidine in N, N-Dimethylformamide (DMF) (5 min x2) followed by washing the resin with DMF (6.5 min). After completion, the N-terminal Fmoc was removed with 20% piperidine in DMF (5 min x 2) followed by washing the resin with DMF (6.5 min). Resins were mixed with 5 mL 50% chloroform in DMF containing 5 g myristic anhydride and kept at 60°C for 1 hr. The crude peptide was then purified on a preparative Kinetex reversed-phase C18 column (Phenomenex, Torrance, CA) using a BioCad Sprint analyzer (Applied Biosystems, Foster City, CA). Elution was performed with a linear gradient from 5% solvent B to 100% solvent B in 30 min. The absorbance of the column effluent was monitored at 230 nm and peak fractions were pooled and lyophilized. The pure peptide fraction was identified by electrospray ionization mass spectrometry (ESI-MS) and lyophilized. Finally, peptides were dissolved in DMSO and 10 mM aliquots stored at −80°C.