Peptide Synthesis on MIMOTOPES Pins

Peptide synthesis was carried out according to a slightly modified Geysen's procedure (PEPSCAN) (Geysen et al., 1987 ; Pyclik et al., 2018 (link)) on the NCP Block of 96 hydroxypropylmethacrylate pins (MIMOTOPES), following the one-plate-one-peptide approach. Stepwise elongation of peptides from C-end to N-end was carried out for 6 h or at night in the presence of coupling solution which contained 60 mM Fmoc corresponding to amino acids diluted in N,N'-dimethylformamide (DMF, Merck KGaA), two coupling reagents: 65 mM of 1-hydroxy-7-azabenzotriazole (Sigma-Aldrich) and 60 mM of diisopropylcarbodiimide (Merck KGaA) and 10 mM bromophenol blue (Sigma-Aldrich). After completion of the synthesis, deprotection was performed. Eventually, pins were subjected to disruption in a buffer containing 1% SDS (Sigma-Aldrich), 0.1% 2-mercaptoethanol (Thermo Fisher) and 0.1 M Na3PO4 (Sigma-Aldrich) of pH = 7.2, heated to 60°C in a sonicator (Branson 2210 DTH Ultrasonic Cleaner) and sonicated for 10 min. The disruption buffer was removed from the pins by submersion in MiliQ water warmed to 60°C for 2 min, followed by washing in MeOH (CHEMPUR) warmed to 60°C for 5 min. Afterwards, the pins were fully dried and kept in dry conditions at 4°C or −20°C.