2.1. Membrane Synthesis
Cellulose-bound peptide arrays were prepared according to standard protocols using a MultiPep-RSi-Spotter (INTAVIS, Köln, Germany) employing Fmoc solid phase peptide synthesis according to the SPOT synthesis method and the manufacturer’s instructions. Peptides were synthesized either on amino-functionalized cellulose membranes (Whatman™ Chromatography paper Grade 1CHR, GE Healthcare Life Sciences #3001-878, Little Chalfont, UK) which were prepared by modifying cellulose paper by introducing Fmoc-β-Alanine as the first spacer residue [21], or on already-derivatized commercially available membranes (Amino-PEG500-UC540 Sheets optimized for use with the MultiPep instruments, INTAVIS). Cellulose-bound peptides were grown on the membranes from their C-terminus by using 0.6 M solutions of Fmoc-amino acid-OPfp (GL Biochem Ltd., Shanghai, China) in N-methyl-2-pyrrolidone (NMP, Sigma-Aldrich, Gillingham, UK, #494496) activated with 1-hydroxibenzotriazol monohydrate (HOBt, AGTC Bioproducts, Hessle, UK, #AGHOBT-H2O) and N-N′-diisopropylcarbodiimide (DIC, Fluka distributed by Sigma-Aldrich, #38370) and spotted on the membrane in 100 nL aliquots per spot using a robotic syringe. Residual amino functions were capped by acetylation between amino-acid block depositions with a 5% solution of acetic anhydride (Fisher Scientific, Loughborough, UK #A/0480/PB08) in NMP. Membranes were then washed several times with ethanol (EtOH) and NMP, before Fmoc groups were cleaved with 20% piperidine in N,N-dimethylformamide (DMF/Piperidine (80/20), AGTC, #AGDMFPIP). These steps were iteratively repeated for each amino acid within each peptide sequence. Following the last coupling step, the acid-labile protection groups of the amino acid side chains were cleaved by using a mixture of 95% trifluoro-acetic acid (TFA, Sigma-Aldrich, #T6508), 3% tri-isopropyl-silane (ACROS Organics distributed by Fisher Scientific, #214920100) and 2% water for 2 h. Membranes were then washed 4 × 30 s with dichloromethane (CH2Cl2, ATGC Bioproducts, Hessle, UK, #AGBC7002), 4 × 2 min with NMP and 2 × 2 min with 100% EtOH (absolute ethanol, Sigma-Aldrich, #32221) and were left to dry overnight. In order to ensure that each membrane had been synthesized properly, ultraviolet light (UV, λ = 280 nM) was used to ensure that all SPOTed peptides were present.