Beh130 prep c18 10 μm column

The peptide GRGDSPC was prepared using a Liberty Blue automated peptide synthesizer (CEM, Mathews, NC) with microwave assisted Fmoc-mediated solid phase synthesis, at 0.25 mmol scale using Rink-Amide resin (ChemPep Inc, Wellington, FL). The peptide was cleaved from the resin in cleavage cocktail (92.5% trifluoroacetic acid, 2.5% 1,2 ethanedithiol, 2.5% triisopropylsilane, and 2.5% H₂O) at room temperature for 20 mins and precipitated in cold diethyl ether. Peptide purification was performed by reverse-phase high-performance liquid chromatography (Waters Corporation, Millford, MA), using a BEH130 Prep C18 10-μm column (XBridge, Waters Corporation). Crude peptides were dissolved in Milli-Q water containing 0.1% (v/v) TFA and were filtered (0.20-μm filter, Corning Inc.) before HPLC injection. The products were subjected to an elution gradient of 100% solvent A (0.1% (v/v) TFA in Milli-Q water) to 70% solvent A within 30 min; solvent B consisted of acetonitrile with 0.1% (v/v) TFA. Peptide fractions were detected using ultraviolet-visible detection at 214 nm (Waters 2489, Waters Corporation) and collected. Target product M/Z was verified using a Waters Xevo G2-XS QTof mass spectrometer (calculated exact mass= 689.7 Da, observed [M+H]⁺= 690.8) and was stored, under argon, as a lyophilized powder at −20°C until use.

Purification was performed via reverse-phase HPLC using a BEH130 Prep C18 10 μm column (XBridge, Waters Corporation, Milford, MA). Crude peptides were dissolved in Milli-Q water containing 0.1 % (by volume) TFA and were filtered (0.20 μm filter, Corning, Inc., Corning, NY) before HPLC injection. Products were subjected to an elution gradient (Quaternary Gradient Module (Waters 2545), Waters Corporation) of 100% Milli-Q water with 0.1 %-vol TFA to 40% Milli-Q water with 0.1 %-vol TFA and 60 % acetonitrile with 0.1 %-vol TFA within 60 min. Fractions were detected using UV-Vis detection at 214 nm (Waters 2489, Waters Corporation) and collected (Waters Fraction Collector III, Waters Corporation). The collected fractions were examined by ESI-mass spectrometry (LCQ Advantage Mass Spectrometer System, Thermo Finnigan, San Jose, CA) with an auto sampler system (Surveyor Autosampler, Thermo Finnigan). Pure fractions were combined and lyophilized followed by analytical UPLC-MS (Waters Xevo G2-S QTof, Waters Corporation) to demonstrate single species purity.