Synthesis and Purification of Citrullinated LL-37 Peptides

LL-37 and citrullinated forms of LL-37: LL-37₇ (1 Cit), LL-37_7,29,34 (3 Cit) and LL-37_{all cit} (5 Cit) were assembled using the Fmoc solid-phase peptide synthesis approach using either model 433A (Applied Biosystems, Foster City, CA, USA) or model Liberty (CEM Corporation, Matthews, NC, USA) automated peptide synthesizers followed by cleavage in trifluoroacetic acid (TFA)/phenol/thioanisole/ethanedithiol/water (10 mL: 0.75 g: 0.5 mL: 0.25 mL: 0.5 mL) mixture at 25°C for 90 min, see Barlow et al. for details (26 (link)). The peptides were purified by RP-HPLC (>98% purity), and their masses were confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Following lyophilisation, the peptides were obtained in the form of their trifluoroacetate salts. Stock solutions (10 mg/mL) were prepared in phosphate-buffered saline stored in aliquots at −20°C. To confirm the purity of synthetic peptides the LAL (Limulus Amebocyte Lysate) test purchased from Lonza (Germany) was performed. The sequences of LL-37 peptides, which were used in this work, are shown in Table I.