LL-37 and citrullinated forms of LL-37: LL-377 (1 Cit), LL-377,29,34 (3 Cit) and LL-37all 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 .
Model liberty
Manufactured by CEM Corporation
Sourced in United States
The Model Liberty is a versatile lab equipment designed for general laboratory use. It features a compact and durable construction with core functionalities to support various experiments and analyses. The specific details and intended applications of this product are not available at this time.
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3 protocols using model liberty
1
Synthesis and Purification of Citrullinated LL-37 Peptides
2
Solid-Phase Peptide Synthesis Protocol
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Peptides were assembled using the Fmoc/tBu solid-phase peptide synthesis approach (39 (link)) using either model 433A (Applied Biosystems, CA, USA) or model Liberty (CEM Corporation, NC, USA) automated peptide synthesizers followed by cleavage in the trifluoroacetic acid (TFA)/phenol/thioanisole/ethanedithiol/water (10:0.75: 0.5:0.25:0.5, w/w) mixture at 25°C for 90 min followed by precipitation with cold diethyl ether. The crude peptides were purified by preparative reversed-phase high-pressure liquid chromatography (RP-HPLC). The peptide purity (>98%) was confirmed by analytical RP-HPLC, and the masses were confirmed by mass spectrometry. Following lyophilization, the purified peptides were obtained in the form of their TFA salts. Stock solutions (5 mg/mL) were prepared in ultrapure DNAse/RNAse free water (Thermo-Fisher, Loughborough, UK) and stored in aliquots at −80°C. The sequences of the peptides used in this study are shown in Table 1 .
3
Solid-Phase Peptide Synthesis of Antimicrobial Peptides
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The peptides were assembled using the Fmoc/tBu solid-phase peptide synthesis approach [38] (link) using either model 433A (Applied Biosystems, CA, USA) or model Liberty (CEM Corporation, NC, USA) automated peptide synthesizers followed by cleavage in the trifluoroacetic acid (TFA)/phenol/thioanisole/ethanedithiol/water (10:0.75:0.5:0.25:0.5, w/w) mixture at 25 °C for 90 min followed by precipitation with cold diethyl ether. The crude peptides were purified by preparative reversed-phase high-pressure liquid chromatography (RP-HPLC). The peptide purity (>98%) was confirmed by analytical RP-HPLC, and the masses were confirmed by mass spectrometry. Following lyophilization, the purified peptides were obtained in the form of their TFA salts; namely: LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), LL-37 analog having “scrambled” sequence (RSLEGTDRFPFVRLKNSRKLEFKDIKGIKREQFVKIL), termed sLL-37 (control peptide). Since porcine Protegrin-1 (PG-1; RGGRLCYCRRRFCVCVGR −amide) was obtained in its reduced form, its two disulfide bridges, connecting Cys-6 and Cys-15, and Cys-8 and Cys-13, were formed by air oxidation of the HPLC purified, all-reduced peptide. SMAP-29 was synthesized in the same way as described above for LL-37. All peptides were dissolved in endotoxin-free ultrapure water (Sigma-Aldrich, UK) and stored at −20 °C until use.
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