Isotope-labeled hIAPP samples were synthesized by Fmoc-based chemistry using an automated microwave peptide synthesizer (LibertyBlue, CEM) on PAL–PEG–PS resin according to published protocols.51,57 (link) Peptides were cleaved off resin with a microwave-assisted cleavage system (Accent, CEM) using TFA : TIS : H2O (18 : 1 : 1 v/v/v) solution. To form a disulfide bond between the N-terminal cysteines, crude peptide was dissolved in DMSO and kept at room temperature for 24 hours. Following the oxidation, the peptide was purified with reversed-phase HPLC using C18 preparative column (Waters XSelect) using a two-buffer purification gradient. Buffer 1 was composed of 0.045% HCl in H2O, and buffer 2 of 80% CH3CN, 20% H2O and 0.045% HCl (v/v). Purification was run with a gradient, increasing the percentage of buffer 2 by 1% per minute. Purity of the peptides were assessed by integrating the 220 nm peptide backbone peak versus all other impurities visible on the HPLC chromatogram; peptide identity was confirmed via MALDI. All peptides used for experiments were >95% purity. All measurements were carried out at 1 mM peptide concentration.
C18 preparative column
Manufactured by Waters Corporation
The C18 preparative column is a liquid chromatography column designed for the purification and separation of a variety of compounds. It features a C18 stationary phase, which is widely used for the separation of non-polar and moderately polar analytes. The column is suitable for preparative-scale applications, allowing for the efficient isolation and concentration of target compounds from complex mixtures.
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2 protocols using c18 preparative column
1
Synthesis of Isotope-Labeled hIAPP Peptides
2
Synthesis and Purification of Isotope-Labeled hIAPP
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Isotope-labeled hIAPP was synthesized by Fmoc solid-phase peptide synthesis on PAL–PEG–PS resin according to published protocols.41 (link),42 Synthesized peptides were cleaved off resin with a microwave-assisted cleavage system (Accent, CEM) using TFA : H2O : TIS (18 : 1 : 1, v/v/v) mixture. Crude peptide was dissolved in dimethyl sulfoxide (DMSO) for 24 hours to form a disulfide bond between Cys2 and Cys7. The oxidized peptide was then purified with reversed-phase HPLC using C18 preparative column (Waters XSelect). Buffer A consisted of 0.045% HCl in H2O and buffer B of 80% acetonitrile, 20% water and 0.045% HCl (v/v). Purification was run with a gradient of 1% buffer B per minute. All measurements were carried out at 1 mM peptide concentration.
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