Symphony x

IRGB10 peptides (1, 2, 3 and scrambles, Supplementary Table. 3) were synthesized in the Hartwell Center of St. Jude Children’s Research Hospital using standard Fmoc chemistry on a Protein Technologies Symphony X instrument. Following synthesis, peptides were cleaved off the resin using TFA/Water/Thioanisole/TIS/Phenol/EDT and precipitated in ice cold diethyl ether. After centrifugation, peptides were dissolved in water and lyophilized. HPLC analysis was performed on a Waters Alliance 2695 separation module and mass spectrometry was recorded using MALDI on a Bruker Microflex instrument. HPLC purification was performed on a Waters Preparative HPLC system.

Sixteen Fc peptides, 15 amino acids in length (Table 1) were synthesized by Fmoc chemistry using a multiplex peptide synthesizer (Symphony X, Protein Technologies Inc., Tucson, AZ). Synthesized peptides were automatically cleaved on the synthesizer using trifluoroacetic acid. The purity of the peptides was ≥ 97% as measured by C18 reverse phase-HPLC, and the identity of the peptides was verified by mass spectrometry. A control peptide, apolipoprotein B (ApoB) 3036–3050 that stimulated Treg in atherosclerosis (11 (link)) was used as a control in experiments involving IgG+ B cell presentation to FACS-sorted, peptide-specific Treg.

Peptides were synthesized by Fmoc chemistry using a multiplex peptide synthesizer (Symphony X, Protein Technologies Inc., Tucson, AZ). Peptides were cleaved automatically on the synthesizer using trifluoroacetic acid. Peptides were ≥97% pure as assessed by C18 reverse phase HPLC, and the identity of the peptides was verified by mass spectrometry. A total of 64 peptides, each 15 amino acids in length with a 10 amino acid-overlap for each peptide, spanning the whole Fc molecule were used to define the fine specificity of Fc-specific nTreg. The amino acid sequences of the 15-mer overlapping peptides are shown in Table 3.