DOI: 10.25345/c5g15tp7c
title: MassIVE MSV000097169 - Large-scale transcriptome mining enables macrocyclic diversification and improved bioactivity of the stephanotic acid scaffold
title: For core peptide analysis, enzyme assays were performed using 50 uL volumes containing either copper(II) chloride (CuCl2) from JT Baker Chemical Co. or water. Assays for BURP-domain proteins comprised 300 uM GheBURP-1xQLFVWGW/100 uM ManBURP-1xQLFFWRY, 1 mM CuCl2, and a citrate:phosphate buffer (pH 7) with final concentrations of 18 mM citrate and 165 mM Na2HPO4. The enzyme reactions were incubated for 24 h at 20 C. Subsequently, 1 ug of trypsin (Sigma-Aldrich, T7575-1KT) in 20 uL of 1 M Tris-HCl (pH 8)/1 ug of LysC (NEB, P8109S) in 10 uL of 10 mM Tris-HCl (pH 8) buffer was added to GheBURP/ManBURP assay, respectively, and further incubated at 37 C for 24 h. Each digest was centrifuged, added to ChromacolTM LC-MS vial, and subjected to LC-MS/MS analysis. LC-MS/MS settings: Injection volume 15 uL, LC - Higgins Analytical PROTO300 C4 5 um 250 x 4.6 mm, solvent A - 0.1% formic acid, solvent B - acetonitrile (0.1% formic acid), 0.7 mL/min, LC gradient: 0 min: 10% B, 10 min: 50% B, 11 min: 95% B, 13 min: 95% B, 13.1 min: 10% B, 20min: 10% B, MS - Positive ion mode, Full MS: Resolution 35000, mass range: 700-1250 m/z for GheBURP digest and 700-1450 m/z for ManBURP digest, dd-MS2: resolution 17500, AGC target 1e5, loop count 5, isolation window 0.4 m/z, collision energy 25 eV, dynamic exclusion 2 s. Scaled enzyme assays for exoproteolytic cyclic peptide formation were conducted on a 2 mL scale using purified GheBURP-1xQLFVWGW/ManBURP-1xQLFFWRY obtained from a 4 L/2 L expression and purification process. These assays included 130-350 uM of the BURP domain cyclase and 1 mM CuCl2 in a citrate:phosphate buffer at pH 7. The assays were incubated for 24 h at 20 C. Following incubation, trypsin/LysC was introduced into the GheBURP/ManBURP reaction mixtures at a protease-substrate molar ratio of 1:250/1:100, with the pH adjusted to 8 using a 1 M Tris-HCl (pH 8) buffer and incubated at 37 C for 24 h. Two digests were subjected to preparative HPLC, respectively (LC settings, Phenomenex Kinetex 5 um C18 100 A LC Column 150 x 21.2 mm, 7.5 mL/min; solvent A, 0.1% TFA; solvent B, acetonitrile (0.1% TFA); LC gradient, 0 min, 10% B; 1 min, 10% B; 36 min, 50% B; 39 min, 95% B; 42 min, 95% B; 42.5 min, 10% B; 60.1 min, 10% B). HPLC fractions were analyzed by LC-MS analysis for target masses of modified core peptides with the following LC-MS parameters: injection volume 2 uL; LC, Phenomenex Kinetex 2.6 um C18 reverse phase 100 A 50 x 3 mm LC column; LC gradient. solvent A, 0.1% formic acid; solvent B, acetonitrile (0.1% formic acid); 0 min, 5% B; 2.5 min, 95% B; 3.0 min, 95% B; 3.1 min, 5% B; 5.0 min, 5% B, 0.5 mL/min; MS, positive ion mode; full MS, resolution 35000, mass range 400-1200 m/z; dd-MS2, resolution 17500; loop count 5, collision energy 25 eV and dynamic exclusion 0.5 s. LC fractions containing target peptides were dried and resuspended in 30 uL DMSO for further analysis. Exopeptidase assays were conducted with approximately 25-50 ug of peptides (with 4% DMSO), carboxypeptidase Y (from S. cerevisiae, Sigma-Aldrich), and aminopeptidase N (from Rat, Sigma Aldrich) at a peptidase:peptides ratio of 1:10 (w/w). These assays were performed in 100 mM Tris-HCl buffer at pH 7 and 37 C for 24 h. Following incubation, the assays were centrifuged and analyzed by LC-MS/MS as follows: Injection volume 15 uL, LC - Higgins Analytical PROTO300 C4 5 um 250 x 4.6 mm, all gradients: solvent A - 0.1% formic acid, solvent B - acetonitrile (0.1% formic acid), 0.7 mL/min, LC gradient: 0 min: 15% B, 60 min: 40% B, 61 min: 95% B, 63 min: 95% B, 63.1 min: 15% B, 80 min: 15% B, MS - Positive ion mode, Full MS: Resolution 35000, mass range: 800-1300 m/z for GheBURP and 700-1450 m/z for ManBURP, dd-MS2: resolution 17500, AGC target 1e5, loop count 5, isolation width 0.4 m/z, collision energy 25 eV, dynamic exclusion 0.6 s. The analysis was compared to purified glechomanin/mercurialin standards.