Fig. 2: Synthesis of all eight neutron-encoded diUbs.
From: Neutron-encoded diubiquitins to profile linkage selectivity of deubiquitinating enzymes
a Synthesis scheme of neutron-encoded isopeptide-linked and linear diUbs using Solid Phase Peptide Synthesis (SPPS), Native Chemical Ligation (NCL) and radical desulfurization (deS). Reagents and conditions: Resin liberation and deprotection: 90.5% TFA, 5% H2O, 2% TIS, 2.5% phenol, 6-40% yield; NCL: 0.1 M TCEP, 0.15 M MPAA, 6 M Gnd∙HCl, 0.15 M sodium phosphate, pH 7.5, 37 °C; deS: 0.25 M TCEP, 0.1 M GSH, 0.075 M VA-044, 6 M Gnd∙HCl, 0.15 M sodium phosphate, pH 7.0, 37 °C, 2.5-16% yield (over two steps). (Full synthetic scheme shown in Supplementary Fig. 1). b Labelling scheme of the eight diUb isoforms. Potentially heavy-isotope labelled amino acid positions in the proximal Ub are red and marked with an asterisk. Nle Norleucine. Positions for linkage-dependent lysines to thiolysine replacements are shown in bold. The table shows the number of introduced neutron-encoded amino acids and the introduced mass difference for each linkage. The isotope labelled amino acids used are shown with the 13C and 15N atoms marked with red asterisks. c Coomassie-stained SDS-PAGE analysis of all eight neutron-encoded diUbs and used internal standards (n = 1). d Representative example of a total ion chromatogram (top), mass- (middle) and deconvoluted mass spectra (bottom) of Lys27-linked neutron encoded diUb (4c). Source data are provided as a source data file.
