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. 2018 May 14;5(7):1800234. doi: 10.1002/advs.201800234

Figure 5.

Figure 5

Synthesis and characterization of K33‐/K11‐linked Mixed Ub chain 17: A) Overall synthetic route; B) analytical HPLC chromatogram and observed ESI‐MS spectrum of purified 17 (34+, 755.6 Da; 32+, 802.8 Da; 30+, 856.3 Da; 28+, 917.4 Da; 26+, 987.8 Da; 24+, 1070.1 Da; 22+, 1167.2 Da; 20+, 1283.9 Da; and 18+, 1426.4 Da); inset: deconvoluted of ESI‐MS spectrum of 17 (observed mass = 25 655.5 Da, calculated mass = 25 657.5 Da, average isotopes); C) FPLC chromatogram; D) CD spectra; E) crystal structure of K33‐/K11‐linked triUb 17; F) Ub molecule within one asymmetric unit; Lys11 is shown as a purple ball, and Lys33 is shown as a red ball. Reactions and conditions: activation: 6 m Gn‐HCl, 10 equiv. NaNO2, pH 3.0; ligation: 6 m Gn‐HCl, 40 equiv. MPAA, pH: 6.4; de‐Acm: 60 equiv. PdCl2, pH 6.4; desulfurization: 6 m Gn‐HCl, 500 × 10−3 m TCEP, tBuSH, VA‐044, pH: 6.9. HPLC (λ = 214 nm).