Figure 1.

ESI-MS spectra of the various steps in the non-enzymatic assembly of K11-linked Ub₂. (a) Preparation of Ub for incorporation as the distal unit in K11-linked Ub₂ ¹⁵N- labeled on the proximal Ub. The molecular weight of wild-type Ub is 8564 Da (black). Reaction with E1 and MESNA adds a C-terminal thioester functional group to Ub, increasing its molecular weight by 125 Da to 8689 Da (blue), and fully converts all Ubs into Ub-SR. Alloc protection of Ub-SR (red) adds a total of nine Alloc groups (each Alloc protecting group is 84 Da). (b) Preparation of ¹⁵N K11Boc Ub for incorporation as the proximal Ub. The molecular weight of ¹⁵N K11Boc Ub relative to WT Ub is increased by 200 Da to 8765 Da (black) as a result of ¹⁵N isotopic enrichment (100 Da) and addition of the Boc protecting group (100 Da). Alloc protection (blue) adds eight Alloc groups to the protein, one less than for Ub-SR because of the Boc protection on residue K11. TFA treatment (red) removes only the Boc group on K11, reducing the molecular weight by 100 Da to 9336 Da. (c) After chemical condensation and complete Alloc deprotection, the expected molecular weight of purified K11-linked Ub₂ is 17210 Da as shown, resulting from the sum of one unlabeled Ub (8564 Da) and one ¹⁵N-labeled Ub (8664 Da) and the loss of one water molecule from the K11 isopeptide linkage. See also Fig. S10.