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. Author manuscript; available in PMC: 2012 Nov 9.
Published in final edited form as: J Am Chem Soc. 2011 Oct 19;133(44):17855–17868. doi: 10.1021/ja207220g

Figure 3.

Figure 3

ESI-MS spectra of various Ub components in the assembly of K11-linked Ub2 with the distal Ub unit both 15N-labeled and K11Boc-protected. (a) Preparation of 15N K11Boc Ub for incorporation as the distal unit in distal-15N- labeled K11-linked Ub2. The molecular weight of 15N K11Boc is 8764 Da (black). Reaction with E1 and MESNA adds a C-terminal thioester functional group to the Ub protein, increasing its molecular weight by 125 Da to 8889 Da (blue), and fully converts all Ubs into 15N-K11Boc-SR. Alloc protection of 15N-K11Boc-Ub-SR (red) adds a total of eight Alloc groups (each Alloc protecting group is 84 Da). (b) Preparation of K11Boc Ub for incorporation as the proximal Ub. The molecular weight of K11Boc Ub is 8665 Da (black) as a result of addition of the Boc protecting group (100 Da). Alloc protection (blue) adds eight Alloc groups to the protein, similar to the distal 15N-K11Boc-SR Ub protein. TFA treatment (red) removes only the Boc group on K11, reducing the molecular weight by 100 Da to 9236 Da. (c) After chemical condensation and complete Alloc deprotection, the expected molecular weight of the purified distal-15N-labeled and distal-K11Boc K11-linked Ub2 is 17310 Da as shown, resulting from the sum of one Ub (8564 Da) and one 15N-labeled K11Boc Ub (8764 Da) and the loss of one water molecule from the K11 isopeptide linkage.