Extended Data Fig. 6 ∣. Identification of TTLL6 MTBH1-2-microtubule interaction sites using cross-linking coupled with tandem mass spectrometry.

a. Chemical reaction showing the cross-linking of primary amine and carboxylic acid in the presence of 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and sulfo N-hydroxysuccinimide (sulfo-NHS). b,c. Representative SDS-PAGE gels from one of two independent experiments showing the cross-linking of MTBH1-2 to microtubules (b) but not to soluble tubulin (c). The cross-linked products are outlined with a magenta rectangle. M, mock. d–h. MS/MS sequencing of the cross-linked products. Peptides were fragmented using EThcD decision tree method, so majority of the MS/MS fragments are c- and z-ions, with a small portion of b- and y-ions. The cross-linked product contains 2 peptides. The sequences of the peptides are shown in the spectrum and labeled as (α) or (β) peptides. When a MS/MS fragment contains cross-linked (α) and (β) peptides, it is labeled as normal c- or z-ion. When a fragment is from (α) or (β) peptides alone, α or β is then included in the labeling of that ion, such as [z12α+1] or c3β. The cross-link sites are highlighted in red. Possible cross-linked residues are underlined when it is not possible to locate the precise site.