Ni nta ff columns

For the human Rab5a construct that was used for maleimide labelling, a WT Rab5a (1–212) fragment was mutated into Q79L, also surface-exposed cysteines were mutated to serines (C19S-C63S) so that one cysteine was left at the C terminus of the protein (plasmid pOP823 His-SUMO-Rab5a(1–212)-Q79L-C19S-C63S). Protein was overexpressed in E. coli C41(DE3)RIPL purified by Ni-NTA FF columns (GE Healthcare 17-5255-01), followed by the removal of the His-tag with SUMO protease, dialysis overnight (10 kDa MWCO, SnakeSkin™ ThermoFisher) and another passage through Ni-NTA resin. The flow-through was concentrated and mixed with 11 molar excess of GTP (Jena bioscience NU-1012) or GDP (Jena bioscience NU-1172) and 18 molar excess of EDTA for 90 min at room temperature. The MgCl₂ was then added to 36 molar excess and incubated for another 30 min. The mixture was loaded on a size-exclusion chromatography and the peak fractions were concentrated to ~0.5–1 mM in 25 mM HEPES pH 7.0, 150 mM NaCl, 0.5 mM tris-(2-carboxyethyl) phosphine (TCEP).
For HXD-MS, a Rab5a-Q79L construct was designed with last 4 residues (CCSN) deleted (plasmid pYO1261 His-SUMO-Rab5a(1-211)-Q79L) and was purified as above.

BrCO6K was synthesised as described above. In contrast to previously reported BrC6K^{24 (link)}, BrCO6K bears a carbamate moiety linking the ε-amino group of lysine to the bromoalkyl functionality, making it stable against the E. coli deacylase CobB. The expression of Rab5a (plasmid STp6, pBAD_Rab5a_Q79L_1-212_C19S_C63S_S84TAG-His6) with site-specifically incorporated BrCO6K was carried out as described previously^{24 (link)}. Protein was overexpressed in DH10B cells (Invitrogen, 18290015) and purified by Ni-NTA FF columns (GE Healthcare 17-5255-01). The protein was concentrated, loaded with nucleotide/MgCl₂ as described above and further purified size-exclusion chromatography. The peak fractions were concentrated to ~0.5–1 mM in 25 mM HEPES pH 8.0, 150 mM NaCl, 0.5 mM tris-(2-carboxyethyl) phosphine (TCEP).