4.6. LC-MS and LC-MS/MS analysis

Protein digests were analysed in a Dionex U3000 nanoLC coupled to a ThermoFisher LTQ FT Ultra mass spectrometer. Samples (2 μl) were injected in load-trapping mode. Peptides were eluted using a 30 min linear gradient from the initial mobile phase A (5 % v/v acetonitrile, v/v 0.1 % formic acid) to 55 % mobile phase B (95 % v/v acetonitrile, 0.1 % v/v formic acid) followed by 5 min at 90 % B and 15 min column (Analytical: Pepmap 300 C18, 0.075×150 mm, nano-column; Trapping: Pepmap 300 C18, 0.3×10 mm, trap cartridge) re-equilibration. The LTQ FT Ultra mass spectrometer was equipped with a nanoelectrospray (nESI) source through which a 1.7 kV voltage was applied to the Picotip emitter. The inlet capillary of the mass spectrometer was held at 275 °C with a tube lens value of 145 V [21]. For labelled peptides and in order to quantify the modification of these peptides by carbine footprinting, samples were separated and data acquired in full scan mode followed by data analysis with PepFoot software [22]. Representative data, visualized with PepFoot, are shown in Fig. S14. For non-labelled protein, nanoHPLC-MS was operated in data directed analysis (DDA) mode, where a low energy survey was used to identify precursors of interest by intensity, charge state and isotope pattern. When precursors of interest were identified, MS/MS was performed on these peptides to obtain fragmentation data. Precursor peptides that had no MS/MS data were targeted in another round of experiments through targeted DDA. The data were submitted to X!Tandem search engine via SearchGUI and searched against a custom database including the wtMOMP and MOMP^T/G268 sequence.