Dionex ultimate 3000 rslc nano hplc system

For MS-based identifications, 250 µg unlabelled proteins were separated by 2D-DIGE and proteins were visualized by MS-compatible silver staining [26 (link)]. Protein spots of interest were excised manually from the gels, de-stained, disulphide was reduced as well as derivatized with iodoacetamide and the proteins were tryptically digested. Subsequently, these peptide samples were applied onto a Dionex Ultimate 3000 RSLC nano-HPLC system (Thermo Scientific) and afterward directly subjected to a QqTOF mass spectrometer oTOF compact (Bruker Daltonics) equipped with a nano-flow CaptiveSpray ionisation device. Detailed analytical conditions were previously described [27 (link)]. The protein identification was obtained with database searches against UniProtKB/Swiss-Prot (2020-06) using Mascot v2.7 server.

Cross-linking experiments were carried out using EDC (Thermo Fisher Scientific) in the presence of N-hydroxysulfosuccinimide (Sulfo-NHS, Thermo Fisher Scientific) as described before17 (link). In brief, the gel bands of cross-linked complexes were processed following standard procedures26 (link) and digested peptides were desalted using C18 StageTips27 (link)28 (link). Peptides were analysed on an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) that was coupled with a Dionex Ultimate 3000 RSLC nano HPLC system, using a high/high strategy29 (link): both MS spectra and MS2 spectra were acquired in the Orbitrap. The mass spectrometric raw data were processed to generate peak lists by MaxQuant (Version 1.5.1.2)30 (link) and cross-linked peptides were matched to spectra using Xi software (ERI, Edinburgh). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE31 (link) partner repository with the dataset identifier PXD004952.