U 3000 nanoflow hplc system

A U3000 nanoflow HPLC system (ThermoFisher, Hemel Hempstead, UK) was directly connected to the mass spectrometer [maXis^TM UHR-Qq-ToF (Bruker Daltonic, Bremen, Germany)], fitted with an EZ Nanoflow Electrospray needle. The U3000 HPLC system was equipped with a nanoLC analytical column [75 μm × 15 cm packed with C18 material, 5 μm, 100 Å particles (LC Packings, CA, USA)] and micro precolumn [300 μm i.d. × 5 packed with C18 material, 5 μm, 100 Å particles (LC Packings, CA, USA)] at flow rates of 300 nl min⁻¹ and 30 μl min⁻¹ respectively. A linear HPLC gradient using buffer A [97% (v/v) HPLC water, 3% (v/v) HPLC acetonitrile with 0.1% (v/v) formic acid] and buffer B [97% (v/v) HPLC acetonitrile, 3% (v/v) HPLC grade water with 0.1% (v/v) formic acid], using a 120 min programme, was applied as follows: 0% B (0–5 min), 0–35% B (5–95 min), 35–100% B (95–101 min), 100% B (101–106 min), 0% B (106–120 min). Acquired data were analysed initially using EasyProt (Gluck et al., 2013 (link)) and then further analysed manually using the mass of precursor/fragment ions predicted from in silico digests using the protein product tool on the Protein Prospector website (http://prospector.ucsf.edu/prospector/mshome.htm).