Proxeon nanospray

‘Bottom-up’ liquid chromatography-coupled tandem MS (LC-MS/MS) was used to elucidate the structure of isolated peptides and to detect post-translational modifications. LC-MS/MS analysis was carried out using an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) coupled to an UltiMate 3000 UHPLC Nano (Thermo Fisher Scientific), Aurora C18 column (IonOpticks), column oven (maintained at 50°C; Sonation) and Proxeon Nanospray ion source (Thermo Fisher Scientific). Peptides (1 µg) were injected onto an Aurora C18 column in buffer A (2% (v/v) acetonitrile, 0.5% (v/v) acetic acid) and eluted with a linear 120-min gradient of 2%–45% (v/v) buffer B (80% (v/v) acetonitrile, 0.5% (v/v) acetic acid). Eluting peptides were ionised in positive-ion mode before data-dependent analysis. A dynamic exclusion window of 30 s was enabled and lockmass was not used.

The procedure was essentially carried out as indicated in earlier work (16 (link)). In brief, following trypsin digestion, peptides were further cleaned using a C18 column and then injected into the UltiMate 3000 RSLC nano HPLC (Thermo Fisher Scientific). This HPLC system is linked to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) using a Proxeon Nanospray source (Thermo Fisher Scientific).