NB4 cell lysate treated with arsenic-biotin was blocked with N-ethylmaleimide, and then the proteins were enriched using streptavidin beads. The peptides were concentrated with streptavidin after trypsin digestion and then analysed by LC-MS/MS.
The nano-LC MS/MS experiments were performed on an LTQ-Orbitrap mass spectrometer (ThermoFisher, San Jose, CA, USA) coupled with an LC-20AD nano-flow HPLC system (Shimadzu, Tokyo, Japan). The sample was separated with a PICOFRIT C18 reverse-phase column (New Objective Inc., Woburn, MA, USA) and the flow rate was 300 nL/min. The mobile phases included phase A (2% acetonitrile with 0.1% formic acid) and phase B (95% acetonitrile with 0.1% formic acid). A 90-min linear gradient from 5% to 45% phase B was used to acquire separation. The mass spectrometer was done in a data-dependent mode. Each cycle of duty consisted of one full MS survey scan in the mass range 350~1800 Da with a high resolution power Orbitrap section, followed by MS2 for the 10 strongest peaks using the LTQ section. Peptides were fragmented in the LTQ section using collision-induced dissociation (CID) with helium, and the normalised collision energy value was set at 35%.
The nano-LC MS/MS experiments were performed on an LTQ-Orbitrap mass spectrometer (ThermoFisher, San Jose, CA, USA) coupled with an LC-20AD nano-flow HPLC system (Shimadzu, Tokyo, Japan). The sample was separated with a PICOFRIT C18 reverse-phase column (New Objective Inc., Woburn, MA, USA) and the flow rate was 300 nL/min. The mobile phases included phase A (2% acetonitrile with 0.1% formic acid) and phase B (95% acetonitrile with 0.1% formic acid). A 90-min linear gradient from 5% to 45% phase B was used to acquire separation. The mass spectrometer was done in a data-dependent mode. Each cycle of duty consisted of one full MS survey scan in the mass range 350~1800 Da with a high resolution power Orbitrap section, followed by MS2 for the 10 strongest peaks using the LTQ section. Peptides were fragmented in the LTQ section using collision-induced dissociation (CID) with helium, and the normalised collision energy value was set at 35%.