Lcq deca xpplus ion trap mass spectrometer

The multistage MS data were acquired on an Agilent 1100 series HPLC system (Agilent Technologies, Waldbronn, Germany) coupled with an LCQ Deca XP^plus ion trap mass spectrometer (IT-MS) (Thermo Finnigan, San Jose, CA, USA) via a commercial ESI interface. The instrument settings of multistage MS experiments were as follows: the scan mass range was set at m/z 100–1500 in both positive and negative ion mode with +4.00 kV and −3.00 kV source voltage, respectively. The capillary temperature was set at 350 °C, with sheath gas N₂ pressure 60 arb and auxiliary gas N₂ pressure 20 arb.
The HR-MS analyses were performed with Waters UPLC (Waters Corp., Milford, MA, USA) equipped with an AB SCIEX Triple TOF 5600^plus System (AB SCIEX, Framingham, MA, USA). The TOF-MS analysis was performed using in both positive and negative ion mode with m/z 100–1500. The conditions of ESI source were as follows: CUR, 30 psi; GS1, 50 psi; GS2, 50 psi; ISVF, −4.5 kV or +5.5 kV; TEM, 550 °C for negative ion mode and 600 °C for positive ion mode. Injection volume for aqueous extracts was set at 10 μL. A margin of error up to ±5 ppm was allowed. Both IT-MS and HR-MS data were acquired during 5–90 min with the same liquid chromatography conditions described above.

DUSP26-binding proteins were affinity-purified from extracts of HEK293T cells stably expressing 3 × Flag-tagged DUSP26. The DUSP26-binding proteins were immunoprecipitated using anti-Flag antibody-conjugated agarose beads (Sigma-Aldrich) from extracts that were washed with buffer containing 20 mM Tris–HCl, pH 7.9, 15% glycerol, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 0.05% Nonidet P40 and 150 mM KCl to remove non-specific contaminants. The bound proteins were eluted by competition with the Flag peptide (0.1 mg ml⁻¹), resolved by SDS–PAGE and prepared for LC-MS/MS analysis. Peptide samples were injected into a column by a Surveyor autosampler (Surveyor; Thermo Finnigan, San Jose, CA, USA) and separated by C18 column. The eluent was directly transferred to the electrospray ionization source of a Thermo Finnigan LCQ DecaXPplus ion trap mass spectrometer. Automated peak recognition, dynamic exclusion, and daughter ion scanning of the two most intense ions were performed and analysed by the XCALIBUR software.