Ltq velos dual pressure linear ion trap mass spectrometer

The experiments were performed using LTQ Velos dual-pressure linear ion trap mass spectrometer equipped with an electrospray ionization source (Thermo Scientific) and interfaced with an Agilent Technologies 1100 Series HPLC system. To record spectra of intact proteins, 1 μg of the protein was loaded onto a C8 cartridge column (Luna 5 μm, 20 × 2.0 mm; Fenomenex) and eluted with a linear 2–100% gradient of acetonitrile/water over 20 min at a flow rate of 0.2 ml/min. Both solvents contained 0.1% formic acid. Intact protein masses were deconvoluted either by use of ProMass for Xcaliber software (Thermo Scientific) or manually for selected peaks.

Triticale stigma proteins (90 μg) extracted with IEF loading buffer were sent to Bioproximity Proteomics Services (http://www.bioproximity.com). Briefly, the protein sample was processed using a filter-assisted sample preparation method (Wiśniewski et al., 2009 (link)), digested at a 1:40 trypsin:substrate ratio and incubated overnight at 37 °C. Digested peptides were desalted using C18 stop-and-go extraction tips (Rappsilber et al., 2007 ) and eluted with 60% acetonitrile, 0.5% acetic acid, and lyophilized. Peptides were then fractionated using an Agilent OFFGEL fractionator (Agilent Technologies) with a 24cm pH 3–10 IPG strip in conjunction with a 24-well tray according to the manufacturer’s instructions. Each fraction was desalted as above and analysed by LC-MS/MS. LC was performed using an Easy Nano-LC II HPLC (ThermoFisher Scientific) using a Zorbax 300SB-C18 column (Agilent Technologies). The LC was interfaced to a LTQ Velos Dual-Pressure Linear Ion Trap mass spectrometer (ThermoFisher Scientific) via nano-electrospray ionization. The mass spectrometer was programmed to acquire, by data-dependent acquisition, tandem mass spectra from the top 15 ions in the full scan from 400 to 1400 m/z. Dynamic exclusion was set to 30 s.