Easy nano lc 2 hplc

Excised pieces of silver stained gels were destained, dried, alkylated using 55 mM iodoacetamide and later rehydrated, followed by In-Gel digestion using 250 ng mass-spectrometry grade trypsin (Promega). Following digestion the reaction mixture was acidified to 1% tri-flouroacetic acid and dried to 5 ul volume which was loaded to a 2 cm×75 um I.D. trap column of Easy nanoLC II HPLC (Thermo) for LC-MS/MS analysis. The LC was interfaced to a dual pressure linear ion trap mass spectrometer (LTQ Velos, Thermo Fisher) via nano-electrospray ionization. An electrospray voltage of 2.0 kV was applied to a pre-column tee. The mass spectrometer was programmed to acquire, by data-dependent acquisition, tandem mass spectra from the top 15 ions in the full scan from 400–1400 m/z. Dynamic exclusion was set to 30 s. Data processing and library searching was performed on Amazon Web Services-based cluster compute instances using the Proteome Cluster interface. All searches required semi-style tryptic cleavage, up to 1 missed cleavage, fixed modification of cysteine alkylation and variable modifications of methionine oxidation. XML output files were parsed and non-redundant protein sets determined using in-house scripts.

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.