2.4 Liquid Chromatography with tandem mass spectrometry (LC-MS/MS)
LC-MS/MS analysis was performed by commercial service of Proteomics International Company, Australia. Protein samples were trypsin digested and peptides extracted according to standard techniques (Bringans et al., 2008). Peptides were analyzed by electrospray ionization mass spectrometry using the Shimadzu Prominence nano HPLC system (Shimadzu, Japan) coupled to a 5600 TripleTOF mass spectrometer (Sciex, USA). Tryptic peptides were loaded onto an Agilent Zorbax 300SB-C18, 3.5 µm (Agilent Technologies, USA) and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v). Spectra were analyzed to identify proteins of interest using Mascot sequence matching software (Matrix Science, UK) with UniProt database.