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.