For each frozen sample, total protein extraction, qualification and digestion were performed as the method described in our previous study [28 (link)]. The digested peptides were labeled following the manufacturer’s protocol with iTRAQ® Reagent 8-plex Kit (AB SCIEX, USA) and subsequently used for LC-MS/MS analyses using an AB SCIEX TripleTOF™ 5600 mass spectrometer (AB SCIEX, USA), coupled with an LC-20AB HPLC Pump system (Shimadzu, Kyoto, Japan).
MS/MS data acquisition was performed with Analyst®QS2.0 software (AB SCIEX, USA), and processed by searching against the database generated from the annotated transcriptome using the Paragon™ Algorithm and the Mascot search engine (Matrix Science, London, UK; version 2.3.02). The relative abundance was analyzed by the report ion peak areas as previously described [71 (link)]. For protein quantitation, it was required that a protein contains at least two unique peptides.
MS/MS data acquisition was performed with Analyst®QS2.0 software (AB SCIEX, USA), and processed by searching against the database generated from the annotated transcriptome using the Paragon™ Algorithm and the Mascot search engine (Matrix Science, London, UK; version 2.3.02). The relative abundance was analyzed by the report ion peak areas as previously described [71 (link)]. For protein quantitation, it was required that a protein contains at least two unique peptides.
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