Mass Spectrometry Analysis

3.4.1. In-gel Digestion

For staining gels, CBB dye was removed by rinsing three times in 60% methanol, 50 mM ammonium bicarbonate, and 5 mM dithiothreitol (DTT) for 15 min and twice in 50% acetonitrile, 50 mM ammonium bicarbonate, and 5 mM DTT for 10min. Gel pieces were dehydrated three times in 100% acetonitrile for 30 min and then rehydrated in an in-gel digestion reagent containing 10 |xg/ml sequencing-grade modified trypsin (Promega, Madison, WI) in 30% acetonitrile, 50 mM ammonium bicarbonate, and 5mM DTT In-gel digestion were performed overnight at 30°C. The samples were rinsed in 30% acetonitrile, 50 mM ammonium bicarbonate, and 5mM DTT for 2hr and lyophilized overnight at -30°C.

3.4.2. Amino Acid Sequencing by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Lyophilized samples were dissolved in 20 |xl 0.1% formic acid and centrifuged at 15,000 g for 5 min. Peptide sequencing of identified protein spots was performed by

LC-MS/MS with a Spectrum Mill MS Proteomics Workbench (Agilent Technologies, Santa Clara, CA).

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