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(2) Add 300 L of protein precipitant methanol-acetonitrile (V:V=2:1, containing mixed internal standard, 4 g/mL), vortex and shake for 1 min

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The samples were subsequently reduced with 4 mM DTT for 30 min at room temperature with shaking at 1,000 rpm, followed by alkylation with 10 mM iodoacetamide for 45 min in the dark at room temperature while shaking at 1,000 rpm

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