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After centrifugation (12,000 g , 15 min, 4 C), protein concentrations were determined using a BCA assay (CWBIO, China)

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The general workflow for peptide sequencing using MS/MS is as follows: Precursor Ion Selection: In the first mass analyzer (MS1), peptides from a complex mixture are separated, and a specific peptide ion (the "precursor ion") is selected based on its mass-to-charge ratio (m/z)

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