In-gel digestion of gel-separated proteins was carried out with trypsin, as previously described (38). After digestion and extraction from the gel pieces, the digested peptides were desalted and concentrated by reversed-phase chromatography onto C18 Stage Tip microcolumns (39). Peptides were then eluted from the stage tips with buffer B (80% acetonitrile and 0.5% acetic acid), lyophilized, resuspended in 0.1% formic acid (FA), and subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis.

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