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Proteomics sample preparation was done according to a published protocol with minor modifications [113]. Approximately 2 × 107 cells were lysed under denaturing conditions in a buffer containing 3 M guanidinium chloride (GdmCl), 5 mM tris (2-carboxyethyl) phosphine, 20 mM chloroacetamide, and 50 mM Tris-HCl pH 8.5. Lysates were denatured at 95 °C for 10 min shaking at 1000 rpm in a thermal shaker and sonicated in a water bath for 10 min. A small aliquot of cell lysate was used for the bicinchoninic acid (BCA) assay to quantify the protein concentration. In total, 50 μg protein of each lysate was diluted with a dilution buffer containing 10% acetonitrile and 25 mM Tris-HCl, pH 8.0, to reach a 1 M GdmCl concentration. Then, proteins were digested with LysC (Roche, Basel, Switzerland; enzyme to protein ratio 1:50, MS-grade) shaking at 700 rpm at 37 °C for 2 h. The digestion mixture was diluted again with the same dilution buffer to reach 0.5 M GdmCl, followed by a tryptic digestion (Roche, enzyme to protein ratio 1:50, MS-grade) and incubation at 37 °C overnight in a thermal shaker at 700 rpm.

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