*2.3. Preparation of Skin Samples for LC–MS/MS Experiments*

Each sample was washed twice with 0.5 mL of phosphate-buffered saline. The samples were homogenized by mechanical disruption in liquid nitrogen. To prepare the protein samples, sodium deoxycholate (SDS) lysis as well as reduction and alkylation buffer pH 8.5, which contained 100 mM TRIS, 1% (*w*/*v*) SDS, 40 mM 2-chloroacetamide, and 10 mM TCEP, were added to the homogenized samples. The samples were sonicated and boiled for 10 min. Then, the protein concentration was determined by Bradford assay and the equal volumes of 1% trypsin solution (*w*/*v*) prepared in 100 mM TRIS pH 8.5 were added.

After overnight digestion at 37 ◦C, peptides were acidified with 1% trifluoroacetic acid (TFA). The samples (2 × 20 µg) were loaded on 14-gauge StageTips containing 2 layers of SDB-RPS discs. Respectively, 2 tips per a sample were used. The tips were consequently washed with equal volumes of ethyl acetate, 100 µL of 1% TFA prepared in ethyl acetate, and 100 µL of 0.2% TFA. After each washing, the excess of liquid was removed by centrifugation (300 g; 1.5 min.) Then, the peptides were eluted with 60 µL of 5% NH4OH prepared in 80% acetonitrile. The eluates were vacuum-dried and stored at −80 ◦C. Prior to the experiment, the vacuum-dried samples were dissolved in 2% acetonitrile/0.1% TFA buffer and sonicated for 2 min.
