Integrated transcriptomic and proteomic analysis of human eccrine sweat glands identifies missing and novel proteins
To maximize the identification of proteins, the sweat grand proteins were extracted by twoTo maximize the identification of proteins, the sweat grand proteins were extracted by twodifferent methods - Filter-aided sample prep (FASP) approach and the guanidine hydrochloride(GuHCl) approach. For the FASP approach, sweat glands were sonicated in 50 mM TEAB/4%SDS/10 mM DTT with Halt protease inhibitor cocktail (Thermo Scientific) for 5 min followedby heating at 95°C for 5 min. After cooling, the samples were alkylated with 30 mMiodoacetamide at room temperature for 30 min followed by centrifugation at 17,000 x g for 10min. SDS in the sample was removed by FAST method . Briefly, the lysate was diluted with50 mM TEAB/8 M urea and concentrated with centricon with 30 KDa MWCO for 40 min at RT.This buffer exchange step was repeated five times. Sweat gland proteins were digested with Lys-C at room temperature for 3 h followed by further digestion with trypsin overnight.For the GuHCl approach, sweat glands were sonicated for 5 min with 35% amplitude in 8 MGuHCl, 50 mM HEPES, pH 7.0, 10 mm dithiothreitol in the presence of Halt protease inhibitorcocktail (Thermo Scientific) followed by heating at 90°C for 3 min and centrifugation at 16,000 xg for 10 min. The pellet was resuspended in 50 mM TEAB/4% SDS/10 mM DTT and subjectedto further protein extraction with barocycler for 60 cycles in which each cycle consisted of45,000 psi at 95°C for 50 sec and atmospheric pressure at 25oC for 10 sec. The proteinsalkylated with 30 mM iodoacetamide at room temperature for 15 min. The proteins weredigested with trypsin at the enzyme to protein ratio of 1:50 at 37oC overnight. Following enzymedigestion, the peptides were desalted using Sep-Pak C18 cartridge. The peptides prepared throughFASP and GuHCl method were fractionated into 24 fractions by basic pH reversed phase liquidchromatography. Briefly, lyophilized samples were reconstituted in solvent A (10 mMtriethylammonium bicarbonate, pH 8.5) and loaded onto XBridge C18, 5 μm 250 × 4.6 mmcolumn (Waters, Milford, MA). Peptides were resolved using a gradient of 3 to 50% solvent B(10 mM triethylammonium bicarbonate in acetonitrile, pH 8.5) over 50 min collecting 96fractions. The fractions were subsequently concatenated into 24 fractions followed by vacuumdrying using SpeedVac. The dried peptides were resuspended in 15 μl 10% FA and the entireamount was injected. The peptides prepared through barocycler were fractionated by SCXStageTip as previously reported .