Tpck trypsin 20233

Cells were grown for 24 h in RH1-free growth media, rinsed 3 times with PBS (37°C), and lysed with 0.5 ml of urea/thiourea lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM DTT, Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Vilnius, Lithuania). The lysates were sonicated for 1 min at the amplitude of 20% and 0.4 s pulsations on/off cycles (Sonopuls HD 2070, Bandelin, Berlin, Germany). Homogenized lysates were centrifuged at 20,000 g for 15 min at 4 °C, and the supernatants were collected. The lysates then were stored at −86 °C.
Trypsin digestion was done according to a modified filter-aided sample preparation (FASP) protocol as described by Wisniewski et al. [99 (link)]. Briefly, proteins were diluted in 8 M urea, and loaded on Amicon Ultra-30 kDa filters. Following two washes with urea, proteins were alkylated with 50 mM iodoacetamide. Filters were washed twice with urea and twice with 50 mM NH₄HCO₃. Proteins digested overnight with TPCK Trypsin 20233 (Thermo Scientific, Vilnius, Lithuania). After overnight digestion, peptides were recovered by centrifugation and then two additional washes using 50% CH3CN were combined, acidified, lyophilized, redissolved in 0.1% formic acid and then fractionated off-line.

The Pro-Q Diamond Phosphoprotein Enrichment kit/reagents and NuPAGE precast 4%–12% Bis–Tris polyacrylamide gels were purchased from Life Technologies. Bradford Protein concentration assay kit and TPCK Trypsin 20233 for protein digestion were obtained from Thermo Fisher. Protease and phosphatase (for serine, threonine, and tyrosine) inhibitors and all other reagents were of analytical grade and quality and were obtained from Sigma (if not stated otherwise).

MenSCs’ culture medium is aspirated and collected in 5 mL tubes and centrifuged for 10 min at 2000× g to remove cells and cell debris. After centrifugation, the supernatant is collected and filtered through a 0.22 μm filter. After filtration, the samples are cleaned and concentrated using ProteoMiner^TM Sequential Elution Large-Capacity kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. The secretome protein concentration was measured using the Pierce Detergent Compatible Bradford Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The secretome proteins were trypsinized according FASP protocol, as described by Wisniewski et al. [32 (link)]. Briefly, proteins were diluted in 8 M urea; following two washes with urea, proteins were alkylated with 50 mM iodoacetamide (GE Healthcare Life Sciences, MA, USA). Protein concentrators were washed twice with urea and twice with 50 mM NH₄HCO₃. Proteins were digested overnight with TPCK Trypsin 20233 (Thermo Scientific, Vilnius, Lithuania). After overnight digestion, peptides were collected from the concentrators by centrifugation at 14,000× g for 10 min and additionally eluted using 20% CH₃CN. The eluates were combined, acidified with 10% CF₃COOH and lyophilized in vacuum centrifuge. The lyophilized peptides were redissolved in 0.1% formic acid.