10 days), cells were detached with trypsin-EDTA, rinsed in sterile PBS, counted, and cryopreserved in FBS supplemented with 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MI, USA) in 1–2 × 106 aliquots. Cryopreserved MSCs (~ 2 × 106 cells) were thawed (P2) and expanded in two T75 cm2 flasks in EM without amphotericin B, supplemented with 50 μg/mL gentamycin (only AT-MSCs). At ~ 90% confluency (10–12 days), the cells were passaged using trypsin-EDTA (0.25%) and replated in ratio 1:3 (P3). P3 cells at ~ 90% confluency (5–6 days) were either used for cell differentiation into mesodermal lineages (chondrogenic and osteogenic), biotinylation of cell surface proteins, or RNA isolation.
Cells were passaged (P4) to 48-well plates at a density of ~ 103 cells/well (9090 cells/cm2) and expanded in EM without amphotericin B, supplemented with 50 μg/mL gentamycin (only AT-MSCs). At ~ 80% confluency, cells were washed with sterile PBS and medium was changed to chondrogenic differentiation medium (DMEM 4.5 g/L glucose, without phenol red, supplemented with 1% (v/v) GlutaMAX (Thermo Fischer Scientific, Waltham, MA, USA), 1% (v/v) pyruvate (Thermo Fischer Scientific, Waltham, MA, USA), 2% (v/v) FBS, 1% (v/v) insulin-transferrin-selenium (ITS) (Thermo Fischer Scientific, Waltham, MA, USA), 100 U/mL penicillin and 100 μg/mL streptomycin, 10−7 M dexamethasone (Sigma-Aldrich, St. Louis, MI, USA), 50 μg/mL ascorbic acid (l-ascorbic acid 2-phosphate) (Sigma-Aldrich, St. Louis, MI, USA), 10 ng/mL TGF-ß3 (recombinant human TGF-ß3) (R&D Systems, Inc., Minneapolis, MI, USA), and 50 μg/mL gentamycin (only AT-MSCs)) or osteogenic differentiation medium (DMEM 1 g/L glucose, without phenol red, supplemented with 1% (v/v) pyruvate (Thermo Fischer Scientific, Waltham, MA, USA), 2% (v/v) FBS, 1% (v/v) ITS, 100 U/mL penicillin and 100 μg/mL streptomycin, 10−7 M dexamethasone, 50 μg/mL ascorbic acid (l-ascorbic acid 2-phosphate), 10 μL/mL 1 M ß-glycerophosphate (ß-glycerophosphate, disodium salt, pentahydrate) (Merck KGaA, Darmstadt, Germany), and 50 μg/mL gentamycin (only AT-MSCs). Differentiation medium was changed every 3 days for 21 days. Confirmation of chondrogenic and osteogenic differentiation was performed by staining for proteoglycans in the extracellular matrix using 0.1% Safranin O, pH 4.6 (Merck KGaA, Darmstadt, Germany) and calcified extracellular matrix deposits using 2% Alizarin red staining, pH 4.2 (Sigma-Aldrich, St. Louis, MI, USA), respectively.
Cell surface proteins were biotinylated and isolated according to the manufacturer’s protocol (Pierce cell surface protein isolation kit, https://www.thermofisher.com/order/catalog/product/89881) with few modifications. In short, EM was removed and cells were washed twice in ice-cold PBS and incubated with a biotin labelling solution for 30 min at 4 °C on a rocking platform. The reaction was quenched with a quenching solution and cells were scraped (cell scraper, Corning Incorporated, Corning, NY, USA) into solution, pooled in a 50 mL conical tube, and washed twice with TBS. Cells were resuspended in lysis buffer supplemented with protease inhibitor (complete protease inhibitor cocktail tablets) (Roche, Basel, Switzerland) 25:1 (v/v), transferred to a 1.5-mL microcentrifuge tube, and incubated on ice 30 min, vortexing every 5 min for 5 s. Cell lysate was centrifuged at 10,000 g for 2 min at 4 °C and the supernatant was transferred to a new tube. The cell lysate was applied to a neutravidin agarose gel column, incubated for 60 min at RT on a rocking platform, and then centrifuged for 1 min at 1000g. The column was washed with wash buffer supplemented with protease inhibitor (25:1 (v/v)), and centrifuged for 1 min at 1000 g. This step was repeated three times. A 4× Laemmli sample buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with 50 mM dithiothreitol was added to the column, and the reaction was incubated for 60 min at RT on a rocking platform followed by centrifugation for 2 min at 1000 g. The eluate was stored at − 80 °C until further processing.
Samples were thawed on ice and the proteins in 25 μL 4× Laemmli buffer were reduced by heating for 7 min at 95 °C, and further processed by SDS-PAGE (10 well, mini protean TGX gel 4–20%) (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 180 V for 10 min, to remove lithium dodecyl sulphate from the sample and separate proteins according to molecular weight allowing the proteins to penetrate the resolving gel for approx. 1 cm. After staining with coomassie blue (Sigma-Aldrich, St. Louis, MI, USA), the visible band was excised into small cubes and transferred to individual 1.5 mL microcentrifuge tubes. The gel pieces were washed with 50 mM triethylammonium bicarbonate buffer (TEAB) (Sigma-Aldrich, St. Louis, MI, USA) for 15 min at RT, followed by addition of acetonitrile (Honeywell-Fluka, Morris Plains, NJ, US) in a 1:1 solution with TEAB and incubation for 15 min at RT, removing of all liquids and shrinking of the gel pieces with acetonitrile. These washing steps were repeated four times. Proteins in the gel pieces were reduced and alkylated by incubation with 1.2 μL 0.5 M tris (2-carboxyethyl)phosphine hydrochlorid (Sigma-Aldrich, St. Louis, MI, USA) per 50 μL TEAB and 6 μL 0.5 M chloroacetamide (Sigma-Aldrich, St. Louis, MI, USA) per 50 μL TEAB for 30 min at 37 °C followed by a fifth washing sequence. Proteins were digested with 12.5 ng trypsin (Thermo Fischer Scientific, Waltham, MA, USA)/μL TEAB at 37 °C overnight, and the supernatant containing the extracted peptides was transferred to a fresh microcentrifuge tube. The gel pieces were then incubated in 1:20 (v/v) formic acid (Honeywell-Fluka, Morris Plains, NJ, USA) in TEAB for 15 min at RT, the supernatant added to the peptide solution, the gel pieces incubated in acetonitrile for 15 min at RT, and the supernatant transferred to the peptide solution. The peptides were concentrated in a vacuum centrifuge and stored at − 80 °C until further processing.
Peptides were resuspended in resuspension buffer (2% acetonitrile, 0.1% triflouroacetic acid (Thermo Fischer Scientific, Waltham, MA, USA), 0.1% formic acid in MilliQ water) and a volume corresponding to ~ 1 μg peptide was analyzed by nanoLC-MS/MS (Thermo Scientific Dionex Ultimate 3000 RSLC) coupled in-line to a Thermo Scientific Q Exactive HF mass spectrometer. The peptide separation was accomplished using a precolumn setup (Acclaim PepMap 100 C18 2 cm 100 μm precolumn; 75 μm 75 cm main column) (Thermo Fischer Scientific, Waltham, MA, USA) and a 35-min gradient from 10% buffer B (99.9% acetonitrile) to 35% buffer B and the buffer A being 99.1% MilliQ with 0.1% formic acid. The mass spectrometer was set to acquire MS1 data from m/z 375–1500 at R = 60 k and MS2 at R = 30 k allowing up to 20 precursor ions per MS1 scan.
Cells were lysed in TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and stored at − 80 °C until further processing. After thawing, the cell lysate was further homogenized through a QIAshredder column (Qiagen, Hilden, Germany) for 2 min at 12,000 g. The homogenate was phase separated by adding 0.2 mL chloroform per mL TRI Reagent, shaked vigorously for 15 s allowing it to stand for 15 min at RT, and centrifuged at 12,000 g for 15 min at 4 °C. The upper phase containing the RNA was transferred to a fresh tube. The RNA was precipitated by adding 0.5 mL 2-propanol per mL TRI Reagent, incubation for 8 min at RT, followed by centrifugation at 12,000 g for 8 min at 4 °C. After removing the supernatant, the RNA pellet was washed by adding 1 mL 75% ethanol per 1 mL TRI Reagent and centrifugation at 7500 g for 5 min at 4 °C. The