Strata x c18 spe column

Following exposure to hypoxic/normoxic PSC-derived exosomes or control medium, PC cells were lysed using 8 M carbamide, 30 mM HEPES, 1 mM PMSF, 2 mM EDTA, and 10 mM DTT. After centrifuging at 8,000 g at 4° C for 15 min, total protein was obtained and digested into peptides. The peptides were resuspended in 0.5 M triethylammonium bicarbonate and labeled using iTRAQ (isobaric tags for relative and absolute quantitation) reagent. After vacuum drying, iTRAQ-labeled peptides were desalted using Strata X C18 SPE columns (Phenomenex, USA) and analyzed on a Q-Exactive mass spectrometer (ThermoFisher Scientific, USA). Log fold-change >0.5 and P-value <0.05 were set as thresholds for differential protein expression.

Cellular debris were first removed from serum samples via a 10-min centrifugation at 12,000×g at 4 °C, and the supernatant transferred to a new centrifuge tube. The top 12 high abundance proteins were then removed by Pierce™ Top 12 Abundant Protein Depletion Spin Columns Kit (Thermo Fisher), and the protein concentration determined using the BCA kit according to the manufacturer’s instructions. For digestion, the protein solution was reduced by treating it with 5 mM dithiothreitol for 30 min at 56 °C, then alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. Next, the prote in sample was diluted by adding 100 mM TEAB to urea concentration less than 2 M. Trypsin, at 1:50 trypsin-to-protein mass ratio, was added for the first digestion overnight, followed by 1:100 trypsin-to-protein for a second 4 h-digestion. After tryps indigestion, peptide was desalted using Strata X C18 SPE columns (Phenomenex), vacuum-dried, then reconstituted in 0.5 M TEAB and labeled for TMT pro11 plexkit according to the manufacturer’s protocol.

Cultured GCs were infected with lentiviral shYthdf1 or shCtrl. After puromycin selection, neurons were rinsed three times with ice‐cold PBS and then lysed with freshly made lysis buffer (8 m urea (Sigma), 0.1 m HEPES (pH 7.4, Invitrogen)) supplemented with protease inhibitors. Cell lysates were then ultrasonicated on ice and centrifuged at 10 000 × g for 10 min at 4 °C. A total of 100 µg protein for each condition was reduced with 5 × 10⁻³m dithiothreitol (Sigma) for 30 min at 56 °C, followed by alkylation with 11 × 10⁻³m iodoacetamide (Sigma) for 15 min at RT in the dark. Then the urea concentration in each sample was diluted to less than 2 m by adding 100 × 10⁻³m triethylammonium bicarbonate (Sigma). Subsequently, protein samples were digested by trypsin (Promega) overnight at 37 °C. Peptides were further desalted by Strata X C18 SPE columns (Phenomenex) and labeled with TMT10plex Mass Tag Labeling kit (Thermo) following the manufacturer's instructions. Finally, the labeled peptides were subjected to high‐performance liquid chromatography fractionation and LC‐MS/MS (liquid chromatography with tandem mass spectrometry) analysis.

Firstly, the chondrocytes were stimulated with LIPUS for 20 min per day for three consecutive days. On the third day, cell samples from each group were added to lysis solution (8 M urea and 1% protease inhibitor cocktail) and lysed by ultrasonication. The supernatant was collected after centrifugation. Proteins were denatured with 5 mM dithiothreitol for 30 min at 56°C, alkylated with 11 mM iodoacetamide for 15 min at room temperature in the dark and then diluted to obtain a urea concentration of less than 2 M by the addition of 100 mM TEAB. Subsequently, trypsin was added at a trypsin:protein mass ratio of 1:50 for the first digestion, which was performed overnight, and at a trypsin:protein mass ratio of 1:100 for the second digestion, which was performed for 4 h. After trypsin digestion, the peptides were desalted on a Strata X C18 SPE column (Phenomenex) and dried under vacuum. The peptides were reconstituted in 0.5 M TEAB and processed with the TMT Mass Tagging Kit (Thermo Fisher, United States) according to the manufacturer’s protocol. In this study, labeling reagents 126, 127N, 127C, 128N, 128C 129N, 129C, 130N and 131 were used to label three biological replicates of the N, L and LP groups. The samples were then fractionated by high-pH reversed-phase HPLC using a Thermo Betasil C18 column (Thermo Fisher, United States).

Brain tissues were manually homogenized in Dounce grinder containing cold lysis buffer (4% SDS, 15 mM Tris–HCl (pH 7.4), 10 mM sodium butyrate and protease inhibitor cocktail [Sigma-Aldrich, St. Louis, MO)]. The homogenized samples were centrifuged at 20,000g for 10 min at 4°C to remove the insoluble pellet. Supernatants were collected and diluted with 8 M urea to a final concentration less than 10 mg/ml. Dithiothreitol (DTT) with the final concentration of 10 mM was added, and the mixture were immersed into a water bath (56°C) for 1 h. To further remove larger proteins, the mixtures were applied to a 10 kDa molecular mass cutoff filter (Millipore, MA) and centrifuged at 14,000g for 15 min at 4°C. The filters were desalted in a Strata X C18 SPE column (Phenomenex) according to the manufacturer’s protocol. Peptides were eluted with acetonitrile (ACN)/water/formic acid (FA) solution (50:49.9:0.1; vol/vol/vol) and dried by vacuum centrifugation. Crude peptide samples were further dissolved in buffer A (5% ACN, 0.1%FA) for LC–MS/MS analysis.

After trypsin digestion, peptides were desalted by Strata X C18 SPE column (Phenomenex), and vacuum-dried. The dried peptides were redissolved in 0.5 M TEAB, and processed according to the manufacturer’s protocol for a 6-plex TMT kit. Briefly, one unit of TMT reagent (It was used to label the tryptic peptides of 100 μg proteins) were thawed and reconstituted in 24 μL acetonitrile (ACN). The peptide mixture was then incubated with the prepared TMT reagent (2 h, room temperature), then TMT-labeled peptide mixtures were pooled equally (1:1:1:1:1:1), desalted, and dried by vacuum centrifugation.