8 m urea

The in-solution digestion of whole milk was adapted from previous methods [14 (link)]. Briefly, a 20 μL milk sample was added to 80 μL of protein lysis buffer (Thermo Fisher Scientific, Rockford, IL, USA) with a protease inhibitor (Thermo Fisher Scientific, Rockford, IL, USA). The whole milk buffer was sonicated for 20 min in a water bath with cold water and put on ice for another 30 min. Impurities were removed by methanol/chloroform protein precipitation and the precipitate was air-dried. The pellet was further dissolved in an 8 M urea (Sigma, St. Louis, MO, USA)/50 mM ammonia bicarbonate (Alfa Aesar, Haverhill, MA, USA) solution, incubated with 10 mM dithiothreitol (Sigma, St. Louis, MO, USA) at 37 °C for 1 h and followed by alkylation with 20 mM iodoacetamide (Sigma, St. Louis, MO, USA) at 37 °C in the dark for 30 min. For digestion, the samples were incubated with a trypsin/lys-C mixture (w:w = 1:50; Promega, Madison, WI, USA) at 37 °C for 16 h and then the digest reaction was quenched with 1% formic acid (Thermo Fisher Scientific, Rockford, IL, USA). The resulting peptides were desalted using a C18 column (Thermo Fisher Scientific, Rockford, IL, USA) prior to lyophilization. The same tryptic peptides for proteomic analysis were directly applied to glycoproteomic analysis without further glycopeptide enrichment.

GBM cells (5 × 10⁶) were seeded in T75 flasks with StemPro^® NSC SFM (Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates were collected in biological triplicates 72 hours postseeding. Cells were lysed in buffer containing 2% SDS (Sigma-Aldrich), EDTA-free protease inhibitor (Roche, Basel, Switzerland) and 100 mM tris-HCl (pH 7.6) [16 (link)]. The lysates were sonicated and incubated for 5 min at 95 °C, protein concentration was quantified, and samples were processed by an in-solution digest protocol. Briefly, 100 µg of cell lysate was precipitated overnight with 5 volumes of ice-cold acetone. The precipitate was collected by centrifugation and resuspended in 8 M urea (Sigma-Aldrich). The samples were reduced with dithiothreitol (Sigma-Aldrich) for 1 h at room temperature, alkylated with iodoacetamide (Sigma-Aldrich) in the dark, diluted to a final concentration of 2 M urea, 10% acetonitrile (Merck, Kenilworth, NJ, USA) and 1 µg of trypsin (Promega, Madison, WI, USA) and digested to peptides overnight at 37 °C. The peptides were desalted on C18 stage tips as described [17 (link)] and resuspended in 0.1% formic acid.

Cell lysates were prepared in 8 M urea (Sigma-Aldrich), protein concentration was determined by Biorad Protein Assay (Bio-Rad, Munchen, Germany). Equal amounts of proteins (40 μg) were subjected to SDS–polyacrylamide gel electrophoresis and were transferred onto a nitrocellulose membrane saturated with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween 20. Membranes were incubated with primary antibody for 18 h at 4°C and subsequently with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Membranes were washed with Tris-buffered saline in 0.1% Tween 20 and developed using the chemiluminescence system Chemidoc MP Imaging System (Bio-Rad). Antibody anti-p21, -p53, -SHMT2, -β-actin and the secondary antibodies anti-goat and -mouse were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Frozen samples of either the total stomach antrum or mesenchymal-enriched stomach antrum fractions were thawed on ice and homogenized directly in 8 M urea (Sigma Aldrich, St. Louis, MO, USA) dissolved in 10 mM HEPES pH 8.0 (Wisent, Saint-Jean-Baptiste, QC, Canada) (100 µL/10 mg wet tissue weight), using the QIAGEN TissueLyser LT (Hilden, Germany). Prior to protein quantification by BCA assay (Pierce Thermo Scientific, Waltham, MA, USA), samples were centrifuged following their homogenization to remove urea-insoluble materials. Following the protocol described by Naba et al., proteins were reduced, alkylated, deglycosylated, and digested, except for the Lys-C digestion, which was omitted [14 (link),29 (link)]. Solutions were prepared using MS-grade water and low protein binding tubes were used for these experiments.

5.0 mg A431 cell lysates were treated with 100 μM PF-06672131 in 1 ml for 1 h at 37 °C. Methanol-chloroform precipitation was performed, and the air-dried pellets were resuspended in 500 μl 8 M urea (Sigma-Aldrich). The samples were sonicated with a bioruptor (Diagenode) for 10 min with 30 s on and 30 s off at high amplitude to fully dissolve all proteins.