Sample grinding kit

Soluble proteins were extracted from the frozen biopsies as previously reported [61 (link)]. Briefly, frozen tissue samples were lysed in 200 µL cold lysis buffer (4% (w/v) CHAPS, 7 M Urea, 2 M Thiourea, 30 mM Tris, pH 8.5) with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), homogenated with a sample grinding kit (GE Healthcare, Uppsala, Sweden) and prepared for two-dimensional electrophoresis (2DE) with 2D Clean-Up Kit (GE Healthcare, Uppsala, Sweden). Protein concentrations were determined with the commercial Bradford reagent (Bio-Rad, Milan, Italy).
Samples were grouped into two groups: “Group I” and “Group II”, including patients with primary tumors classified as either “T1 or T2”, or “T3 or T4”, respectively. Other comparison groupings were based on: (i) anatomical subsite (corpus or antrum); (ii) histological type (diffuse or intestinal); (iii) age (< or >50 years); and (iv) sex (male or female).

The grain samples were homogenized in ten volumes of lysis buffer containing 10 mM Tris–HCl pH 8.0, 7 M urea, 2 M thiourea, 5 mM (CH₃COO)₂Mg, 4% (w/v) CHAPS, and protease inhibitors (11697498001, Roche Diagnostics) using the Sample Grinding Kit (GE Healthcare Bio–Sciences). The homogenate was then centrifuged at 20,000 × g for 30 min at 4 °C. The supernatant was added to 100 µL reduction processing solution (100 mM NH₄HCO₃ containing 0.15% (w/v) DTT) and incubated at 57 °C for 30 min. After incubation, the supernatant was added to 100 µL alkylation process solution (100 mM NH₄HCO₃ containing 1% (w/v) IAA) and then incubated at room temperature. After adding 100 µL modified trypsin and 50 mM NH₄HCO₃, the mixture was incubated at 30 °C overnight. The fluid was then dried using a centrifugal concentrator (CC–105, TOMY SEIKO) and dissolved into 30 µL 0.1% formic acid. The resulting solution was then centrifuged at 20,000 × g for 10 min. The resulting supernatant was then used in nanoLC–MS/MS analyses.
Mass spectrometric analysis was carried out using nanoLC–MS/MS. The samples were loaded onto the column (75 µm internal diameter and 500 mm length: L–column2 ODS, CERI) using nano–HPLC (UltiMate 300, Dionex). The eluted peptides were analyzed by Q–Exactive Plus MS (Thermo Scientific) while the nano–LC and MS were controlled using Xcalibur (Thermo Scientific).

Adult worms (45 males or 40 females per biological replicate) were separately grinded for 5 min in chilled 1.5 mL microcentrifuge tubes containing resin (Sample Grinding Kit, GE Healthcare) and 200 μL of 40 mM Tris base with 0.1% Triton X-100. Samples containing L1 larvae (~ 120,000 larvae per biological replicate) were re-suspended in 250 μL of 40 mM Tris-HCl pH 6.8 with 0.1% Triton X-100 in 1.5 mL microcentrifuge tubes containing abrasive resin. Protein extraction was performed by a combination of grinding for 5 min on ice, followed by 10 freeze-thaw cycles in liquid nitrogen and room temperature. Cell debris were removed by centrifugation at 16,000 x g for 10 min at 4°C and the protein content in the supernatant was measured by BCA Protein assay (Thermo Scientific, Schwerte, Germany), using bovine serum albumin as standard.

Cell pellets were mechanically broken down in RIPA buffer (Wako, Osaka, Japan), which contained protease inhibitor (Wako), and centrifuged at 15,000 rpm for 5 min at 4 °C. Supernatants were collected and analyzed with the immuno-wall assay. In order to lyse the tumor tissues, the tissues were placed in 1.5 ml tubes containing 200 μl RIPA buffer, a protease inhibitor, and resin beads, which were then collectively ground using pestles from a sample-grinding kit (GE Healthcare, Little Chalfont, UK). The lysate was then centrifuged at 15,000 rpm for 5 min at 4 °C and the supernatants were collected and analyzed. Approximately 100 μg of protein was extracted from 10⁵ cells. 

Fifty (50) mg of IM samples was solubilized with 0.5 or 1 mL of lysis buffer (4% SDS, 0.1 M Tris-HCl, pH 7.6). The samples were ground in tubes containing resin on ice using pestle (Sample Grinding Kit, GE Healthcare Life Science, 80648337, Piscataway, NJ, USA). Then, 1 M Dithiothreitol was added to the extraction solution, to obtain a final concentration of 0.1 M. Tubes were centrifuged for 10 min at maximum speed to remove resin and cellular debris. The supernatants were collected and heated at 95 °C for 5 min and then stored at −20 °C until further use.