Sample Homogenization

For optimization of homogenization protocol we have used porcine gastric tissue and several conditions were tested. In particular, manual homogenization with sonication was compared with automatic homogenization using Precellys system (Bertin Technologies, Montigny-le Bretonneux, France). For the extraction of proteins from porcine gastric tissue, 50 mg (wet weight) sample tissue was weighted, 1 mL of solubilisation buffer was added to the tube (7 M urea, 2 M thiourea, 40 mM Tris and a protease inhibitor cocktail at the dilution 1:100 (Sigma-Aldrich)) and the tissue was homogenized using a VDI-12 homogenizer (VWR), 4 × 15 sec, or using a Precellys 24, 2 × 30 sec cycles, samples were kept on ice between the cycles. Homogenization was followed by sonication (MSE Soniprep 150, 4 × 15 sek, 16 micron amplitude setting) at 4 °C for one batch of the samples but for second batch sonication was not performed to verify if this step was essential for efficient tissue homogenization. Further, different concentrations of detergent have been tested (0.5%, 1% and 2%). After homogenization, detergent (CHAPS or SDS) and 10 mM DTT were added to the samples to facilitate solubilisation. Following overnight incubation at 4 °C on a rotor, 25 mM IAA was added and incubated at RT in the dark for 40 min. Glycan release, desalting and LC-MS analysis of O-linked glycans was performed using the same protocol (as described in the subsequent sections) for all the samples.

The snap-frozen and heat stabilized human tissue samples were weighted (50 mg, wet weight) into 2 mL reinforced homogenization tubes (Bertin Technologies) and loaded with 6 zirconium oxide beads (3 mm) from Bertin Technologies. During weighing, the samples were kept on ice to ensure temperature control during sample handling. The tissue was then homogenized in 500 μL solubilisation buffer (7 M urea, 2 M thiourea, 40 mM Tris and a protease inhibitor cocktail at the dilution 1:100 (, Sigma-Aldrich)) at power 5500 using a Precellys 24, 2 repeated 30-second cycles. Following the homogenization, another 500 μL of solubilisation buffer was added including SDS and DTT (final concentration 0.5% SDS and 10 mM DTT). The extraction of up to 2 × 24 tubes was then continued at 4 °C overnight on a rotor. After overnight solubilisation, 25 mM IAA was added and incubated for 40 min at RT in the dark. The tissue extracts were centrifuged for 20 min at 13,200 rpm and the supernatants were transferred to a new tube and stored at −20 °C until further analysis.