br Acknowledgments This research was supported by

Acknowledgments This research was supported by funds (341256) provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) to Joe Quadrilatero. Troy Campbell is a recipient of a NSERC postgraduate scholarship.
Data This data article includes the N- and O-glycome of MKN45 cells and assesses the glycosylation alterations induced by ST3GAL4 overexpression. In addition, we provide the data on the proteins with significant increased sialylated N-glycans upon ST3GAL4 overexpression.
Experimental design, materials and methods Glycomic and sialoproteomic analyses were performed as described below comparing MKN45 cells stably transfected with the full-length ST3GAL4 gene or empty vector (mock). Glycome data and statistical evaluation are shown in Tables 1, 2, 3 and 4. Sialoproteome data and statistical evaluation are shown in Tables 5 and 6.
Frozen cell pellets (107 cells) of mock or ST3GAL4 transfected MKN45 cells [2] were directly resuspended in 7M urea, 2M thiourea, 40mM Tris, 2% CHAPS, 10mM DTT and 1% protease inhibitor (Sigma-Aldrich, St. Louis, MO). The cell membranes were disrupted by 10 times 10s sonication with 16 amplitudes and 1min on ice in between, and subsequent shaking at 4°C overnight. To reduce the viscosity of the lysates, the DNA was degraded by adding 1µl benzonase® nuclease (250units, Sigma-Aldrich) and 30min incubation at 37°C. In order to impair refolding of proteins, 25mM iodoacetamide were added for alkylation during 1h in the dark. The lysates were centrifuged for 30min at 14,000rpm and the supernatants were transferred to a fresh tube. Then, solubilized proteins were concentrated by adding 150µl of supernatant on a 10kDa cut-off spinfilter (PALL, Port Washington, NY), spinning down for 5min with 12,000xg and washing 3 times with 100µl 50mM NH4HCO3, pH 8.4. N-linked oligosaccharides were released in the spinfilter using 20µl 50mM NH4HCO3 and PNGase F (5mU, Prozyme, Hayward, CA) with incubation at 37°C overnight. Subsequently, the N-glycans were collected by washing 3 times with 20µl H2O and dried in Speedvac. Reactions were quenched with 1µl of glacial acetic Sulfo-NHS-Biotin and N-glycan samples were desalted and dried as previously described [3]. N-glycan samples were subjected to LC-ESI-MS/MS analysis using a 10cmx250µm I.D. column, prepared in-house, containing 5µm porous graphitized carbon (PGC) particles (Thermo Scientific, Waltham, MA). Glycans were eluted using a linear gradient from 0% to 40% acetonitrile in 10mM NH4HCO3 over 40min at a flow rate of 10μl/min. The eluted N-glycans were detected using a LTQ ion trap mass spectrometer (Thermo Scientific) in negative-ion mode with an electrospray voltage of 3.5kV, capillary voltage of −33.0V and capillary temperature of 300°C. Air was used as a sheath gas and mass ranges were defined dependent on the specific structure to be analyzed. The data were processed using the Xcalibur software (version 2.0.7, Thermo Scientific) and manually interpreted from their MS/MS spectra. Optionally prior to analysis N-glycan were digested in 50mM sodium phosphate, pH 6.0 at 37°C overnight using sialidase S (4mU, ProZyme) that releases α2-3 linked non-reducing terminal sialic acids (recombinant sialidase from Streptococcus pneumoniae, expressed in Escherichia coli) and sialidase A (5mU, ProZyme) that releases α2-3/6/8 linked non-reducing terminal sialic acid (recombinant gene from Arthrobacter ureafaciens, expressed in Escherichia coli) to confirm sialic acid linkage. All analyzes were performed in three independent replicates and results were subjected to statistical analyses (Average, standard deviation and unpaired t-test) Table 1.
Frozen cell pellets (107 cells) of mock or ST3GAL4 transfected MKN45 cells were directly resuspended in 2mL of lysis buffer (50mM Tris–HCl, 100mM NaCl, 1mM EDTA and protease inhibitor at pH 7.4) and stored on ice for 20min. The cells were lysed using a Polytron homogenizer for at least three times for 10s in a cold room. Cellular debris and unlysed cells were sedimented by centrifugation at 2000g for 20min at 4°C. The supernatant was collected and the pellets resuspended in 1mL of lysis buffer and centrifuged again at 2000g for 20min at 4°C. All the supernatants were combined, diluted in 20mM Tris–HCl (pH 7.4) and ultracentrifuged at 120,000g for 90min at 4°C. The supernatant was separated from the pellet containing the cell membrane proteins. The membrane proteins were resuspended with 150μL of 100mM ammonium bicarbonate buffer and lyophilized overnight. The dried samples were solubilized in 50μL of 8M urea and 10µL aliquots were dot-blotted onto PVDF membranes as described previously [4]. N- and O-glycan release as well as PGC-nanoLC ESI MS/MS analysis on an amaZon ETD Speed ion trap (Bruker, Bremen, Germany) were performed as described in detail previously [4,5] (Table 2).