Glycosylation Analysis of Feline Small Intestine Following Toxoplasma gondii Infection- raw datas 3
Glycosylation Analysis of Feline Small Intestine Following Toxoplasma Gondii Infection- raw datas, Rawdata_7.tar, Rawdata_8.tar, Rawdata_9.tar,
Steps to reproduce
Total Protein Extraction and Digestion The sample was mixed with 5 mm magnetic beads and lysis buffer 3 (containing 1 mM PMSF, 2 mM EDTA, and 10 mM dithiothreitol) and centrifuged to obtain the supernatant, and then 10 mM dithiothreitol was added and the sample was incubated in a water bath at 56 °C for 1 h. After adding 55 mM IAM at room temperature (Avoid light incubation for 45 min), 4 times the volume of acetone (-20 °C, 2 h) was added, and the same volume of acetone (-20 °C, 2 h) was added again two to three times until the supernatant was colorless. The precipitate was collected after centrifugation, mixed with 5 mm magnetic beads with lysis buffer 3, shaken for 2 min, centrifuged, and used for quantification. Quantification of the protein was carried out using the Bradford Kit (Bradford Assay Kit, Bio-Rad, USA) and was analyzed by SDS–PAGE, as described in the manufacturer’s protocol. A total of 500 μg of protein solution was taken from each sample, and the enzyme hydrolysis activity was determined according to the protein solution: trypsin enzyme = 40:1 (37 °C, 4 h). A Strata X column was utilized to remove salt and then the enzymatic peptides were subjected to vacuum drying. A small portion of the peptides was used to detect the enzymatic hydrolysis effect by using mass spectrometry. Enrichment of N-glycosylated Peptides and Desugaring Three hundred microliters of qualified peptides were dissolved in 60% ACN and 0.1% FA, and the optimized HILIC method was then used for enrichment and grouping fractionation by HPLC (Merck, 5 µm, 150×4.6 mm) . Fractionated product collection started at the 30th min and ended at the 54th min, with one tube per minute. A total of 24 tubes were collected and labeled and then pumped by a freezer pump. Each adjacent 4 tubes were reconstituted with a total of 50 µl of 50 mM NH4HCO3, combined into one tube, and finally divided into 6 components. Then, 2.5 µl of PNGase F was added to each component, vortexed, mixed well, centrifuged immediately and incubated overnight at 37 °C. Then, it was frozen and drained.