2.6 Protein enzymatic hydrolysis
Appropriate amount of protein sample was transferred into a 1.5ml centrifuge tube and added with 5mm steel bead and appropriate amount of Lysis Buffer 3, added PMSF with a final concentration of 1 mM, and EDTA with a final concentration of 2 mM. Vortex and let stand for 5 minutes, add DTT with a final concentration of 10 mM. Oscillate with a tissue grinder for 2 minutes (Frequency=50HZ). 25000g centrifugation at 4 °C for 20 minutes and add 10 mM DTT to the supernatant and water bath at 56 °C for 1 hour. After returning to room temperature, 55 mM IAM was added and incubated in dark room for 45 minutes. Add cold acetone by 4 times volume of the sample, and stand at -20 °C for 2h. Repeat previous step until the supernatant is colorless. 25000g centrifugation at 4 °C for 20 minutes and discard the supernatant. Add an appropriate amount of Lysis Buffer 3 to precipitate, followed by ultra-sonication to dissolve the precipitated proteins. After centrifugation at 25000 g * 4 °C for 20 minutes, the supernatant was taken for quantification.
For protein extraction quality control, standard protein (0.2 μg / μl BSA) was added sequentially to the 96-well plate A1 to A10. 0, 2, 4, 6, 8, 10, 12, 14, 16, 18μl, then add pure water 20, 18, 16, 14, 12, 10, 8, 6, 4, 2μl, add each well MAS Bright Blue G-250 Quantitative Working Solution 180 μl. The OD595 was measured with a microplate reader, and a linear standard curve was prepared based on the OD595 and protein concentration. Dilute the protein solution to be tested several times, add 180 μl of the quantitative working solution to 20 μl of the protein solution and read OD595. The sample protein concentration was calculated based on the standard curve and sample OD595. Each 10 μg of protein solution was mixed with an appropriate amount of loading buffer, heated at 95 ℃ for 5 minutes, centrifuged at 25,000 g for 5 minutes, and the supernatant was poured into a well of a 12% SDS polyacrylamide gel. 120V constant pressure electrophoresis for 100 minutes; After electrophoresis, the gel was stained with Coomassie brilliant blue for 2 hours, then decolored with appropriate amount of decoloring solution (40% ethanol 10% acetic acid) on the shaker every 30 minutes, in total 3~5 times.
100 μg of protein solution per sample and dilute with 50mM NH4HCO3 by 4 times volumes. Add 2.5 μg of Trypsin enzyme in the ratio of protein: enzyme =40:1, and digest for 4 hours at 37 °C. Add Trypsin once more in the above ratio and continue to digest for 8 hours at 37 ℃. Enzymatic peptides were desalted using a Strata X column and vacuumed to dryness (Gillet et al., 2012; Rost et al., 2014).