2.4. Sample preparation based on iST kit
Glioma and BrM tissues from patients as well as HeLa cells were prepared according to the iST kit manufacturer’s instructions [20]. Briefly, tissue samples were cut and weighed, and 1 mg of each tissue sample was placed into a clean 1.5-mL protein LoBind tube followed by the addition of 100 μL of LYSE and incubation at 95 °C for 30 min with shaking (1000 rpm). HeLa cells for the experimental control were treated simultaneously. The sample was sheared using a sonicator (10 cycles; 30 sec ON/OFF), and protein concentration was determined using the PierceTM BCA Protein Assay Kit. An equal amount of protein (100 μg) for each sample was used for digestion. Then, 210 μL of RESUSPEND was added to DIGEST followed by shaking at room temperature for 10 min at 500 rpm. Then, 50 μL of DIGEST was added to the sample and heated using a pre-heated heating block at 37 °C for 3 h at 500 rpm. After digestion, 100 μL of STOP was added to the sample followed by shaking at room temperature for 10 min at 500 rpm. The sample was then centrifuged at 14,000 rpm for 1 min, and the sample was then serially washed with 200 μL of WASH 0, WASH 1, and WASH 2 for desalting. Then, 100 μL of ELUTE was added twice to elute peptides, and the peptides were lyophilized to dryness and stored at −20 °C or redissolved in 0.1% FA for nano-LC-MS/MS analysis.
2.5. Nano-LC-MS/MS analysis
Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed on a timsTOF mass spectrometer with PASEF (Bruker Daltonics, Bremen, Germany) coupled to a Nano Elute liquid chromatograph (Bruker Daltonics, Bremen, Germany) with a 75 μm × 25 cm-long column (1.6 μm id, Dr. Maisch GmbH, Germany). Peptide separation was performed at a flow rate of 200 nL/min with mobile phases A (0.1% FA in water) and B (0.1% FA in ACN) in the following 90 min gradient: 0 - 70 min, 2% - 22% B; 70 - 80 min, 22 - 37% B; 80 - 82 min, 37% - 80% B; and 82 - 90 min, 80% B.
Peptides were acquired in the data-dependent acquisition mode (DDA). The following parameters were used: mass range, 100 to 1700 m/z; 1/K0 start at 0.7 V⋅s/cm2 and end at 1.3 V⋅s/cm2; capillary voltage, 1500 V; dry gas, 3 L/min; and dry temp, 180 °C. The PASEF settings were as follows: 4 MS/MS scans (total cycle time 0.53 sec); charge range, 0 – 5; active exclusion, 0.4 min; scheduling target intensity, 20000; intensity threshold, 2500; and CID collision energy started at 27 eV and ended at 45 eV.