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.