2.3 Global proteomic analysis of glioma samples
One microgram of the digested desalted peptide was run in the Q-Exactive
Orbitrap Mass Spectrometer (Thermo Fisher Scientific, USA) coupled with
an automated Easy-nLC 1200 system with a gradient of solvent A (0.1%
formic acid) to solvent B (0.1% FA, 80% Acetonitrile) for 120 min in a
positive mode with blanks after every sample.
For MS OT, detector type was set to orbitrap and resolution was set to
60,000. A scan range of 375−1700 m/z with a maximum injection time of
125 ms was set. Monoisotopic peak determination was on peptide mode,
charge state was set to 2-6. For dynamic exclusion, mass tolerance was
10 ppm and exclusion duration was set to 40 s. For ddMS2 OT HCD,
resolution was set to 30,000, isolation window was 2, HCD collision
energy was 30 %, mass range was normal and AGC target was set to
standard.
The MS acquired data in .raw format was analysed using Maxquant
(Version 1.6.12.0) against the
UniProt Human Proteome Database (downloaded on 07092021) for protein
identification. Trypsin was used as the enzyme for protein digestion
allowing up to two missed cleavages. Carbamidomethylation of cysteine
(+57.021464 Da) was set as fixed modification whereas oxidation of
methionine (+15.994915) was set as dynamic modifications. The false
discovery rate (FDR) for proteins and peptides identification was set to
0.01 to ensure high reliability.