2.6 Multiple reaction monitoring (MRM) based targeted proteomic
analysis
Proteins showing differential expression in HGG and LGG were further
validated using MRM experiment. The Transition list of the selected
proteins was prepared using Skyline daily version 22.2.1.501 (19) with
human database (downloaded on 07092021). The peptide uniqueness was
checked using neXtProt (20) and imported to Skyline-daily. Proteins
which had 3 or more than 3 unique peptides with 8-20 amino acid length
and peptides which had 4 or more than 4 transitions were selected. The
missed cleavage criteria was set to 0, precursor charge and product
charge was selected as +2, +1 respectively. All y ions from ion 2 to
last ion -2 were monitored. Initial optimization of transition list was
carried out in sample pools to select the best peptides and their
transitions for every protein. For each sample, 1.5 µg peptide was
injected in a triple quadrupole mass spectrometer Altis (Thermo Fisher
Scientific) coupled with an Ultimate 3000 UHPLC system. A binary buffer
system (Buffer A = 0.1% Formic acid in water and Buffer B = 0.1%
Formic acid in Acetonitrile) allowed for the separation of peptides. The
data was acquired using the same methodology mentioned in the previous
study (21). BSA was run to check the instrument’s performance on
different days. PROSIT library was used as the spectral library (22).
Data analysis was performed using MSstats tool (23) in Skyline daily to
identify the significant peptides (p value < 0.05) and to
calculate the fold changes keeping a confidence interval of 95%.
Protein abundances were exported from the skyline daily and after log 2
transformation, violin plots showing differential expression were
plotted.