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.