3.4 Multiple Reaction Monitoring based validation of proteins identified by global proteomics analysis
Differentially regulated significant proteins were validated using multiple reaction monitoring assay. For Initial optimizations, 20 proteins where 185 peptides and 1508 transitions were monitored to identify the best flying peptides and their transitions using sample pools. Further, data was refined based on peak shape, peak intensity and peak area and final transition list was prepared with 14 proteins comprising of 69 peptides and 447 transitions. For the MRM experiment, the data was acquired for total 19 tissue samples, including 14 high grade glioma samples and 5 low grade glioma samples. To monitor the system suitability, an equal amount of heavy labelled synthetic peptide (ENQTCDIYNGEGR) was spiked in each sample and observed CV was 10%.(Figure 4). We observed overexpression of 10 proteins including, FN1, FGA, FGB, TGF-BI, VTN, RPN1, RPN2, FLNA, VIM and DDOST in HGGs while 1 protein TNR showed downregulation during MRM experiment.
Three subunits of OST complex; RPN1, RPN2 and DDOST showed similar trend as discovery data, high abundance in HGG samples compared to LGG. The cumulative log2 fold change (cLog2FC) identified from MSstat analysis was 1.12, 1.56 and 2.64 respectively with a confidence level of 95%. Proteins related to ECM remodelling such as vitronectin (cLog2FC - 3.27), fibrinogen alpha chain (cLog2FC - 3.25), fibrinogen beta chain (cLog2FC - 4.32), filamin-A (cLog2FC -) also showed over expression in HGG which corelates with the global proteomics data. Mesenchymal markers such as fibronectin (cLog2FC - 2.51), vimentin (cLog2FC - 5.95) and transforming growth factor-β (cLog2FC- 2.59) also showed high expression in high grade gliomas compared to LGG there by validating the results of LFQ experiment. (Figure 4, Figure S4) . The final list of the proteins and their corresponding peptides along with p value and Log2 FC was given in Table S1.