Quantitative mass spectrometry identifies perturbed ‘response to metal ions’ and EMT markers, including NCAM1, affected in PrP-deficient cells.

<p>(a) Design of quantitative global proteome comparisons giving rise to datasets I to III. (b) Workflow of global proteome analyses conducted by comparative mass spectrometry. Note that this workflow was executed 3 times to generated datasets I to III, with the ‘x’ being replaced by the respective condition specified at the top of this panel. To facilitate comparison of datasets, the three experiments differed in the biological samples which were labeled with even-numbered TMT reagents. All three datasets shared the use of wt NMuMG cell extracts following 48 h TGFB1 exposure as reference samples labeled with odd-numbered TMT reagents. (c) Example graph depicting post-acquisition filtering of datasets and benchmarks of mass spectrometry analysis (shown for dataset I). (d) Profound overlap amongst top 200 proteins whose levels are most changed during EMT or following stable PrP kd. (e) Exposure of NMuMG cells to TGFB1 causes changes to proteins whose KEGG annotations identify them as players in pathways that contribute to ‘focal adhesion’ formation and ‘actin cytoskeleton regulation’. (f) Direct comparison of global proteomes of wt and stable PrP kd NMuMG cells following TGFB1 exposure identifies highly significant perturbations in biological processes with ‘response to inorganic substance’ and ‘response to metal ions’ GO annotations.