Phosphoprotein signaling networks from multiplex, quantitative single-cell proteomics. All data represented are uniquely measured at the single-cell level. (a) A Monte-Carlo simulation of fluctuations that represent the copy numbers per cell of an activated (such as phosphorylated) form of a protein, as that protein is involved in increasing numbers of regulatory processes. On the right are the experimentally measured fluctuations of HIF-1α from model GBM cancer cells as these cells are exposed to different O2 partial pressures. The increasingly important role of HIF-1α under hypoxic conditions is evident. Reproduced from . (b) Scatter plot showing protein-protein correlations for two phosphoproteins. The black and red dots represent measurements from 0-cell and 1-cell SCBC microchambers, respectively. Reproduced from . (c) A protein-protein correlation network for model GBM cancer cells following epidermal growth factor (EGF) stimulation (top), and following EGF stimulation + erlotinib (anti-EGF receptor) inhibition (bottom). The weight of the network edges reflects the correlation strength, and a red edge indicates an anti-correlation. Reproduced from . (d) Collective signaling modes, as determined by the eigenvectors of the single-cell protein-protein covariance matrix. Shown are the eigenvectors associated with mTORC1 signaling in model GBM cells, as pO2 is varied. The composition of the green, red, and blue eigenvectors (top plot) is given in the pie charts below for each value of pO2 investigated. The amplitude of the mTORC1 associated eigenvectors shows a minimum between 1.5% and 2% pO2, indicating the loss (and undruggability) of that signaling within this narrow window of pO2 values. Note that HIF-1α is strongly associated with mTORC1 signaling above 2% pO2, but not below 2% pO2, indicating a switch in the structure of the signaling network. The cells studied were model GBM cell lines containing the EDFR variant III (vIII) oncogene (U87 EGFRvIII; panels a, b, d) or the EGRFvIII oncogene plus loss of the phosphatase and tensin homolog (PTEN) tumor suppressor gene (EGFRvIII PTEN). Reproduced from  with permission.