Fig. 2.

Workflows for analyzing proteome changes and DNA sensor protein interactions during immune signaling. (Top) Preparation and lysis of cells prior to immunoaffinity purification and proteome analysis. Cells subjected to different experimental conditions, i.e., different time points of viral infection, are collected and lysed using either cryogenic or detergent-based techniques. Lysates are then used for either mass spectrometry proteome analysis or immunoaffinity purification of DNA sensor. (Middle) nLC-MS/MS analysis and relative quantification of protein abundances. Peptides from whole-cell lysates are produced using in-solution digestion then analyzed using either label-free or labeling quantification, such as by labeling with tandem mass tags. (Bottom) Investigation of protein-protein interactions using immunoaffinity purification coupled to nLC-MS/MS analysis. Purification of protein complexes is performed by incubating antibody-conjugated beads with cell lysates. If targeting the endogenous protein, the control IP can be performed with IgG; if the DNA sensor bait is tagged, cells expressing the tag alone can serve as a control. Eluted protein complexes are digested either in-solution or in-gel, and peptides are analyzed by nLC-MS/MS. The specificity of protein interactions can be assessed using the Significance Analysis of INTeractome (SAINT), and associations that pass the specificity filtering can be assembled into functional protein interaction networks.