To survive in diverse niches, bacteria must adapt to changes in their local environment by sensing and responding to environmental cues. External cues are transduced through complex signaling networks throughout a cell and drive diverse changes in cellular behavior. In bacteria, cyclic di-guanosine-monophosphate (c-di-GMP) is a nucleotide-derived second messenger that mediates signal transduction of important biological processes for bacterial growth and survival e.g. motility, biofilm formation and metabolism. These biological processes are also crucial in clinical settings as they underlay antibiotic resistance in important pathogenic bacteria. Recent advances in MS-based proteomics have provided different tools to investigate the proteome of an organism in a systematic and global manner. Especially, thermal proteome profiling (TPP) and limited-proteolysis MS (LiP-MS) are pioneering methods to study change of protein states proteome-wide. I aim to employ these proteomics based approaches to achieve the global map of the c-di-GMP signaling network in two different bacteria, Escherichia coli and Caulobacter crescentus, both are model organisms of Gram-negative bacteria. Furthermore, I will apply these methods to investigate signaling network of another important second messenger, (p)ppGpp, and explore how the networks of these two messenger molecules interact in bacteria to dictate cellular physiology.