Decoding protein phosphorylation signaling networks by mass spectrometry

Phosphorylation is one of the most important post-translational modifications of proteins. Phosphorylation-mediated signaling networks play key roles in regulating cellular responses under changing environmental conditions. Such cellular signaling is highly complex and comprises thousands of protein kinases, substrates, and phosphorylation sites. Deciphering these signaling networks has attracted increasing attention among researchers. In the past decades, numerous methods have been developed to identify and quantify the dynamic changes of phosphorylation sites under various cellular conditions. Pertinent phosphoproteins/phosphopeptides have been predicted and identified with gel-based or antibody-based techniques. However, these analyses only revealed a few phosphorylation sites and fewer kinase candidate substrates. Furthermore, these approaches are limited in their ability to resolve basic or hydrophobic phosphoproteins and also limited in their resolution to identify phosphoproteins present at low levels. In contrast, with rapid advances in mass spectrometric instrumentation, shotgun phosphoproteomics has become a powerful alternative to gels for analyzing complex protein samples. The breakthrough in efficient proteome-wide analysis of phosphorylation sites enabled marked progress in mapping protein kinase substrates, leading to a better understanding of cellular signaling networks. In this review, we summarize recent breakthroughs of mass spectrometric methodologies adopted to identify phosphoproteins and signaling components. We briefly describe phosphoprotein/phosphopeptide enrichment methods, and compare their advantages and limitations in phosphoproteomic studies. We show that successive enrichment of phosphopeptides using a two-step method termed tandem MOAC can markedly improve the depth of phosphoproteomic coverage. We then highlight recent advances of targeted and untargeted proteomic technologies in global studies of protein phosphorylation sites. Moreover, we summarize recent applications of untargeted phosphoproteomic studies in plants, including in research on phytohormone signaling, stress responses, and nutrient assimilation. These studies have led to the functional mapping of thousands of protein phosphorylation sites and various protein kinase substrates. Targeted proteomic approaches provide a powerful option to detect and quantify selected phosphoproteins or sites of interest. We therefore describe their principles and dynamic ranges in the detection and quantification of phosphorylated peptides. Moreover, we show that the methods of data-dependent and -independent acquisition are complementary in the global analysis of phosphoproteins. In addition, proteome-wide analysis of protein-protein interactions provides key information on signal transduction and perception in plants. Mass spectrometry-based interactomics has been increasingly used to analyze protein complexes and interaction partners in plant cells. Thus, we compare the methods that are currently most widely used in studies of protein interactomics. In conclusion, we summarize strategies that are currently used for phosphoprotein analysis and kinase substrate mapping. We also discuss their relative strengths and weaknesses, and how these methods are applied to gene functional analysis at the proteome-wide level. Overall, we conclude that decoding protein phosphorylation signaling networks could provide new insights that deepen our understanding of plant development and metabolism.