Phosphoproteomic Analysis of Soybean Roots Under Salinity by Using the iTRAQ Labeling Approach

Protein phosphorylation is one of the most important posttranslational modifications. The phosphorylation and dephosphorylation of proteins regulate almost every cellular process, and the understanding of their functions can provide insights into the regulation of living systems at the molecular level. In recent years, both the rapid developments of enrichment approaches for phosphoproteins and MS techniques have improved the research scope and depth of phosphoproteomics. Using NaCl-treated soybean roots as the experimental materials, this chapter introduces the protein extraction, digestion with filter-aided sample preparation (FASP), eight-plex iTRAQ labeling, TiO₂-based enrichment of phosphopeptides, LC-MS/MS analysis, as well as bioinformatic methods and protocols.