Abstract

Plant phosphoproteomics provides a global view of phosphorylation-mediated signaling in plants; however, it demands high-throughput methods with sensitive detection and accurate quantification. Although protein precipitation is indispensable for removing contaminants and improving sample purity, it limits the sensitivity and throughput of plant phosphoproteomic analysis. The multiple handling steps involved in protein precipitation lead to sample loss and process variability. Herein, we developed an approach based on suspension trapping (S-Trap), termed tandem S-Trap-IMAC (immobilized metal ion affinity chromatography), by integrating an S-Trap micro column with an Fe-IMAC tip. Compared with a precipitation-based workflow, the tandem S-Trap-IMAC method deepened the coverage of the Arabidopsis (Arabidopsis thaliana) phosphoproteome by more than 30%, with improved quantification accuracy and short sample processing time. We applied the tandem S-Trap-IMAC method for studying abscisic acid (ABA) signaling in Arabidopsis seedlings. We thus identified 24,055 phosphopeptides and quantified several key phosphorylation sites on core ABA signaling components across four time points. Our results show that the optimized workflow aids high-throughput phosphoproteome profiling of low-input plant samples.