Suspension Trapping-Based Sample Preparation Workflow for In-Depth Plant Phosphoproteomics.

Plant phosphoproteomics provides a global view of phosphorylation-mediated signaling in plants; however, it demands high-throughput methods with sensitive detection and accurate quantification. Despite the widespread use of protein precipitation 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 a Fe-IMAC tip. Compared with a precipitation-based workflow, the tandem S-Trap-IMAC method deepened the coverage of the Arabidopsis () phosphoproteome by more than 30%, with improved number of multiply phosphorylated peptides, 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 discovered that a significant proportion of the phosphopeptides induced by ABA are multiply phosphorylated peptides, indicating their importance in early ABA signaling 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.