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

Plant phosphoproteomics provides a global view of phosphorylation-mediatedsignaling in plants; however, it demands high-throughput methods withsensitive detection and accurate quantification. Despite the widespreaduse of protein precipitation for removing contaminants and improvingsample purity, it limits the sensitivity and throughput of plant phosphoproteomicanalysis. The multiple handling steps involved in protein precipitationlead to sample loss and process variability. Herein, we developedan approach based on suspension trapping (S-Trap), termed tandem S-Trap-IMAC(immobilized metal ion affinity chromatography), by integrating anS-Trap micro-column with a Fe-IMAC tip. Compared with a precipitation-basedworkflow, the tandem S-Trap-IMAC method deepened the coverage of theArabidopsis (Arabidopsis thaliana)phosphoproteome by more than 30%, with improved number of multiplyphosphorylated peptides, quantification accuracy, and short sampleprocessing time. We applied the tandem S-Trap-IMAC method for studyingabscisic acid (ABA) signaling in Arabidopsis seedlings. We thus discoveredthat a significant proportion of the phosphopeptides induced by ABAare multiply phosphorylated peptides, indicating their importancein early ABA signaling and quantified several key phosphorylationsites on core ABA signaling components across four time points. Ourresults show that the optimized workflow aids high-throughput phosphoproteomeprofiling of low-input plant samples.

Automated summary

Plant phosphoproteomics provides a global view of phosphorylation-mediated signaling in plants, but high-throughput methods with sensitive detection and accurate quantification are needed. In this study, we developed a tandem S-Trap-IMAC method based on suspension trapping, which improved the coverage and quantification accuracy of the Arabidopsis phosphoproteome compared to precipitation-based workflow. We applied this method to study ABA signaling in Arabidopsis seedlings and quantified key phosphorylation sites on core ABA signaling components across different time points, revealing the importance of multiply phosphorylated peptides in early ABA signaling. Our optimized workflow enables high-throughput phosphoproteome profiling of low-input plant samples.