A Suspension Trapping-based Sample Preparation Workflow for Sensitive Plant Phosphoproteomics

ABSTRACT: Plant phosphoproteomics reveals a global view of phosphorylation-mediated signaling in plants, but it remains demanding a high-throughput method with detection sensitivity and quantification accuracy. In general, the indispensable protein precipitation step requiring for interferences removal limited the sensitivity and throughput in plant phosphoproteomics analysis. Though protein precipitation improves the sample purity, the multiple handling steps cause sample loss and process variability. Here we developed a suspension trapping (S-Trap)-based approach, termed tandem S-Trap-IMAC, by integrating a S-Trap micro column with a Fe-IMAC tip for plant phosphoproteomics. In comparing to precipitation-based approach, tandem S-Trap-IMAC method deepened the coverage of Arabidopsis phosphoproteome more than 30% with improved quantification accuracy and short sample processing time compared to the precipitation-based workflow. The tandem S-Trap-IMAC method was further applied to study the abscisic acid (ABA) signaling in Arabidopsis seedlings. Among a total of 24,055 identified phosphopeptides, several key phosphorylation sites on core ABA signaling components were quantified across four time points. The optimized workflow aids in high-throughput phosphoproteome profiling of low-input plant samples.

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: Chuan-Chih Hsu 

PROVIDER: PXD039933 | JPOST Repository | Mon Aug 28 00:00:00 BST 2023

REPOSITORIES: jPOST