Unveiling Plant Proteomic Mysteries: Tandem S-Trap-IMAC Strategy Revolutionizes Phosphorylation Analysis

In our daily encounters with nature, plants are everywhere. Yet, within their seemingly ordinary structures lies a profound question: how do they sustain life? Fueled by rapid technological advancements, scientists now utilize unprecedented precision tools to explore the mysteries of plants, aiming to uncover their structures and growth mechanisms. In this mystical realm, understanding the process of plant protein phosphorylation has become one of the keys to unlocking the secrets of plant life. These tiny proteins, adorned with intricate phosphorylation modifications, control vital physiological functions in plants, regulating growth and responses to environmental stresses. As we delve deeper into how plants adapt to diverse environments, understanding the phosphorylation process of plant proteins emerges as a crucial aspect.

Proteomics, the comprehensive study of all proteins within organisms, delves into aspects such as protein types, quantities, interactions, and post-translational modifications (PTMs). PTMs refer to a series of chemical modifications that occur after the synthesis of protein, with phosphorylation being a common type of modification. Plant phosphoproteomics focuses on understanding phosphorylation processes in plants, where protein phosphorylation, a crucial regulatory mechanism, modulates protein function by adding phosphate groups. This dynamic process enables rapid signal transmission within plant cells, offering insights into how plants adapt to changes like drought, extreme temperatures, and salinity. By influencing plant responses to stimuli, such as nutrients, stress and growth hormones, protein phosphorylation not only advances our understanding of plant biology but also holds potential for improving crop yield and resilience.

The pursuit of understanding phosphorylated proteins within plant cells has led scientists to employ mass spectrometry (MS), a pivotal tool in proteomic analysis. At the core of this groundbreaking technique is the identification of phosphorylation sites on proteins. This process enables researchers to pinpoint which amino acids on the proteins are phosphorylated, offering deeper insights into how phosphorylation affects protein functionality. Initially, proteins in the sample are broken down into smaller peptide fragments using protease. Subsequently, these phosphorylated peptide fragments are enriched and injected into the mass spectrometer for analysis. With MS’s high sensitivity and accuracy, scientists can precisely identify phosphorylation sites, providing a comprehensive view of protein phosphorylation status under various physiological conditions within plant cells. This analytical approach illuminates the intricate phosphorylation networks within plant cells, revealing the changes brought about by different physiological conditions.

While MS has become a powerful tool for studying phosphorylation systematically, the challenge of detecting phosphorylated peptides directly due to their low abundance has emerged. Recently, Dr. Chuan-Chih Hsu’s team at the Proteomics Core Lab (PCL) of the Institute of Plant and Microbial Biology, Academia Sinica, introduced a solution named “Tandem S-Trap-IMAC.” This innovative method effectively reduces the loss of phosphorylated proteins during sample preparation, addressing limitations of traditional approaches.

The tandem S-Trap-IMAC workflow is built upon the suspension trapping (S-Trap) sample pre-processing method. It integrates a S-Trap micro-column with a Fe-IMAC tip. The operational procedure involves directly loading digested peptides from the S-Trap into the Fe-IMAC tip, eliminating the need for additional processing steps. This results in the direct enrichment of phosphorylated peptides in plant cells. Compared to traditional sample processing methods, this technique simplifies procedures such as desalting and buffer exchange, reducing sample preparation time and significantly increasing experimental analysis throughput. Moreover, unlike conventional protein precipitation-based workflows, where the separation of proteins and plant pigments may lead to sample loss, the new technique prevents such losses. It improves the identification and quantification accuracy of multiply phosphorylated peptides. Thus, the development of this new approach has become a valuable analytical tool in the field of plant biology research.

Figure 1: Experimental design of the tandem S-Trap-IMAC workflow
for plant phosphoproteomics.

Interfering contaminants in the plant lysate are removed by washing the S-Trap, and proteins are digested within the S-Trap. Phosphopeptides are enriched using an Fe-IMAC tip via a direct loading strategy and analyzed using a Fusion Lumos mass spectrometer in data-dependent acquisition mode.

To demonstrate the scalability of the tandem S-Trap-IMAC method for plant phosphoproteomic analysis, PCL research team applied it to study abscisic acid (ABA) signaling in Arabidopsis seedlings. ABA plays a crucial role in plant growth and responses to environmental stress, such as water shortage and nutrient deficiency. Studying changes in ABA signaling contributes to a deeper understanding of plant adaptation mechanisms. The experiment quantitatively analyzed phosphorylation sites on core ABA signaling enzymes at four time points (0, 10, 30, and 60 minutes). Results showed that 60% of early ABA-induced phosphopeptides were multiply phosphorylated, indicating their importance in early ABA signaling. This underscores the significance of multiple phosphorylation events in regulating enzymes involved in early ABA signaling.

Figure 2: Phosphoproteomic analysis of ABA-dependent signaling in Arabidopsis.
A) Schematic representation of the experimental workflow.
B) Principal component analysis of the phosphorylation sites identified across all replicates of four groups.
C) The number of singly and multiply phosphorylated peptides significantly increased after 10, 30, and 60 min of ABA treatment.
D)  The number of singly and multiply phosphorylated peptides significantly down-regulated after 10, 30, and 60 min of ABA treatment.
E) Heatmap showing the relative intensity of key phosphorylation sites during time-dependent ABA signaling.

The development of tandem S-Trap-IMAC approach has provided a new perspective for the analysis of phosphorylated proteins in plants. This innovation lies not only in its high-throughput sample processing but also in its enhancement of the identification and quantification accuracy of multiply phosphorylated peptides. Being able to directly and more comprehensively explore crucial phosphorylation events within plant cells will contribute to a deeper understanding of the fundamental mechanisms in plant biology.

The pursuit of cutting-edge analytical methods persists as scientists relentlessly innovate to unveil captivating revelations about the intricacies of plant growth. This effort not only holds the potential to revolutionize agricultural practices but also offers unprecedented opportunities for environmental resilience and sustainability.


Chin-Wen Chen, Chia-Feng Tsai, Miao-Hsia Lin, Shu-Yu Lin, Chuan-Chih Hsu*
Suspension trapping-based sample preparation workflow for in-depth plant phosphoproteomics.
Anal. Chem., 2023,