Universal Sample Preparation Workflow for Plant Phosphoproteomic Profiling.

Area of Science:

• Plant biology
• Biochemistry
• Proteomics

Background:

• Mass spectrometry (MS)-based phosphoproteomics is crucial for cell signaling research.
• Studying plant phosphoproteomes is challenging due to low cell lysis yield and complex plant lysates.

Purpose of the Study:

• To develop a streamlined, high-throughput sample preparation workflow for plant phosphoproteomics.
• To address low yield and interference issues in plant lysate analysis.
• To achieve in-depth phosphoproteomic coverage and build a comprehensive signaling network resource.

Main Methods:

• A streamlined sample preparation workflow was developed, optimizing lysis and reducing pigment/metabolite interference.
• Chemical labeling and high pH reverse phase fractionation were integrated for enhanced phosphoproteomic coverage.
• The workflow's scalability was demonstrated through analysis of cold stress effects on the tomato phosphoproteome.

Main Results:

• The workflow successfully addressed low yield and interference in plant lysate analysis.
• In-depth phosphoproteomic analysis identified over 30,000 phosphopeptides from tomato leaves.
• Over 5,000 kinase-substrate pairs were identified in Arabidopsis, establishing the largest phosphoproteomic and signaling network resource to date.

Conclusions:

• The developed workflow significantly advances high-throughput plant phosphoproteomics.
• This approach provides an unprecedented resource for understanding plant signaling networks and responses to environmental stress.
• The method is scalable and applicable to diverse plant species and research questions.