Preface
Contents
Contributors
Chapter 1: Phosphorylation Site Motifs in Plant Protein Kinases and Their Substrates
1 Introduction
2 Relationship Between Kinases Families and Biological Functions
3 Phosphorylation Motifs and Functional Classification in Arabidopsis
3.1 P-Motifs in Kinase Substrates
3.2 Frequently Identified P-Motifs Functions
4 Frequently Identified Motifs in Kinase Families
5 Conclusion
References
Chapter 2: Protein Phosphorylation Response to Abiotic Stress in Plants
1 Introduction
2 Protein Phosphorylation
3 Ion Stress Signaling
3.1 Protein Phosphorylation and the SOS Pathway
3.2 Protein Phosphorylation for Ion Homeostasis Is Crucial for Plant Response to Abiotic Stress
4 Phosphorylation and the ROS-Mediated (Osmotic) Stress Response
5 Phytohormone-Linked Protein Phosphorylation
5.1 ABA-Responsive Genes
5.2 GA-Responsive Genes
5.3 Brassinosteroids-Responsive Genes
6 Nonstress Factor That Affects Protein Phosphorylation
7 Conclusion
References
Chapter 3: Protein Phosphorylation in Plant Cell Signaling
1 Introduction
2 Protein Kinases and Phosphatases in Plants
3 Protein Phosphorylation in Plant Hormone Signaling
3.1 Protein Phosphorylation Regulates the Biosynthesis and Signaling of Auxin, Ethylene, and Gibberellins
3.2 Protein Phosphorylation Modulates ABA, CK, and BR Signaling as Well as Their Crosstalk
3.3 Protein Phosphorylation in Defense Phytohormone (JA and SA) Signaling
4 Protein Phosphorylation in Plant Stress Responses
4.1 Protein Phosphorylation of Abiotic Stress Sensors in Different Organelles
4.2 Protein Phosphorylation Modulates the Signal Transduction of Abiotic Stresses
4.2.1 Heat Stress
4.2.2 Cold Stress
4.2.3 Salt Stress
4.2.4 Drought Stress
5 Conclusion and Perspective
References
Chapter 4: Phosphoproteomics Profiling of Receptor Kinase Mutants
1 Introduction
2 Materials
2.1 Buffers and Solutions
2.2 Other Materials
2.3 Specific Equipment
3 Methods
3.1 Sample Preparation
3.2 In-Solution Trypsin Digestion
3.3 TiO2 Phosphopeptide Enrichment
3.4 Peptide Desalting Using C18 StageTips
3.5 LC-MS/MS Analysis and Peptide Identification
3.6 Label-Free Relative Quantification Analysis
4 Notes
References
Chapter 5: Phosphoproteomic Analysis of Soybean Roots Under Salinity by Using the iTRAQ Labeling Approach
1 Introduction
2 Materials
2.1 Plant and Plant Growth Materials
2.2 Protein Extraction
2.3 Protein Digestion with Filter Aided Sample Preparation (FASP)
2.4 Eight-Plex iTRAQ Labeling and Phosphopeptide Enrichment
2.5 NanoRPLC-MS/MS Analysis of Phosphopeptides
3 Methods
3.1 Seed Sterilization (See Note 1)
3.2 Plant Growth (See Note 2)
3.3 Protein Extraction (See Note 4)
3.4 Protein Digestion with FASP
3.5 Eight-Plex iTRAQ Labeling and Phosphopeptide Enrichment
3.6 NanoRPLC-MS/MS Analysis of Phosphopeptides (See Note 5)
3.7 Phosphopeptide Identification and Quantitative Analysis
3.8 Bioinformatic Analysis
4 Notes
References
Chapter 6: Universal Sample Preparation Workflow for Plant Phosphoproteomic Profiling
1 Introduction
2 Materials
2.1 Plant Tissue Lysis
2.2 Protein Precipitation
2.3 Protein Digestion
2.4 Peptide Labeling
2.5 Phosphopeptide Enrichment Using PolyMAC-Ti
2.6 Phosphopeptide Fractionation
2.7 Mass Spectrometry and Data Analysis
3 Methods
3.1 Plant Tissue Lysis
3.2 Protein Precipitation
3.3 Protein Digestion
3.4 Peptide Dimethyl Labeling
3.5 Phosphopeptide Enrichment Using PolyMAC-Ti
3.6 Phosphopeptide Fractionation Using Hp-RP StageTip (See Note 10)
3.7 Mass Spectrometry and Data Analysis
4 Notes
References
Chapter 7: Mapping Plant Phosphoproteome with Improved Tandem MOAC and Label-Free Quantification
1 Introduction
2 Materials
2.1 Plant Growth
2.2 Total Protein Extraction
2.3 Al(OH)3-Based MOAC Enrichment of Phosphoproteins
2.4 In-Solution Digestion
2.5 TiO2-Based MOAC Enrichment of Phosphopeptides
2.6 Nano-RPLC-MS Analysis
3 Methods
3.1 Plant Growth
3.2 Protein Extraction
3.3 Al(OH)3-Based MOAC Enrichment of Phosphoproteins
3.4 In-Solution Digestion
3.5 TiO2-Based MOAC Enrichment of Phosphopeptides
3.6 Nano-RPLC-MS Analysis
3.7 Peptide Identification and Label-Free Quantification (LFQ)
4 Notes
References
Chapter 8: SILIA-Based 4C Quantitative PTM Proteomics
1 Introduction
2 Materials
2.1 SILIA Growth Medium
2.2 Growth Container
2.3 Urea-Based Protein Extraction Buffer (UEB)
2.4 Precipitation Solution
2.5 Protein Resuspension Buffer (PRB)
2.6 Trypsin Digestion Solution (TDS)
2.7 TiO2-Binding Solution
2.8 TiO2-Washing Solution
2.9 IMAC-Binding Buffer
2.10 Kinase Extraction Buffer (KEB)
2.11 Kinase Assay Buffer (KAB)
2.12 SCX Column Loading Buffer (SLB)
2.13 SCX Column Elution Buffer (SEB)
2.14 Affinity Beads for Phosphopeptide Enrichment
3 Methods
3.1 Labeling Plants with Heavy Nitrogen
3.2 Tissue Harvesting
3.3 Extraction of Plant Protein
3.4 Peptide Preparation
3.5 TiO2 Bead Affinity Enrichment
3.6 Fe+3IMAC Bead Affinity Enrichment
3.7 SCX Chromatographic Separation
3.8 LC-MS/MS Analysis
3.9 Identification of PTM Peptides
3.10 Quantitation of PTM Peptides
3.11 Motif Analysis
3.12 String Analysis
3.13 Gene Ontology Analysis
3.14 Double In Vivo Substrate and Kinase Assay (DISKA)
3.15 AQUIP Analysis of PTM Occupancy
3.16 Molecular and Cellular Biological Validation
4 Notes
References
Chapter 9: Phosphoproteomics Analysis of Plant Root Tissue
1 Introduction
2 Materials
2.1 Buffers and Solutions
2.2 Other Materials
2.3 Specific Equipment
3 Methods
3.1 Protein Extraction
3.2 Protein Precipitation
3.3 In-Solution Trypsin Digestion
3.4 Phosphopeptides Enrichment
3.5 Desalting
3.6 LC-MS/MS Analysis
4 Notes
References
Chapter 10: Plant Phosphopeptides Enrichment by Immobilized Metal Ion Affinity Chromatography
1 Introduction
2 Materials
2.1 Plant Sample
2.2 Protein Extraction
2.3 Resuspension, Reduction, Alkylation, and Digestion of Plant Proteins
2.4 Desalting of Tryptic Peptides
2.5 Prefractionation of Tryptic Peptides by SCX-HPLC
2.6 Immobilized Metal Ion Affinity Chromatography
2.7 Desalting of Phosphopeptides by StageTips
2.8 Mass Spectrometry
2.9 Other Materials
3 Methods
3.1 Protein Extraction
3.2 Resuspension, Reduction, Alkylation, and Digestion of Plant Proteins
3.3 Desalting of Tryptic Peptides
3.4 Prefractionation of Tryptic Peptides by SCX-HPLC
3.5 Immobilized Metal Ion Affinity Chromatography (Fig. 1)
3.6 Desalting of Phosphopeptides by StageTips
3.7 Mass Spectrometry
4 Notes
References
Chapter 11: Two-Dimensional Gel Electrophoresis and Pro-Q Diamond Phosphoprotein Stain-Based Plant Phosphoproteomics
1 Introduction
2 Materials
2.1 Protein Extraction
2.2 Two-Dimensional Electrophoresis (2-DE) Gel Preparation
2.3 Gel Staining and Image Acquisition
2.4 In-Gel Digestion and MS
3 Methods
3.1 Plant Total Protein Preparation Using TCA/Acetone Extraction Method
3.2 Protein Separation by 2-DE Gel Electrophoresis
3.3 Gel Analysis by Pro-Q Diamond Staining
3.4 Phosphoproteins Identification by MALDI-TOF MS
4 Notes
References
Chapter 12: 2-D DIGE Combined with Pro-Q Diamond Staining for the Identification of Protein Phosphorylation for Chlamydomonas ...
1 Introduction
2 Materials
2.1 Buffers
2.2 Equipment/Chemicals
3 Methods
3.1 Plant Material Cultivation
3.2 Extraction of Total Protein
3.3 Protein CyDye Labeling
3.4 Preparation of Traditional 2-DE for Pro-Q Diamond Staining
3.5 Electrophoresis
3.6 DIGE Analysis
3.7 Pro-Q Diamond Staining and Phosphoproteins Profiling
4 Notes
References
Chapter 13: Plant Phosphopeptide Identification and Label-Free Quantification by MaxQuant and Proteome Discoverer Software
1 Introduction
2 Materials
2.1 MaxQuant
2.2 Proteome Discovery
3 Methods
3.1 Use of MaxQuant
3.2 Use of Proteome Discoverer (PD)
4 Notes
References
Chapter 14: PhosPhAt 4.0: An Updated Arabidopsis Database for Searching Phosphorylation Sites and Kinase-Target Interactions
1 Introduction
2 Search of the Phosphorylation Sites with PhosPhAt 4.0
3 Phosphorylation Site Prediction by PhosPhAt 4.0
4 Motif Search by PhosPhAt 4.0
5 Kinase-Target Relationships Retrieval by PhosPhAt 4.0
6 Retrieval of Kinase-Substrate Relationships by Family Search´´ Mode
7 Conclusion
8 Notes
References
Chapter 15: Computational Phosphorylation Network Reconstruction: An Update on Methods and Resources
1 Introduction
2 Basis of Phosphorylation Network Reconstruction
2.1 (De-)Phosphorylation Substrate Specificity
2.2 Protein-Protein Interaction Network
2.3 Quantitative Phosphorylation Level Profiling
3 (De-)Phosphorylation Substrate Prediction
3.1 Kinase-Generic Phosphorylation Site Prediction
3.2 Kinase-Specific Phosphorylation Site Prediction
3.3 Phosphatase-Substrate Relationships (PSRs) Prediction
4 Phosphorylation-Mediated Protein Interaction Network Reconstruction
4.1 Protein-Protein Interactions (PPI) Mapping
4.2 Inference Methods Exploiting Quantitative Proteomics Data
4.3 Heterogeneous Data-Based Integration Method
5 Summary and Future Challenges
References
Chapter 16: The Application of an R Language-Based Platform cRacker for Phosphoproteomics Data Analysis
1 Introduction
2 Materials
3 Methods
3.1 Loading the Data
3.2 Parameters and Settings
3.2.1 TheMain´´ Tab
3.2.2 The PeptidesProteins´´ Tab
3.2.3 TheExtra´´ Tab
3.2.4 The Quantitation Mode´´ Tab
3.2.5 TheStatistics´´ Tab
3.2.6 The Plotting´´ Tab
3.2.7 ThePaths´´ Tab
3.3 Output
4 Notes
References
Chapter 17: Kinase Activity Assay Using Unspecific Substrate or Specific Synthetic Peptides
1 Introduction
2 Materials
2.1 Purifying Kinase-GFP Complex
2.2 Collecting the (Phosphorylated) Peptides from the Kinase Reaction
2.3 Kinase Assay
3 Methods
3.1 Purification of Kinase-GFP Complex
3.2 In Vitro Peptide Phosphorylation Assay (See Note 5)
3.3 Kinase Activity Assay by Using Unspecific Substrate
4 Notes
References
Correction to: Plant Phosphopeptide Identification and Label-Free Quantification by MaxQuant and Proteome Discoverer Software
Index