Plant Proteomics: Methods and Protocols (Methods in Molecular Biology (2139))

Preface
Contents
Contributors
Chapter 1: What Is New in (Plant) Proteomics Methods and Protocols: The 2015-2019 Quinquennium
	1 Introduction
	2 Novelties in the 2015-2019 Period
	3 Proteomics Data Validation, and Integration into Other Classic and -Omics Approaches in the Systems Biology Direction
	References
Chapter 2: Multiple Biomolecule Isolation Protocol Compatible with Mass Spectrometry and Other High-Throughput Analyses in Mic...
	1 Introduction
	2 Materials
		2.1 Cell Culture Materials
		2.2 Sampling and Extraction Materials
		2.3 Sampling and Extraction Reagents and Solutions
	3 Methods
		3.1 Sampling Method
		3.2 Metabolite Extraction Method
		3.3 Pigment Extraction Method
		3.4 Lipid Extraction Method
		3.5 Nucleic Acid Purification Method
		3.6 Protein Extraction and Purification Methods
	4 Notes
	References
Chapter 3: Protein Interaction Networks: Functional and Statistical Approaches
	1 Introduction
	2 Materials
	3 Methods
		3.1 Selection of Differential Expression Proteins (for Targeted Networks)
			Workflow
		3.2 Integration Tools
			3.2.1 Statistical Integration Networks: Dynamic Protein-Protein Interaction Networks
				Workflow
			3.2.2 Statistical Interaction Networks between Proteins with Other Omics Datasets
				Partial Least Square Regression (PLS) and Variates
					Workflow
				Data-Driven Integration and Differential Network Analysis (xMWAS)
					Workflow
				DIABLO
					Workflow
		3.3 Biological Interaction Network Enrichment
			3.3.1 STRING
				Workflow
			3.3.2 ShinyGO
				Workflow
		3.4 Merged Functional and Statistical Interaction Networks
			Create a STRING Network for Merged Networks
			3.4.1 Cytoscape Merged Functional and Statistical Interaction Network
				Workflow
			3.4.2 Manually Merged Functional and Statistical Interaction Network
				Workflow
		3.5 Network Visualization Tools
			3.5.1 Cytoscape
				Workflow
			3.5.2 Gephi
				Workflow
		3.6 Future Perspectives
	4 Notes
	References
Chapter 4: Specific Protein Database Creation from Transcriptomics Data in Nonmodel Species: Holm Oak (Quercus ilex L.)
	1 Introduction
	2 Materials
		2.1 Nucleotide Sequences
			2.1.1 Required Software
		2.2 Raw Reads Quality Control
		2.3 Preprocessing Raw Data
		2.4 Assembling Raw Data
		2.5 Removing Redundant Transcripts
		2.6 Evaluating the Assembly Structure and Completeness of a Transcriptome
		2.7 Annotation of a Transcriptome
		2.8 Construction of a Custom Protein Database
	3 Methods
		3.1 Nucleotide Sequences
		3.2 Raw Reads Quality Control (See Note 2)
		3.3 Preprocessing Raw Data
		3.4 Assembling Raw Data
		3.5 Removing Redundant Transcripts
		3.6 Evaluating the Assembly Structure and Completeness of a Transcriptome
		3.7 Annotation of a Transcriptome
		3.8 Construction of a Custom Protein Database
	4 Notes
	References
Chapter 5: Subcellular Proteomics in Conifers: Purification of Nuclei and Chloroplast Proteomes
	1 Introduction
	2 Materials
		2.1 Nuclei Isolation
		2.2 Chloroplast Isolation
		2.3 Protein Extraction
	3 Methods
		3.1 Nuclei Isolation
		3.2 Chloroplast Isolation
		3.3 Protein Extraction
	4 Expected Results
	5 Notes
	References
Chapter 6: Apoplastic Fluid Preparation from Arabidopsis thaliana Leaves Upon Interaction with a Nonadapted Powdery Mildew Pat...
	1 Introduction
	2 Materials
		2.1 Plant Growth and Pathogen Inoculation
		2.2 Apoplastic Fluid Preparation
		2.3 Protein Purification by Chloroform-Methanol Precipitation
	3 Methods
		3.1 Plant Growth and Pathogen Inoculation
		3.2 Apoplastic Fluid Preparation
		3.3 Protein Purification by Chloroform-Methanol Precipitation
	4 Notes
	References
Chapter 7: Shotgun Proteomics of Plant Plasma Membrane and Microdomain Proteins Using Nano-LC-MS/MS
	1 Introduction
	2 Materials
		2.1 Plasma Membrane Purification Components
		2.2 Detergent-Resistant Membrane Extraction Components
		2.3 In-Gel Tryptic Digestion
			2.3.1 SDS-Polyacrylamide Gel Components
			2.3.2 Tryptic Digestion Components
		2.4 In-Solution Tryptic Digestion
		2.5 Peptide Purification Components
	3 Methods
		3.1 Plasma Membrane Purification
		3.2 Detergent-Resistant Membrane Extraction
		3.3 In-Gel Tryptic Digestion
			3.3.1 SDS-Polyacrylamide Gel Electrophoresis
			3.3.2 In-Gel Tryptic Digestion for Nano-LC-MS/MS
		3.4 In-Solution Tryptic Digestion
		3.5 Peptide Purification
		3.6 Nano-LC-MS/MS Analysis
	4 Notes
	References
Chapter 8: A Protocol for the Plasma Membrane Proteome Analysis of Rice Leaves
	1 Introduction
	2 Materials
		2.1 Plant Material
		2.2 Reagents, Equipment, and Software
		2.3 Buffers
	3 Methods
		3.1 Extraction of Plasma Membrane Proteins
		3.2 In-Solution Trypsin Digestion by Filter-Aided Sample Preparation (FASP)
		3.3 Desalting of Peptides
		3.4 Mass Spectrometry
		3.5 Data Processing Using MaxQuant Software
	4 Notes
	References
Chapter 9: Isolation, Purity Assessment, and Proteomic Analysis of Endoplasmic Reticulum
	1 Introduction
	2 Materials
		2.1 Isolation of ER Fraction
		2.2 Immunoblot Analysis for ER Purity Assessment
		2.3 Enzymatic Analysis for ER Purity Assessment
		2.4 Concentration Measurement of Proteins
		2.5 Proteomic Analysis of ER Proteins
		2.6 Search Engine, Software, and Database for Proteomic Analysis
	3 Methods
		3.1 Isolation of Total ER Fraction
		3.2 Isolation of Rough ER Fraction
		3.3 Immunoblot Analysis for ER Purity Assessment of ER Fraction
		3.4 Enzymatic Analysis for ER Purity Assessment of ER Fraction
		3.5 Proteomic Analysis of ER Proteins
			3.5.1 Preparation of Peptides for Gel-Free/Label-Free Proteomic Analysis
			3.5.2 Mass Spectrometry Analysis
			3.5.3 Protein Identification from Acquired Mass Spectrometry Data
			3.5.4 Analysis of Relative Protein Abundance Using Acquired Mass Spectrometry Data
			3.5.5 Analysis of Absolute Protein Amount Using Acquired Mass Spectrometry Data
			3.5.6 Visualization of Protein Abundance
			3.5.7 Protein Localization Prediction
	4 Notes
	References
Chapter 10: Dimethyl Labeling-Based Quantitative Proteomics of Recalcitrant Cocoa Pod Tissue
	1 Introduction
	2 Materials
		2.1 Sample Preparation and Labeling
		2.2 Sample Analysis by LC-MS/MS
	3 Methods
		3.1 Sample Preparation and Labeling
			3.1.1 Protein Extraction
			3.1.2 Sample Precipitation
			3.1.3 Sample Trypsin Digestion
			3.1.4 Sample Dimethyl Labeling
			3.1.5 Sample Cleaning
		3.2 Sample Analysis by LC-MS/MS
		3.3 Protein Identification and Quantitation
			3.3.1 LC-MS and MS/MS Processing with Progenesis QI for Proteomics
			3.3.2 Protein Identification
			3.3.3 Protein Identification Refinement with Progenesis QI for Proteomics
			3.3.4 Protein Quantitation in Proteolabels
	4 Notes
	References
Chapter 11: Quantitative Profiling of Protein Abundance and Phosphorylation State in Plant Tissues Using Tandem Mass Tags
	1 Introduction
	2 Materials
		2.1 Protein Extraction
		2.2 Filter-Aided Sample Preparation (FASP) and on Filter Digestion
		2.3 C18 Desalting
		2.4 TMT Labeling
		2.5 Phosphopeptide Enrichment
	3 Methods
		3.1 Protein Extraction
		3.2 FASP
		3.3 C18 Desalting
		3.4 TMT Labeling
		3.5 Phosphopeptide Enrichment
	4 Notes
	References
Chapter 12: Optimizing Shotgun Proteomics Analysis for a Confident Protein Identification and Quantitation in Orphan Plant Spe...
	1 Introduction
	2 Material
		2.1 Plant Material
		2.2 Protein Extraction
		2.3 SDS Polyacrylamide Gel
		2.4 Protein Digestion
		2.5 Peptide Desalting
		2.6 Solutions for LC-MS/MS
		2.7 Equipment and Software
	3 Methods
		3.1 Protein Extraction by TCA/Acetone/Phenol
		3.2 SDS-PAGE Electrophoresis
		3.3 Sample Preparation for MS Analysis
			3.3.1 Protein Digestion
			3.3.2 Peptides Extraction
			3.3.3 Peptides Desalting
		3.4 nLC-MS/MS
		3.5 Protein Identification
		3.6 Protein Quantification
	4 Anticipated Results
	5 Notes
	References
Chapter 13: Combining Targeted and Untargeted Data Acquisition to Enhance Quantitative Plant Proteomics Experiments
	1 Introduction
	2 Materials
		2.1 Creation of Inclusion Lists and TDA/DDA LC-MS/MS Methods
	3 Methods
		3.1 Creation of Inclusion Lists
		3.2 Creation of TDA/DDA LC-MS/MS Methods
	4 Notes
	References
Chapter 14: A Phosphoproteomic Analysis Pipeline for Peels of Tropical Fruits
	1 Introduction
		1.1 Fractionation Methods
		1.2 Enrichment Methods
		1.3 Quantification Strategies
		1.4 Plant Phosphoproteome Tools and Databases
	2 Materials
		2.1 Total Protein Extraction
		2.2 SDS-Polyacrylamide Gel Electrophoresis
		2.3 Reduction, Alkylation, and Digestion
		2.4 Direct Phosphopeptide Enrichment
		2.5 High pH Reversed-Phase (RP) Fractionation
		2.6 SCX-RP Fractionation and Enrichment
		2.7 Other Materials
	3 Methods
		3.1 Tissue Protein Extraction
		3.2 Subject the Extract to SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) According to Laemmli
		3.3 Reduction, Alkylation, and Digestion
		3.4 Direct Fe-NTA Enrichment
		3.5 Fractionation Prior to Fe-NTA Enrichment
			3.5.1 High RP Fractionation and Enrichment
			3.5.2 SCX-RP Fractionation and Enrichment
		3.6 LC/MS-MS Analysis
	4 Notes
	References
Chapter 15: Label-Free Quantitative Phosphoproteomics for Algae
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Protein Extraction
		2.3 Reduction, Alkylation, and Digestion
		2.4 Desalting
		2.5 Phosphopeptide Enrichment
		2.6 Sample Purification
		2.7 LC-MS/MS
		2.8 Data Analysis
	3 Methods
		3.1 Culturing
		3.2 Protein Extraction
		3.3 Reduction, Alkylation, and Digestion
		3.4 Desalting
		3.5 Phosphopeptide Enrichment
		3.6 Sample Purification
		3.7 LC-MS/MS
		3.8 Data Analysis
	4 Notes
	References
Chapter 16: Targeted Quantification of Phosphopeptides by Parallel Reaction Monitoring (PRM)
	1 Introduction
	2 Materials
	3 Methods
		3.1 Sample Preparation for DDA and PRM Measurements
		3.2 DDA Measurement Using an Orbitrap Mass Spectrometer
		3.3 DDA Data Processing with MaxQuant Software
		3.4 Target List Construction with Skyline Software Using the MaxQuant Output File
		3.5 PRM Data Acquisition
		3.6 PRM Data Analysis
		3.7 Target List Construction with the Skyline Software by In Silico Digest
	4 Notes
	References
Chapter 17: Enrichment of N-Linked Glycopeptides and Their Identification by Complementary Fragmentation Techniques
	1 Introduction
	2 Materials
		2.1 Microsomal Preparation
		2.2 Digestion and Hydrophilic Interaction Liquid Chromatography (HILIC) Enrichment of N-Glycopeptides
		2.3 Identification of N-Glycopeptides by Tandem Mass Spectrometry
		2.4 Spectral Data Interrogation and Matching
	3 Methods
		3.1 Preparation of Microsomal Fraction and Peptide Digestion
		3.2 Digestion and N-Glycopeptide Enrichment
		3.3 Identification of N-Glycopeptides by Tandem Mass Spectrometry
		3.4 Spectral Data Interrogation
	4 Notes
	References
Chapter 18: High-Resolution Lysine Acetylome Profiling by Offline Fractionation and Immunoprecipitation
	1 Introduction
	2 Materials
		2.1 Protein Extraction
		2.2 Filter-Aided Sample Preparation (FASP)
		2.3 Desalting and Dimethyl Labeling of Peptides
		2.4 ZIC-HILIC Offline Fractionation
		2.5 Enrichment of Lysine-Acetylated Peptides
		2.6 Desalting of Peptides on SDB-RPS Stop-and-Go-Extraction Tips (Stage Tips)
	3 Methods
		3.1 Protein Extraction
		3.2 Filter-Aided Sample Preparation (FASP)
		3.3 Desalting and Dimethyl Labeling of Peptides on C18 Columns
		3.4 ZIC-HILIC Offline Fractionation
		3.5 Enrichment of Lysine-Acetylated Peptides
		3.6 Desalting of Peptides on SDB-RPS Stop-and-Go-Extraction Tips (Stage Tips)
		3.7 Guidelines for LC-MS/MS Analysis
	4 Notes
	References
Chapter 19: A Versatile Workflow for the Identification of Protein-Protein Interactions Using GFP-Trap Beads and Mass Spectrom...
	1 Introduction
	2 Materials
		2.1 Plant Tissue
		2.2 Immunoaffinity Pull-Down Procedure
		2.3 SDS-Polyacrylamide Gel Electrophoresis
		2.4 Western Blot
		2.5 Mass Spectrometry
	3 Methods
		3.1 Pull-Down Procedure
			3.1.1 Extraction of Intact Protein Complexes
			3.1.2 GFP-Trap a Capture of Protein Complexes
		3.2 Validation of the Pull-Down Procedure
			3.2.1 SDS-PAGE
			3.2.2 Western Blot Analysis
		3.3 Sample Preparation for Mass Spectrometry Analysis
		3.4 LC-MS/MS Data Acquisition, Data Processing, and Statistical Analysis
	4 Notes
	References
Chapter 20: In Vivo Cross-Linking to Analyze Transient Protein-Protein Interactions
	1 Introduction
	2 Materials
		2.1 Plant Material
		2.2 Formaldehyde Cross-Linking of Cells
		2.3 Preparation of Cell Lysates
		2.4 Immunoaffinity Purification
		2.5 In-Solution Trypsin Digestion
		2.6 Peptide Desalting with C18-StageTips
	3 Methods
		3.1 Formaldehyde Cross-Linking and Preparation of Cell Lysates
		3.2 Covalent Coupling of Antibodies to Magnetic Beads
		3.3 Immunoprecipitation (Antigen Binding to Ig-Coated Beads)
		3.4 In-Solution Digestion
		3.5 Peptide Desalting and Purification
	4 Notes
	References
Chapter 21: Proteome Analysis of 14-3-3 Targets in Tomato Fruit Tissues
	1 Introduction
	2 Materials
		2.1 Plant Material
		2.2 Immunoprecipitation of 14-3-3 Complexes from Tomato Fruit Tissue
		2.3 SDS-PAGE and SYPRO Ruby Stain
		2.4 In-Gel Trypsin Digestion
		2.5 LC-MS/MS Analysis
	3 Methods
		3.1 Immunoprecipitation of 14-3-3 Complex from Tomato Fruit Tissue
		3.2 SDS-PAGE and SYPRO Ruby Staining
		3.3 In-Gel Trypsin Digestion
		3.4 LC-MS/MS Analysis and Protein Identification
	4 Notes
	References
Chapter 22: The Use of Proteomics in Search of Allele-Specific Proteins in (Allo)polyploid Crops
	1 Introduction
	2 Methods
		2.1 Workflow 1: No Resources Available for mRNA Seq (Fig. 2)
		2.2 Workflow 2: Resources Are Available to Generate mRNA Seq Libraries (Fig. 4)
	3 Notes
	References
Chapter 23: Methods for Optimization of Protein Extraction and Proteogenomic Mapping in Sweet Potato
	1 Introduction
	2 Materials
		2.1 Materials for Phenol Procedure, Method 1 (M1)
			2.1.1 Stock Solutions
		2.2 Materials for Polyethylene Glycol (PEG) Procedure 4000, Method 2 (M2)
			2.2.1 Stock Solutions
	3 Methods
		3.1 Overview of the Protein Extraction and Optimization Methodology
			3.1.1 Tissue Collection
			3.1.2 Protein Extraction Using Phenol Procedure (M1) (See Notes 1-4)
			3.1.3 Protein Extraction Using Polyethylene Glycol Procedure 4000 (M2)
		3.2 LC-MS/MS and Peptide Identification
		3.3 Proteogenomic Analysis Workflow
		3.4 Proteogenomic Analysis Method (See Note 5)
			3.4.1 Data Input Processing for Proteogenomic Analysis
			3.4.2 Blast Peptides against Genome and Transcriptome Annotations
			3.4.3 Classify Peptides and Generate New Annotations (See Note 6)
		3.5 Novel Peptide Analysis and Validation
	4 Notes
	References
Chapter 24: In Silico Analysis of Class III Peroxidases: Hypothetical Structure, Ligand Binding Sites, Posttranslational Modif...
	1 Introduction
	2 Materials
		2.1 Amino Acid Sequences
			2.1.1 PeroxiBase (RedOxiBase)
		2.2 Physicochemical Properties
			2.2.1 ProtParam v. 1.0
		2.3 Topology
			2.3.1 TMHMM v. 2.0
			2.3.2 HMMTOP v. 2.0
		2.4 Signal Peptides and Localization
			2.4.1 SignalP v. 4.1
			2.4.2 PSORT v. 1.0)
		2.5 Posttranslational Modifications
			2.5.1 Pyrrolidone Carboxylic Acid Modification (PROSITE v. 20.0)
			2.5.2 N-Glycosylation (NetNGlyc v. 1.0)
			2.5.3 Palmitoylation (CSS-PALM v. 2.0)
			2.5.4 GPI-Anchor (GPI-SOM)
		2.6 Tertiary Structure
			2.6.1 Modeling of Protein Structure
				SwissModel
				Phyre2 v. 2.0
		2.7 Interactive Visualization of Structures
			2.7.1 PyMol v. 2.2
				APBS Plugin
			2.7.2 UCSF Chimera v. 1.13.1
		2.8 Docking Analyses
			2.8.1 3DLigandSite v. 1.0
			2.8.2 PatchDock Server v. 1.3
			2.8.3 SwissDock Server
				Target Templates
				Substrate Templates (ZINC v. 12)
	3 Methods
		3.1 Amino Acid Sequences
			3.1.1 PeroxiBase
		3.2 Physicochemical Properties
		3.3 Topology
			3.3.1 TMHMM
			3.3.2 HMMTOP
		3.4 Signal Peptides and Localization
			3.4.1 SignalP
			3.4.2 PSORT
		3.5 Posttranslational Modifications
			3.5.1 Pyrrolidone Carboxylic Acid (PCA)
			3.5.2 N-Glycosylation
			3.5.3 Palmitoylation
			3.5.4 GPI-Anchor
		3.6 Tertiary Structure
			3.6.1 Modeling of Protein Structure
				SwissModel
				Phyre 2
		3.7 Interactive Visualization of Structures
			3.7.1 PyMOL
				Tertiary Structure
				Alignment
				APBS Plugin
			3.7.2 UCSF Chimera
				Tertiary Structure
				Active Center
				Surface by Properties of Residues
		3.8 Docking Analyses
			3.8.1 3DLigandSite
			3.8.2 Protein-Heme Docking
			3.8.3 SwissDock Analysis
				Protein-Substrate Docking
			3.8.4 Visualization of Docking Results
	4 Notes
	References
Chapter 25: MALDI Mass Spectrometry Imaging of Peptides in Medicago truncatula Root Nodules
	1 Introduction
	2 Materials
		2.1 Embedding Nodules
		2.2 MALDI-MSI Sample Preparation
	3 Methods
		3.1 Embedding Nodules
		3.2 MALDI-MSI Sample Preparation
		3.3 MSI Data Acquisition on the MALDI LTQ Orbitrap XL
		3.4 Data Processing
	4 Notes
	References
Chapter 26: Cystatin Activity-Based Protease Profiling to Select Protease Inhibitors Useful in Plant Protection
	1 Introduction
	2 Materials
		2.1 Biotinylated Plant Cystatins
		2.2 Insect Midgut Proteins
		2.3 Laboratory Tools and Materials
		2.4 Media, Buffers and Other Solutions
	3 Methods
		3.1 Capture of Target Proteases with Biotinylated Cystatins
			3.1.1 Heterologous Expression and Purification of the AviTagged Cystatins
			3.1.2 Binding of AviTagged Cystatins to NeutrAvidin Agarose Beads
			3.1.3 Extraction of Target Proteases
			3.1.4 Target Protease Capture on Cystatin-Embedded Agarose Beads
		3.2 Mass Spectrometric Analysis of Captured Proteases
			3.2.1 Sample Preparation for Mass Spectrometry
			3.2.2 LC-MS/MS Analysis
			3.2.3 Identification of Captured Proteases
			3.2.4 Quantitation of Captured Protease Peptides
		3.3 Working Examples
			3.3.1 Example 1: The Protease Capture Approach as a Decision Tool to Select Cystatins Useful in Herbivore Pest Control
			3.3.2 Example 2: The Protease Capture Approach as an Analytical Tool to Address Basic Questions on the Evolution and Protease ...
	4 Notes
	References
Chapter 27: A Pipeline for Metabolic Pathway Reconstruction in Plant Orphan Species
	1 Introduction
	2 Materials
		2.1 Datasets
			2.1.1 Transcriptomics Datasets
			2.1.2 Proteomics Datasets
			2.1.3 Metabolomics Datasets
		2.2 Integration Tools
	3 Methods
		3.1 Functional Plant Categorization
		3.2 KEGG Metabolic Pathways
		3.3 MapMan Metabolic Representation
	4 Notes
	References
Chapter 28: Detection of Plant Low-Abundance Proteins by Means of Combinatorial Peptide Ligand Library Methods
	1 Introduction
	2 Plant Proteins: a Minor Component of Plant Extracts with Specific Properties
	3 Pretreatments of Plant Extracts to Eliminate Interfering Material
	4 The Reduction of Protein Dynamic Range with Low-Abundance Protein Enhancement
	5 Materials and Methods
	6 Protein Capture with Concomitant Dynamic Range Reduction
		6.1 General Capture Method under Physiological Conditions
		6.2 Protein Capture in Low-Ionic Strength
		6.3 The Capture of Dominantly Acidic Proteins
		6.4 The Capture of Dominantly Cationic Proteins
		6.5 Focus on Hydrophobic Protein Capture
	7 Recovery Protocols of Plant Protein from CPLLs
		7.1 Global Protein Harvesting
			7.1.1 Global Protein Elution with SDS-Containing Buffers
			7.1.2 Global Protein Elution with Guanidine Hydrochloride Solutions
		7.2 Fractionated Elution Approaches
			7.2.1 Two-Step Elution with Increased Stringency
			7.2.2 Three-Step Elution with Increased Stringency (Option 1)
			7.2.3 Three-Step Increased Stringency Elution (Option 2)
		7.3 Direct on-Bead Protein Digestion
	8 Compatibility Between Protein Elution from CPLLs and Analysis
	9 Practical Application Examples of CPLL-Treated Plant Extracts
	10 Notes
	References
Chapter 29: iTRAQ-Based Proteomic Analysis of Rice Grains
	1 Introduction
	2 Materials
		2.1 Plant Material
		2.2 Protein Extraction and Quantification
		2.3 Protein Digestion and iTRAQ Labeling
		2.4 Cation Exchange Liquid Chromatography and Peptide Desalting
		2.5 Mass Spectrometry
	3 Methods
		3.1 Sample Preparation
		3.2 Protein Extraction
		3.3 Protein Digestion
		3.4 iTRAQ Peptide Labeling
		3.5 Cation Exchange Chromatography
		3.6 C18 Spin Columns and Peptide Desalting
		3.7 Mass Spectrometry
	4 Notes
	References
Index