Peptide Microarrays: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Applications of Peptide Microarrays in Autoantibody, Infection, and Cancer Detection
	1 Introduction
	2 Autoimmunity
	3 Infection
		3.1 Bacterial Infection
		3.2 Viral Infection
	4 Autoantibody Signatures in Cancer
	5 Conclusions
	References
Chapter 2: Peptide Microarrays for Studying Autoantibodies in Neurological Disease
	1 Introduction
	2 Peptide Microarrays for Studying B-Cell Response
		2.1 Continuous Epitopes: Immunogenicity, Ai-Guided Reduction, and High-Density Display
		2.2 Mimicking Discontinuous Epitopes: Structure- and Paratope-Based Predictions
		2.3 Improving Method Robustness: Filtering and On-Chip Validation
	3 Conclusion and Outlook
	References
Chapter 3: A Bifunctional Polymeric Coating for the Co-Immobilization of Proteins and Peptides on Microarray Substrates
	1 Introduction
	2 Materials
		2.1 Synthesis of Copoly Mix
		2.2 Coating of Si-SiO2 Slides
		2.3 Immobilization of Probes on the Coated Slides
		2.4 Slide Incubation and Fluorescence Analysis
	3 Methods
		3.1 Synthesis of Copoly Mix
			3.1.1 Synthesis of Parent Polymer MCP-4
			3.1.2 Partial Post-Polymerization Modification
		3.2 Coating of Silicon Slides
			3.2.1 Surface Pretreatment
			3.2.2 Adsorption of Copolymer on the Substrates
		3.3 Immobilization of Probes
			3.3.1 Immobilization of Antibodies
			3.3.2 Immobilization of Alkyne-Modified Peptide
		3.4 Slides Incubation and Fluorescence Detection
	4 Notes
	References
Chapter 4: Manufacturing of Peptide Microarrays Based on Catalyst-Free Click Chemistry
	1 Introduction
	2 Materials
		2.1 Slides
		2.2 Slide Functionalization Components
		2.3 Azide Modification Components
		2.4 Peptide Spotting Components
		2.5 Immunoassay Components
		2.6 Buffer Solutions
	3 Methods
		3.1 Slide Functionalization
		3.2 Slide Functionalization Control
		3.3 BSA Protein Azide Derivative
		3.4 Peptide Spotting
		3.5 Immunoassay
	4 Notes
	References
5: Hybrid Peptide-Agarose Hydrogels for 3D Immunoassays
	1 Introduction
	2 Materials
		2.1 Peptide Synthesis and Purification: Resin Loading
		2.2 Peptide Coupling
		2.3 Cleavage from the Resin
		2.4 Work-Up, RP-HPLC Analysis, and Purification
		2.5 Microarrays
	3 Methods
		3.1 Peptide Synthesis
		3.2 Hydrogel Formation
		3.3 Microarrays: General Procedure
		3.4 3D Immunoassay Test
	4 Notes
	References
6: Low-Cost Peptide Microarrays for Mapping Continuous Antibody Epitopes
	1 Introduction
	2 Materials
		2.1 Array Peptide Synthesis Components
		2.2 Peptide Microarray Printing and Usage Components
			2.2.1 Peptide Dilution
			2.2.2 Plate Setup
			2.2.3 Printing
			2.2.4 Humidification/Immobilizing
			2.2.5 Numbering, Blocking, and Storage
			2.2.6 Sample Incubation
	3 Methods
		3.1 Synthesis of Array Peptides
			3.1.1 Preparation of Fmoc-β-Alanine Derivatized Cellulose Membrane
			3.1.2 Preparation of Reagents for Peptide Synthesis
			3.1.3 Synthesis of Array Peptides
		3.2 Peptide Microarray Printing and Usage
			3.2.1 Dissolving Peptides for Stock Solution
			3.2.2 Printing Plate Setup
			3.2.3 Printing
			3.2.4 Humidification/Immobilizing
			3.2.5 Numbering, Blocking, and Storage
			3.2.6 Sample Incubation
			3.2.7 Scanning and Data Analysis
	4 Notes
	References
7: Fast Protocols for Characterizing Antibody-Peptide Binding
	1 Introduction
		1.1 Arrays
		1.2 Traditional Protein Affinity Assays
		1.3 Quantitative Assays of Peptide Binding to Antipeptide Antibodies or to Other Proteins
		1.4 Binding Theory
	2 Materials
	3 Methods
		3.1 Peptides Design and Modifications
		3.2 Fast Assessment of Peptide-Binding Properties Using OCTET N1
		3.3 Data Analysis: Fitting Dissociation Rate
		3.4 Data Analysis: Evaluating the Association Rate
	4 Notes
	References
Chapter 8: Peptides and Anti-peptide Antibodies for Small- and Medium-Scale Peptide and Anti-peptide Affinity Microarrays
	1 Introduction
	2 Materials
		2.1 Selection of Peptides for Anti-peptide Antibody Development
		2.2 Affinity Peptidomics: Antibody Microarrays
	3 Methods
		3.1 Selection of Peptides for Anti-peptide Antibody Development
		3.2 Affinity Peptidomics: Antibody Microarrays
		3.3 Proteolysis and Labeling of Serum Protein Samples
		3.4 Microarrays for Fluorescent Detection and Quantification of Peptides (See Note 25)
	4 Notes
	References
Chapter 9: One-Shot Generation of Epitope-Directed Monoclonal Antibodies to Multiple Nonoverlapping Targets: Peptide Selection...
	1 Introduction
	2 Materials
		2.1 Oligonucleotide Design and Gene Synthesis
		2.2 Subcloning of Synthetic Antigen Peptide into pET-32a
		2.3 Bacterial Transformation
		2.4 Bacterial Expression of Thioredoxin-Tripeptide Fusion
		2.5 Purification of Thioredoxin-Tripeptide Fusion by IMAC
		2.6 Epitope Mapping by Alanine Scanning
	3 Methods
		3.1 Linear Epitope Prediction
		3.2 Selection of the Predicted Epitopes
		3.3 Deriving the Oligonucleotide Sequence Encoding the 3-Copy Peptide with Linkers
		3.4 In-Frame Fusion of Antigen Sequence into Active Site of Thioredoxin
		3.5 Expression and Purification of Antigen
			3.5.1 Bacterial Induction
			3.5.2 Bacteria Protein Expression Profiling
			3.5.3 Antigen Purification by IMAC
		3.6 Epitope Mapping by Alanine Scanning
	4 Notes
	References
Chapter 10: Peptide Microarray-Based Protein Interaction Studies Across Affinity Ranges: Enzyme Stalling, Cross-Linking, Deple...
	1 Introduction
	2 Microarray Synthesis in μSPOT Format
		2.1 Production of Functionalized Cellulose Disks
		2.2 Automated Peptide Synthesis on Cellulose Solid Support
		2.3 LC-MS or MALDI Quality Control
		2.4 Cellulose Disk Work-up
		2.5 Peptide Microarray Printing
	3 Microarray Evaluations: Protein-Protein Interaction Profiling Using Recombinant Protein & Lysates
	4 Enhancing Sensitivity in Microarray-Based Binding Assays
		4.1 Microarray Surface Blocking
		4.2 Protein Cross-Linking to Identify Weak and Transient Binders
		4.3 Enhancing Sensitivity by Removing Strong Binders that Deplete the Protein of Interest from the Incubated Solution
		4.4 Stall/Catch Transient Binders Exemplified for HDACs Using Hydroxamic Acid-Modified Peptides
		4.5 ``In-situ on-chip validation´´ by Neutralization with the Putative Positive Binders
	5 Quantification of Interaction Strengths in Microarray Format and Complementary Techniques
		5.1 Titration of Microarrays for EC50 Determination
		5.2 KD Determination with In-Solution Temperature-Related Intensity Change (TRIC) Measurements
	References
Chapter 11: Profiling of Multiple Matrix Metalloproteinases Activities in the Progression of Osteosarcoma by Peptide Microarra...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of ZnONR@P(GMA-HEMA) Decorated Slides
		3.2 Preparation of Cell Culture Medium
		3.3 Preparation of Serum and Tissue Homogenate
		3.4 Fabrication of Peptide Microarray and Profiling of MMP Activities
			3.4.1 Detection of MMP Activities Secreted by OS Cells
			3.4.2 Detection of MMP Activities in Serum and Tissue Homogenate
			3.4.3 Studying the Sensitivity of the As-Proposed Method
			3.4.4 Studying the Specificity of the As-Proposed Method
	4 Notes
	References
Chapter 12: Peptide Array-Based In Situ Fluorescence Assay for Profiling Multiple Matrix Metalloproteinase Activities
	1 Introduction
	2 Materials
	3 Methods
		3.1 Determination of Secreted MMP Activities in Cell Monolayer
		3.2 Screening of MMP Inhibitors
		3.3 Profiling MMP Activities of Thyroid Tissue
		3.4 Fluorescence Imaging of MMP Activities in Tissue
		3.5 Immunofluorescence Staining for MMP-2 and MMP-14
	4 Notes
	References
Chapter 13: Peptide-Based Sensor and Microfluidic Platform for IgG Antibody Detection by Differential Impedance Sensing
	1 Introduction
	2 Materials
		2.1 Surface Functionalization
		2.2 Impedance Detection
	3 Methods
		3.1 Surface Functionalization
		3.2 Differential Impedance Sensing
	4 Notes
	References
Chapter 14: Peptide Microarrays for Flavivirus Diagnosis
	1 Introduction
	2 Materials
	3 Methods
		3.1 Designing Specific YFV-WT and YFV-VAC Peptides
			3.1.1 Creating Your Own Peptide Database
			3.1.2 Exploring the Antigenic Potential of the Selected Peptides
		3.2 Microarray Procedures
			3.2.1 Strategy Using Slides Coated by MCP-6
			3.2.2 Strategy Using Slides Coated by Nitrocellulose
	4 Notes
	References
Chapter 15: Epitope Mapping on Microarrays Highlights a Sequence on the N Protein with Strong Immune Response in SARS-CoV-2 Pa...
	1 Introduction
	2 Materials
		2.1 Serum Samples
		2.2 Proteome-Wide Microarray
		2.3 Peptide Synthesis and Purification
			2.3.1 Resin Loading and Peptide Coupling
			2.3.2 Cleavage
			2.3.3 Work-up, RP-HPLC Analysis, and Purification
		2.4 Peptide Microarray
		2.5 Bioassays
	3 Methods
		3.1 Peptide Synthesis
		3.2 Epitope Mapping: Preliminary Screening on SARS-CoV-2 Proteome
		3.3 In-house Peptide Microarrays and Epitope Validation Screening
		3.4 Diagnostic Potential Probes: N Protein Epitope 155-171 (AM57)
	4 Notes
	References
Chapter 16: IgE and IgG4 Epitope Mapping of Food Allergens with a Peptide Microarray Immunoassay
	1 Introduction
	2 Materials
		2.1 Microarray Printing
		2.2 Microarray Hybridization
		2.3 Data Acquisition and Analysis
	3 Methods
		3.1 Peptide Microarray Printing
		3.2 Microarray Hybridization
			3.2.1 Rehydration and Blocking
			3.2.2 Serum Incubation
			3.2.3 IgE and IgG4 Detection
		3.3 Data Acquisition and Analysis
			3.3.1 Microarray Scanning
			3.3.2 Gridding, Spot Finding, and Quantitation
			3.3.3 Data Analysis and Visualization
	4 Notes
	References
Chapter 17: Extracellular Vesicle Molecular Profiling for Diagnostic Purposes: An Application of Phage Display Technology
	1 Introduction
	2 Materials
		2.1 Immunoplate
		2.2 Buffers
		2.3 Media
	3 Methods
		3.1 Cell Culture and EV Insolation from Cell Medium
		3.2 Panning Procedure
		3.3 Preparing Individual Clones for Sequencing
		3.4 Sequence Reading
	4 Notes
	References
Chapter 18: Membrane-Sensing Peptides for Extracellular Vesicle Analysis
	1 Introduction
	2 Materials
		2.1 Peptide Synthesis and Purification: Resin Loading
		2.2 Peptide Coupling
		2.3 Cleavage from the Resin
		2.4 Work-Up, RP-HPLC Analysis, and Purification
		2.5 Microarrays
		2.6 EV Analyses
	3 Methods
		3.1 Peptide Synthesis
		3.2 Peptide Microarrays
		3.3 EV Analyses with ExoView
	4 Notes
	References
Index