Peptide Microarrays: Methods and Protocols (Methods in Molecular Biology, 570)

✍ Scribed by Marina Cretich (editor), Marcella Chiari (editor)

• Publisher: Humana
• Year: 2009
• Tongue: English
• Leaves: 426
• Category: Library

✦ Synopsis

Due to their versatility, along with the diminishing costs of library synthesis and the growth of commercial support, peptide microarrays will likely expand beyond being just a research tool into an adaptable and powerful platform to be harnessed for wider drug discovery and point-of-care applications. In Peptide Microarrays: Methods and Protocols, experts in the field provide a cutting-edge view of peptide array technology, its applications, and technical issues. After examining how peptides can characterize proteins and clarify, at the amino acid level, the molecular recognition events in which they are involved, the volume goes on to cover topics such as the production and use of peptide arrays for enzyme and binding motifs characterization, epitope mapping, and diagnostics, the newest technological advancements, as well as software and web tools for the design of peptide arrays and for the analysis of output data. As a volume in the highly successful Methods in Molecular Biology™ series, chapters include brief introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls.

Authoritative and easy-to-use, Peptide Microarrays: Methods and Protocols promises to serve scientists with its unique insights and novel solutions in peptide array technology in order to advance this vital field.

✦ Table of Contents

METHODS IN MOLECULAR BIOLOGY TM
Peptide Microarrays
Preface
Contents
Contributors
Section I: Introduction
Exploring and Profiling Protein Function with Peptide Arrays
1. Introduction
2. Advancement of Peptide Arrays
3. Preparation of Peptide Arrays
3.1. In Situ Peptide Synthesis
3.2. Immobilization of Pre-synthesized Peptides
4. Applications of Peptide Arrays
References
Peptide Arrays for Enzyme Profiling
1. Introduction
2. Chemistry of Peptide Array Preparation
3. Library Types
4. Assays and Detection
5. Substrate Identification
6. Substrate Optimization
7. Miscellaneous
References
Using Peptide Array to Identify Binding Motifs and Interaction Networks for Modular Domains
1. Introduction
2. In Situ Synthesis of Peptide Arrays
3. Immobilization of Pre-made Peptides
4. Applications
4.1. Peptide Walking
4.2. Oriented Peptide Array Library
4.3. Peptide Array Target Screen (PATS)
5. Perspectives
References
Molecular Simulations of Peptides: A Useful Tool for the Development of New Drugs and for the Study of Molecular Recognition
1. Introduction
2. Overview
3. Simulation Methods
3.1. Introduction
3.2. General Aspects
3.3. Molecular Dynamics
3.3.1. Setting Up and Running a Molecular Dynamics Simulation
3.3.2. Integrating the Equations of Motion
3.3.3. Temperature and Pressure Control
3.3.4. Temperature Control
3.3.5. Pressure Control
3.3.6. Summary
3.4. Simplifying the Conformational Search Problem. The Coarse-Grained Monte Carlo for Protein and Peptide Simulations
3.4.1. The Coarse-Grained Monte Carlo Method
3.5. Molecular Docking
4. Rational Development of Shepherdin
4.1. Introduction Molecular Determinants of Cancer
4.1.1. Survivin and Hsp90
4.1.2. Simulation Setup
4.1.3. Results
4.2. Discussion
5. Rational Identification of a New Non-peptidic Anticancer Lead, Based on Peptide-Hsp90 Recognition
5.1. Introduction
5.2. Materials and Methods
5.2.1. Simulation Setup, Docking Experiments, and Molecular Dynamics (MD) Refinement of Hsp90-Shepherdin Complex
5.2.2. Pharmacophore Generation
5.2.3. Small Molecule - Hsp90 Docking and MD Refinement of the Complex
5.2.4. Recombinant Proteins and Binding Studies
5.2.5. Cell Lines and Cultures
5.2.6. Growth Inhibition and Apoptosis Assays
5.2.7. Analysis of Hsp90 Chaperone Function
5.3. Results
5.3.1. Characterization of Hsp90/Shepherdin Binding Interface
5.3.2. Pharmacophoric Hypotheses and Small Molecule Identification
5.3.3. Molecular Interactions Between AICAR and Hsp90: Molecular Dynamics Simulation Results
5.3.4. AICAR Induces Tumor Cell Death
5.3.5. AICAR Inhibits Hsp90 Chaperone Function
5.4. Discussion
6. Peptide Self-Organization
6.1. Introduction
6.2. Computer Simulations of Peptide Self-Assembly
6.3. Simulations of the Properties of Monomers and the Formation of Oligomers
6.4. Simulations of Preformed Supra-molecular Structures
6.5. Combining Coarse Grained and All-Atom Simulations for the Ab Initio Simulation of Peptide Aggregation
6.6. Results
6.7. Discussion
7. Conclusions
References
Section II: Applications
Synthesis of Peptide Arrays Using SPOT-Technology and the CelluSpots-Method
1. Introduction
2. Materials
2.1. Solvents
2.2. Spot Synthesis of Macroarrays
2.2.1. Preparation of the Membrane
2.2.2. Preparation of Activated Amino Acid Solutions
2.2.3. Spot Synthesis on Planar Cellulose Membranes
2.3. Preparation of the Peptide Microarrays (CelluSpots Arrays)
2.3.1. Synthesis of Peptide - Cellulose Conjugates for CelluSpots Microarrays
3. Methods
3.1. Spot Synthesis of Macroarrays
3.1.1. Preparation of a Cellulose Membrane for Spot Synthesis
3.1.2. Preparation of Activated Amino Acid Solutions
3.1.3. Spot Synthesis on Planar Cellulose Membranes
3.2. Preparation of the Peptide Microarrays (CelluSpots Arrays)
3.2.1. Synthesis of Peptide - Cellulose Conjugates for CelluSpots Microarrays
3.2.2. Preparation of Peptide-Cellulose Conjugate Solutions
3.2.3. Preparation of CelluSpots Microarrays
4. Notes
References
Rapid Identification of Linear Protein Domain Binding Motifs Using Peptide SPOT Arrays
1. Introduction
2. Materials
2.1. SPOT Blot Synthesis
2.2. SPOT Blot Analysis
3. Methods
3.1. Designing the Peptide Array
3.2. SPOT Blot Synthesis
3.3. Analysis of Peptide Binding on SPOT Blot
3.4. Stripping the SPOT Blot
4. Notes
References
Characterization of Kinase Target Phosphorylation Consensus Motifs Using Peptide SPOT Arrays
1. Introduction
2. Materials
2.1. Peptide SPOT Array Synthesis
2.2. Kinase Assay
3. Methods
3.1. SPOT Array Synthesis
3.2. Design of the Peptide Array
3.3. Preparation of SPOT Membrane and Reaction Mixture
3.4. Kinase Assay
4. Notes
References
CelluSpotstrade: A Reproducible Means of Making Peptide Arrays for the Determination of SH2 Domain Binding Specificity
1. Introduction
2. Materials
2.1. Synthesis of Peptide Arrays by SPOT
2.2. Producing (pTyr) OPALs by the CelluSpotstrade Technology
2.3. Screening of a pTyr OPAL by an SH2 Domain
2.4. Screening of a Targeted Peptide Array by an SH3 Domain
3. Methods
3.1. Synthesis of Peptide Arrays (e.g. SH3 Ligand Array) by SPOT
3.2. Producing pTyr OPALs by the CelluSpotstrade Technology
3.3. Screening of a pTyr OPAL Using an SH2 Domain
3.4. Peptide Array Target Screening (PATS) Assay for SH3 Domains
4. Notes
References
High-Density Peptide Microarrays for Reliable Identification of Phosphorylation Sites and Upstream Kinases
1. Introduction
2. Materials
2.1. Equipments
2.2. Radioisotopic Readout
2.3. Fluorescence Readout
3. Methods
3.1. Radioisotopic Readout
3.2. Fluorescence Readout
4. Notes
References
Epitope Mapping of Human Chromogranin A by Peptide Microarrays
1. Introduction
2. Materials
2.1. Synthesis of the Copoly(DMA-NAS-MAPS)
2.2. Derivatization of Glass Slide with Copoly(DMA-NAS-MAPS)
2.3. Preparation and Spotting of Peptides
2.4. Blocking and Incubation of Microarrays
2.5. Fluorescent Scanning and Data Analysis
3. Methods
3.1. Synthesis of the Copoly(DMA-NAS-MAPS)
3.2. Derivatization of Glass Slides with Copoly(DMA-NAS-MAPS)
3.3. Synthesis of Peptides and Preparation for Spotting
3.3.1. Synthesis of Peptides
3.3.2. Preparation of Peptides for Spotting
3.4. Spotting
3.5. Blocking and Incubation of Microarrays
3.6. Fluorescent Scanning
3.7. Data Analysis
4. Notes
References
Antimicrobial Peptide Arrays for Detection of Inactivated Biothreat Agents
1. Introduction
2. Materials
2.1. Preparation of Patterning and Assay Templates
2.2. Cleaning of Slides
2.3. Activation of the Glass Slide Surface (for Section 3.3)
2.4. Immobilization of Antimicrobial Peptides (for Section 3.4)
2.5. Fluorescent Labeling
2.5.1. Labeling of Cells, Viruses (for Section 3.5.1)
2.5.2. Labeling of Antibodies (for Section 3.5.2)
2.6. Assays
2.6.1. Direct Assays (for Section 3.6.1)
2.6.2. Sandwich Assays (for Section 3.6.2)
3. Methods
3.1. Preparation of Patterning and Assay Template
3.2. Cleaning of Slides
3.3. Surface ‘‘Activation’’
3.3.1. Silanization
3.3.2. Attachment of Crosslinker
3.4. Immobilization of Antimicrobial Peptides
3.5. Fluorescent Labeling
3.5.1. Labeling of Cells, Viral Particles
3.5.2. Labeling of Antibodies
3.6. Assays
3.6.1. Direct Assays
3.6.2. Sandwich Assays
3.7. Data Collection and Analysis
3.8. Examples of Bacteria, Viruses Detected
3.8.1. Detection of Gram-Negative Threat Agents
3.8.2. Detection of Gram-Positive Threat Agents
3.8.3. Detection of Enveloped Viruses
4. Notes
References
Mapping Functional Prion-Prion Protein Interaction Sites Using Prion Protein Based Peptide-Arrays
1. Introduction
2. Materials
2.1. MBP-PrP Construction and Verification
2.2. MBP-PrP Expression and Analysis
2.3. Peptide-Array Analysis
3. Methods
3.1. MBP-PrP Construction and Verification
3.2. MBP-PrP Expression and Analysis
3.3. Peptide-Array Analysis
4. Notes
References
A Designed Peptide Chip: Protein Fingerprinting Technology with a Dry Peptide Array and Statistical Data Mining
1. Introduction
2. Materials
2.1. Construction of a Designed Peptide Library
2.2. Dry Peptide Array System and Assay
2.3. Generation of PFP from Raw Data and Data Analyses from PFPs
3. Methods
3.1. Construction of a Designed Peptide Library
3.2. Dry Peptide Array System and Assay
3.3. Generation of PFP from Raw Data
3.4. Data Analyses from PFPs
4. Notes
References
Section III: Technological Advancements
Peptide Microarrays on Bisphenol A Polycarbonate
1. Introduction
2. Materials
2.1. Preparation of Silica Nanoparticles
2.2. Preparation of PC Slides
2.3. Printing
2.4. Incubations and Scanning
3. Methods
3.1. Preparation of Silica Nanoparticles
3.2. Preparation of PC Slides
3.3. Printing
3.4. Incubations (see Note 15)
4. Notes
References
Self-Assembly of PNA-Encoded Peptides into Microarrays
1. Introduction
2. Materials
2.1. Optimized Codon System for Libraries
2.2. Microarray Spotting
2.3. Library Synthesis
2.4. Microarray Hybridization
2.5. Microarray Scanning and Analysis
2.6. Separation of Enzyme-Bound Inhibitor-PNA Adduct by Gel Electrophoresis
2.7. Separation of Enzyme-Bound Inhibitor-PNA Adduct from the Rest of the Library
3. Methods
3.1. Microarray Spotting
3.2. Library Synthesis
3.3. Microarray Hybridization and Detection
3.4. Gel Isolation of Enzyme-Bound PNA-Encoded Inhibitors
3.5. Enzyme Activity Profile from Crude Cell Lysates
4. Notes
References
A Novel Combinatorial Approach to High-Density Peptide Arrays
1. Introduction
2. Materials
2.1. Chips
2.2. Chip Surface Modification
2.3. Amino Acid Microparticles
2.4. Peptide Synthesis
2.5. Immunostaining
3. Methods
3.1. Graft Polymerization of Poly(ethylene glycol) methacrylate (PEGMA) on the Chip Surface
3.2. Surface Modification for SPPS
3.3. Amino Acid Microparticles
3.4. Solid-Phase Peptide Synthesis on Chip’s Pixel Electrodes
3.5. Immunostaining with Fluorescently Labelled Antibodies
4. Notes
References
Polypyrrole-Peptide Microarray for Biomolecular Interaction Analysis by SPR Imaging
1. Introduction
2. Materials
2.1. Synthesis of N-(2-Mercaptoethyl)-6-(1H-pyrrole-1-yl) hexanamide (=Pyrrole-SH)
2.2. Preparation of Pyrrolylated Peptides
2.3. Immobilization of the Peptides on the Chip
2.4. SPRi Interaction Monitoring
3. Methods
3.1. Synthesis of N-(2-Mercaptoethyl)-6-(1H-pyrrole-1-yl) hexanamide (=Pyrrole-SH)
3.2. Preparation of Pyrrolylated Peptides
3.3. Immobilization of the Peptides on the Chip (‘‘Electrospotting’’)
3.4. SPRi Interaction Monitoring
3.5. Application to Antibody Detection
4. Notes
References
The Peptide Microarray-Based Assay for Kinase Functionality and Inhibition Study
1. Introduction
2. Materials
3. Methods
3.1. Preparation of Gold Nanoparticle Probes
3.2. Peptide Microarray Fabrication and Phosphorylation
3.2.1. Preparation of Assays for RLS Detection
3.2.2. Preparation of Assays for SERS Detection
3.3. PKA Inhibition and IC50 Assays
4. Notes
References
An Advanced Application of Protein Microarrays: Cell-Based Assays for Functional Genomics
1. Introduction
2. Materials
2.1. Slides
2.2. Cell Transfection, Virus Biotinylation and Concentration
2.3. Slide Functionalization
2.4. Target Cells
2.5. Immunofluorescence Experiment, Acquisition and Analysis
3. Methods
3.1. Viral Preparation
3.1.1. Day 1: Plating of Cells for Transfection of Retroviral Vector DNA
3.1.2. Day 2: Transfection of Retroviral Plasmid PINCO and PINCO-NPM
3.1.3. Day 3: Retroviral Biotinylation
3.1.4. Day 4: Retroviral Collection
3.1.5. Day 5: Retroviral Precipitation, Concentration and Storage
3.2. Retroviral Array Preparation
3.3. Cell Plating
3.4. DAPI Staining and Image Acquisition
3.5. Image Analysis
4. Notes
References
Profiling the Autoantibody Repertoire by Screening Phage-Displayed Human cDNA Libraries
1. Introduction
2. Materials
2.1. Construction of ORF-Enriched cDNA Libraries
2.2. Cre Recombination
2.3. Phage Production and Titration
2.4. Immunoprecipitation with Magnetic Beads
2.5. Subcloning Antigenic Fragments in Bacterial Expression Vector
2.6. High-Throughput Expression and Purification of Recombinant Polypeptides
2.7. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
3. Methods
3.1. Construction of ORF-Enriched cDNA Libraries
3.2. Cre Recombination for Removal of the beta-Lactamase Gene
3.3. Phage Production and Titration
3.4. Phage Selection by Magnetic Beads Coated with Serum Antibodies
3.5. Subcloning of cDNA Fragments in Bacterial Expression Vector
3.6. High-Throughput Expression and Purification of Recombinant Polypeptides
3.7. Analysis of Recombinant Proteins by SDS-PAGE and Coomassie Staining
4. Notes
References
Section IV: Software/Web Tools and Data Analysis
Visualisation and Pre-processing of Peptide Microarray Data
1. Introduction
1.1. Data Example
1.2. Slide Structure
1.3. Digitised Peptide Microarray Data
2. Preparing the Computing Environment
2.1. Reading GPR Data Files
3. Quality Control
3.1. Inspection of Flags
3.2. Visual Inspection
4. Choice of Response Measure and Construction of a Working Dataset
5. Data Reduction
5.1. Identification and Removal of Peptides with Non-detectable Response
5.2. Identification of False Positives
6. Preparation of Data for Use in Comparative and Predictive Analyses
7. Normalization
References
Web-Based Design of Peptide Microarrays Using muPepArray Pro
1. Introduction
2. Overviews of Peptide Array Design Process and muPepArray Pro
2.1. Peptide Array Design vs. Oligonucleotide Array Design
2.2. muPepArray Pro Features and a Dissection of the Design Flow
2.3. TBPG Construction Explained
3. Methods
3.1. Constructing a TBPG
3.1.1. Selecting Seed Peptides
3.1.2. Generating Derived Peptides
3.1.3. Re-evaluating Peptides
3.2. Assembling ALPL
3.3. Making Array Layout
References
Qualitative and Quantitative Analysis of Peptide Microarray Binding Experiments Using SVM-PEPARRAY
1. Introduction
2. SVM-PEPARRAY Overview
3. Methods
3.1. Establishing a Qualitative or Quantitative Model
3.1.1. Selecting or Uploading a Microarray Dataset
3.1.2. Constructing a Qualitative SVM Model
3.1.3. Constructing a Quantitative SVM Model
3.2. Examining a Newly Constructed Qualitative or Quantitative Model
3.3. Making Predictions Using an Established Qualitative or Quantitative Model
4. Notes
References
PASE: A Web-Based Platform for Peptide/Protein Microarray Experiments
1. Introduction
2. Materials: PASE Minimum Requirements
2.1. Web Server
2.2. Personal Computer
3. Methods
3.1. Installation and First Use of PASE
3.2. Describing Reporters
3.3. Describing Additional Experiments Information (Array LIMS)
3.4. Describing Biomaterials
3.5. Analyze Data
3.6. Additional Analysis Tools
3.6.1. Rule Mining
3.6.2. Statistical Analysis
4. Notes
References
Index