Mass Spectrometry-Based Proteomics (Methods in Molecular Biology, 2718)

✍ Scribed by Kris Gevaert

• Tongue: English
• Leaves: 337
• Category: Library

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Increasing the Overall Proteome Coverage by Combining Protein Digestion by Tryp-N and Trypsin
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 Digestion Buffers
2.3 S-Trap Protocol
2.4 Mass Spectrometry
2.5 Equipment
3 Methods
3.1 Sample Preparation and Loading
3.2 Peptide Elution
3.3 Mass Spectrometry
3.4 Data Analysis
4 Notes
References
Chapter 2: Cell Surface Biotinylation Using Furan Cross-Linking Chemistry
1 Introduction
2 Materials
2.1 Furan-Biotin Synthesis
2.2 Surfaceome Labeling
2.3 Biotin Pull-Down
2.4 Cleanup of Purified Peptides
3 Methods
3.1 Furan-Biotin Synthesis
3.2 Surfaceome Labeling
3.3 Biotin Pull-Down
3.4 Cleanup of Peptides
3.5 Data Analysis
3.5.1 Search Settings for Peptides and Proteins
4 Notes
References
Chapter 3: Studying Cellular Dynamics Using Proximity-Dependent Biotinylation: Somatic Cell Reprogramming
1 Introduction
2 Materials
2.1 Generating Lentiviral Expression Constructs
2.2 Generating Secondary Reprogramming MEFs
2.3 Generating Lentivirus for Bait Expression
2.4 Generating Bait-Expressing Reprogramming MEFs
2.5 BioID and MS
3 Methods
3.1 Generating Lentiviral BioID Expression Constructs
3.2 Generating a Chimeric Secondary Reprogramming MEF Line
3.2.1 Chimeric Embryo Generation
3.2.2 MEF Isolation
3.2.3 MEF Expansion
3.2.4 Analysis of Chimeric Contributions
3.2.5 Large-Scale Batch Generation
3.3 Generating Lentiviral Particles to Transduce BioID Baits
3.4 Generating and Validating Bait-Expressing Secondary Reprogramming MEFs
3.4.1 Generating BioID Bait-Expressing MEFs
3.4.2 Preparing Cell Pellets for BioID
3.4.3 Parallel Quality Control of Bait Expression, Labeling, and Localization
3.5 BioID
3.5.1 Lysis and Streptavidin Bead Capture
3.5.2 On-Bead Trypsin Digestion
3.5.3 MS and Data Analysis
4 Notes
References
Chapter 4: Virotrap: Trapping Protein Complexes in Virus-Like Particles
1 Introduction
2 Materials
2.1 Transfection for VLP Production
2.1.1 Reagents
2.1.2 Materials and Equipment
2.2 VLP Harvest
2.2.1 Reagents
2.2.2 Materials and Equipment
2.3 Sample Preparation for Mass Spectrometry
2.3.1 Reagents
2.3.2 Materials and Equipment
2.4 LC-MS/MS Analysis
2.5 Data Analysis
3 Methods
3.1 Transfection for VLP Production for MS (and WB Pilot Study) *
3.2 VLP Harvest for MS (and WB Pilot Study) *
3.3 Sample Preparation for Mass Spectrometry
3.4 LC-MS/MS Analysis
3.5 Data Analysis
4 Notes
References
Chapter 5: Thermal Proteome Profiling for Drug Target Identification and Probing of Protein States
1 Introduction
2 Materials
2.1 Preparation of Input Material
2.1.1 Cell Culture
2.1.2 Crude Lysate from Cell Pellets
2.2 Compound Treatment
2.2.1 Adherent Cells
2.2.2 Suspension Cells
2.3 Heat Treatment
2.4 Live Cell Format: Cell Lysis
2.5 Collection of Soluble Protein Fraction
2.5.1 DNA Digest
2.5.2 Removal of Aggregated Proteins
2.6 Mass Spectrometry-Based Proteomic Analysis
2.6.1 Sample Preparation and TMTpro Labeling
2.6.2 Offline Reverse Phase Fractionation at Basic pH
2.6.3 LC-MS/MS Analysis and Data Analysis
3 Methods
3.1 Preparation of Input Material
3.1.1 Cell Culture
3.1.2 Crude Lysate from Cell Pellets
3.1.3 Quality Control of Crude Lysates
3.2 Compound Treatment
3.2.1 Compound Treatment of Live Adherent Cells
3.2.2 Compound Treatment of Live Suspension Cells
3.2.3 Compound Treatment of Crude Lysate
3.3 Heat Treatment
3.4 Live Cell Format: Cell Lysis
3.5 Collection of Soluble Protein Fraction
3.5.1 DNA Digest
3.5.2 Removal of Aggregated Proteins (Live Cell Format)
3.5.3 Removal of Aggregated Proteins (Crude Lysate Format)
3.6 Mass Spectrometry-Based Proteomics Analysis
3.6.1 Sample Cleanup and Proteolytic Digest
3.6.2 Peptide Labeling Using Tandem Mass Tags
3.6.3 Final Peptide Purification
3.6.4 Offline Fractionation by High pH Reversed-Phase Chromatography
3.6.5 LC-MS/MS Analysis
3.7 Peptide and Protein Identification and Quantification
3.8 Data Analysis
4 Notes
References
Chapter 6: Improved Coverage of the N-Terminome by Combining ChaFRADIC with Alternative Proteases
1 Introduction
2 Materials
2.1 Cell Lysis, Reduction, and Alkylation
2.2 Protein Concentration Determination
2.3 Labeling of Primary Amines on Protein Level Using iTRAQ 8-Plex
2.4 Proteolytic Digestion and Peptide Cleanup
2.5 Desalting
2.6 Enrichment of N-Terminal Peptides Using ChaFRAtip
2.7 Nano-LC-MS/MS Analysis (See Note 5)
2.8 Data Analysis (See Note 5)
3 Method
3.1 Cell Lysis, Reduction, and Alkylation
3.2 Protein Concentration Determination
3.3 Labeling of Primary Amines on Protein Level Using iTRAQ 8-Plex
3.4 Proteolytic Digestion and Peptide Cleanup
3.5 Desalting
3.6 Enrichment of N-Terminal Peptides Using ChaFRAtip
3.7 Nano-LC-MS/MS Analysis
3.8 Data Analysis
4 Notes
References
Chapter 7: Sensitive and High-Throughput Exploration of Protein N-Termini by TMT-TAILS N-Terminomics
1 Introduction
2 Materials
2.1 Digestion with Test Protease (Optional)
2.2 Protein Denaturation, Reduction, and Alkylation
2.3 Labeling
2.4 Acetone Precipitation
2.5 Digestion
2.6 N-Termini Retrieval
2.7 Sample Desalting
2.8 High-pH Fractionation (Optional)
2.9 EvoTip Sample Loading (Optional)
2.10 LC-MS/MS Analysis
2.11 Data Analysis
3 Methods
3.1 Test Protease Digestion
3.2 Reduction and Alkylation
3.3 Labeling
3.4 Acetone Precipitation
3.5 Digestion
3.6 N-Terminal Peptide Retrieval
3.7 Sample Desalting
3.8 Fractionation (Optional)
3.9 Evotip Loading
3.10 LC-MS/MS Analysis
3.11 Data Analysis
3.12 Experimental Design, Post-Processing, and Downstream Analysis
4 Notes
References
Chapter 8: The Global Acetylation Profiling Pipeline for Quick Assessment of Protein N-Acetyltransferase Specificity In Cellulo
1 Introduction
2 Materials
2.1 Materials
2.2 Main Chemicals
2.3 Buffers and Solutions
2.4 NAT Expression Plasmids
3 Methods
3.1 Preparation of N-Acetoxy-[2H3]-Succinimide
3.2 E. coli Cell Extract Preparation
3.3 Protein Extraction
3.4 Protein Denaturation and Labeling
3.5 Protein Digestion and Desalting Step
3.6 Strong Cation Exchange (SCX) Peptide Fractionation
3.7 LC-MS/MS Analysis
3.8 Data Processing
4 Notes
References
Chapter 9: Systems-Wide Site-Specific Analysis of Glycoproteins
Abbreviations
1 Introduction
2 Materials
2.1 Sample Preparation, Protein Reduction, and Alkylation
2.2 In-Solution Protease Digestion
2.3 SP3-Based Protease Digestion
2.4 HILIC Enrichment
2.5 LC-MS/MS Analysis of Glycopeptides
3 Methods
3.1 Sample Preparation, Protein Reduction, and Alkylation
3.2 In-Solution Protease Digestion
3.3 SP3 Sample Preparation
3.4 Hydrophilic Interaction Chromatographic Enrichment of Glycopeptides
3.5 LC-MS/MS Analysis of Glycopeptides
3.6 Data Analysis
4 Notes
References
Chapter 10: Targeted Profiling of Protein Phosphorylation in Plants
1 Introduction
2 Materials
2.1 General Equipment
2.2 Plant Growth Material
2.3 Cloning and Agrobacterium Transformation
2.4 Tobacco Growth and Leaf Infiltration
2.5 Protein Extraction, Immunoprecipitation, and Protein Digestion
2.6 Desalting
2.7 Data Analysis
3 Methods
3.1 Cloning
3.2 Agrobacterium Transformation
3.3 Tobacco Infiltration
3.4 Protein Expression Checking and Sample Collection
3.5 Protein Extraction, Immunoprecipitation, and Protein Digestion
3.6 Desalting
3.7 LC-MS/MS Data Acquisition
3.8 MaxQuant Search for Identification and Quantification
3.9 Data Analysis and Statistics
3.10 Data Visualization and Interpretation
4 Notes
References
Chapter 11: Automated Sample Preparation for Mass Spectrometry-Based Clinical Proteomics
1 Introduction
1.1 Types and Requirements of Clinical Samples
1.2 Protein Extraction
1.3 Protein Cleanup and Digestion (AutoSP3)
2 Materials
2.1 Specimen Lysis, DNA Shearing, and Protein Extraction
2.2 Protein Quantification
2.3 Protein Reduction and Alkylation
2.4 AutoSP3 Processing and Protein Digestion
2.4.1 Hardware Equipment and Accessories
2.4.2 Consumables
2.4.3 Solvents and Reagents
2.5 Peptide Recovery
2.6 LC-MS Data Acquisition and Processing
3 Methods
3.1 Specimen Lysis, DNA Shearing, and Protein Extraction
3.1.1 Selection of a Suitable Sample Lysis Method
3.1.2 Handling of Liquid Specimen/Biopsies
3.1.3 Handling of Cells
3.1.4 Handling of Fresh-Frozen Tissue
3.1.5 Handling of Formalin-Fixed and Paraffin-Embedded (FFPE)
3.2 Protein Quantification
3.3 AutoSP3 Processing and Protein Digestion
3.3.1 AutoSP3 Installation
3.3.2 AutoSP3 Deck Setup
3.3.3 Supplying the Deck with SP3 Reagents
3.3.4 Running AutoSP3
3.4 Peptide Recovery After Digestion
3.4.1 Peptide Recovery Deck Setup and Supplying Reagents
3.4.2 Running Peptide Recovery
3.4.3 Peptide Quantification for FFPE Material
3.5 Compatibility with Downstream Applications
3.6 Quality Control of Acquired MS Data
4 Notes
References
Chapter 12: Proteomics-Based Analysis and Diagnosis of Formalin-Fixed Paraffin-Embedded Amyloidosis Samples
1 Introduction
2 Materials
2.1 Protein Extraction and Solubilization from FFPE Samples
2.2 Protein Concentration Determination and Reduction/Alkylation of Proteins
2.3 S-Trap, Digestion of Proteins and Peptide Purification
2.4 LC-MS/MS Analysis
3 Methods
3.1 Protein Extraction and Solubilization from FFPE Samples
3.1.1 Protein Extraction from FFPE Samples Using a Probe Sonicator
3.1.2 Protein Extraction from FFPE Samples Using a PIXUL Ultrasonicator
3.2 Protein Concentration Determination and Reduction/Alkylation of Proteins
3.3 S-Trap, Digestion of Proteins, and Peptide Purification
3.4 LC-MS/MS Analysis
3.5 Data Analysis
4 Notes
References
Chapter 13: A Robust and Clinically Applicable Sample Preparation Protocol for Urinary Extracellular Vesicle Isolation Suitabl...
1 Introduction
2 Materials
2.1 Urine Collection
2.2 Urine Concentration
2.3 Urinary Extracellular Vesicle Isolation
2.4 Urinary Extracellular Vesicle Lysis
2.5 Sample Preparation for Mass Spectrometry
2.5.1 Protein Fractionation of Urinary Extracellular Vesicle Lysates
2.5.2 In-Gel Digestion
3 Methods
3.1 Urine Collection
3.2 Urine Concentration
3.3 Urinary Extracellular Vesicle Isolation
3.4 Urinary Extracellular Vesicle Lysis
3.5 Sample Preparation for Mass Spectrometry (See Note 28)
3.5.1 Protein Fractionation of Urinary Extracellular Vesicle Lysates
3.5.2 In-Gel-Digestion (See Note 33)
3.6 Mass Spectrometry
3.7 Data Analysis
4 Notes
References
Chapter 14: Density-Based Fractionation of Cell-Conditioned Medium to Prepare Proteomics Grade Extracellular Vesicles
1 Introduction
2 Materials
2.1 Cell Culture and Cell-Conditioned Medium Preparation
2.2 Collection and Concentration of Cell-Conditioned Medium
2.3 Preparation of Size-Exclusion Chromatography Columns
2.4 Concentrated CM Fractionation by Bottom-Up Density Gradient Ultracentrifugation
2.5 Recovery of EV by Size-Exclusion Chromatography and Ultrafiltration
3 Methods
3.1 Cell Culture and Conditioned Medium Preparation
3.2 Collection and Concentration of Cell-Conditioned Medium
3.3 Preparation of Size-Exclusion Chromatography Columns
3.4 Concentrated Conditioned Medium Fractionation by Bottom-Up Iodixanol Density Gradient
3.5 Recovery of EV using Size-Exclusion Chromatography and Ultrafiltration
4 Notes
References
Chapter 15: Mass Spectrometry-Based Analysis of Histone Posttranslational Modifications from Laser Microdissected Samples
1 Introduction
2 Materials
2.1 Tissue Staining
2.2 Laser Microdissection
2.3 Histone Enrichment
2.4 SDS-PAGE
2.5 PRO-PIC In-Gel Digestion
2.6 High-Performance Liquid Chromatography-Mass Spectrometry
2.7 Data Acquisition and Analysis
3 Methods
3.1 Laser Microdissection (LMD)
3.2 Histone Extraction
3.3 ``PRO-PIC´´ In-Gel Digestion
3.4 LC-MS/MS and Data Analysis
4 Notes
References
Chapter 16: Phosphoproteomics After Guanidinium Thiocyanate Extraction of Tissue Biopsies
1 Introduction
2 Materials
2.1 Biopsies
2.2 Cryosectioning
2.3 DNA, RNA, and Protein Extraction
2.4 Protein Isolation Control
2.5 Protein Reduction, Alkylation, and Trypsin Digestion
2.6 Desalting of Peptide Mixtures
2.7 Phosphopeptide Enrichment
3 Methods
3.1 Biopsies
3.2 Cryosectioning
3.3 DNA, RNA, and Protein Extraction
3.4 Protein Isolation Control
3.5 In-Solution Protein Reduction, Alkylation, and Trypsin Digestion
3.6 Desalting of Peptide Mixtures
3.7 Phosphopeptide Enrichment with the AssayMAP Bravo Platform
4 Notes
References
Chapter 17: Probing Antibody Structures by Hydrogen/Deuterium Exchange Mass Spectrometry
1 Introduction
2 Materials
3 Methods
3.1 Tuning Digestion Conditions
3.2 Hydrogen/Deuterium Exchange
3.3 Mass Spectrometric Analysis
3.4 HDX-MS Data Interpretation
3.5 Data Presentation and Visualization
3.6 Preparation of the 3D Structure for HDX Data Visualization
4 Notes
References
Index