Preface
Acknowledgments
Contents
Contributors
Part I: Shotgun Proteomics of Extracellular Vesicles and Subcellular Structures
Chapter 1: Mesenchymal Stem Cell-Derived Extracellular Vesicle Isolation and Their Protein Cargo Characterization
1 Introduction
2 Materials
2.1 EV Extraction from Culture Medium by Serial Ultracentrifugation
2.2 Particle Number Characterization by Nanoparticle Tracking Analysis (NTA)
2.3 Morphology Characterization by Transmission Electron Microscopy (TEM)
2.4 Protein Extraction and Quantification
2.5 Protein Digestion and TMT Labeling
2.6 Peptide Basic-Reversed-Phase Fractionation
2.7 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
3 Methods
3.1 EV Extraction from Culture Medium by Serial Ultracentrifugation
3.2 Particle Number Characterization by Nanoparticle Tracking Analysis (NTA)
3.3 Morphology Characterization by Transmission Electron Microscopy (TEM)
3.4 Protein Extraction and Quantification
3.5 Protein Digestion and TMT Labeling
3.6 Peptide Basic-Reversed-Phase Fractionation
3.7 LC-MS/MS Analysis and Data Processing
4 Notes
References
Chapter 2: Clinical Proteomics for the Analysis of Circulating Extracellular Vesicles
1 Introduction
2 Materials
2.1 Blood Collection
2.2 EV Isolation
2.3 Gel-Based Proteomics: 2D-DIGE
2.4 Gel-Free Proteomics: Label-Free
3 Methods
3.1 Blood Collection
3.2 EV Isolation
3.3 EV Characterization
3.4 Gel-Based Proteomics: 2D-DIGE Labeling
3.5 2D-Electrophoresis
3.6 Differential Image Analysis
3.7 MS Analysis
3.8 Gel-Free Proteomics: Label-Free
3.9 FASP Digestion
3.10 LC-MS/MS Analysis
3.11 Differential Analysis for Label-Free
3.12 System Biology
4 Notes
References
Chapter 3: Utilization of Laser Capture Microdissection Coupled to Mass Spectrometry to Uncover the Proteome of Cellular Protr...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Preparation for LCM
2.3 Fixation
2.4 Laser Capture Microdissection
2.5 Protein Extraction
2.6 Mass Spectrometry Sample Preparation
2.7 Mass Spectrometers
3 Methods
3.1 Cell Culture
3.2 Differentiated CAD Cells
3.3 Preparation of MMI Live Chamber Dish
3.4 Fixation
3.5 Laser Capture Microdissection (LCM)
3.6 Protein Extraction
3.7 Sample Preparation for Mass Spectrometry Using Limited Gel
3.8 Sample Preparation for Mass Spectrometry Using iST Sample Preparation Kit from PreOmics
3.9 Data Analysis
4 Notes
References
Part II: Shotgun Proteomics of Non-model Organisms
Chapter 4: Isolation of Apoplastic Fluid from Woody Plant Leaves: Grapevine and Coffee as a Case Study
1 Introduction
2 Materials
2.1 Plant Material
2.2 Apoplastic Fluid Isolation Buffers and Equipment
2.3 Apoplast Protein Concentration and Quantification
3 Methods
3.1 Infiltration
3.2 Isolation of Apoplastic Fluid by Centrifugation
3.3 Apoplast Protein Concentration and Quantification
4 Notes
References
Chapter 5: Shotgun Proteomics for L3 and L4 Anisakis simplex Development Stages
1 Introduction
2 Materials
2.1 Laboratory Equipment
2.2 Parasites Isolation and In Vitro Culture
2.3 Protein Extraction and Precipitation
2.4 SDS-Polyacrylamide Gel Electrophoresis
2.5 Protein Digestion
2.6 TMT Labeling
2.7 Peptide Fractionation and Cleanup
2.8 LC-MS/MS Analysis
2.9 MS Data Processing
3 Methods
3.1 Parasite Isolation and In Vitro Culture
3.2 Protein Extraction and Precipitation
3.3 SDS-Polyacrylamide Gel Electrophoresis
3.4 Protein Digestion (See Note 22)
3.5 TMT Labeling
3.6 Peptide Cleanup and Fractionation
3.6.1 Conditioning of the Spin Columns
3.6.2 Fractionation of Peptide Samples
3.7 LC-MS/MS Analysis (See Note 32)
3.8 MS Data Processing
4 Notes
References
Chapter 6: A Primer and Guidelines for Shotgun Proteomic Analysis in Non-model Organisms
1 Introduction
2 Materials
2.1 Total Protein Extraction and Quantification
2.2 1-D SDS-PAGE
2.3 Gel Staining and Fractionation
2.4 In-Gel Protein Digestion
2.5 Isobaric-Labeling Using TMT-Multiplex
2.6 Peptides Resuspension and LC-MS/MS Analysis
3 Methods
3.1 Experimental Design: Preliminary Considerations
3.2 Total Protein Extraction and Quantification
3.3 Label-Free Quantitative Proteomic Analysis: 1-D SDS-PAGE
3.4 Gel Staining and Fractionation
3.5 In-Gel Protein Digestion and Mass Spectrometry Analysis
3.6 Quantitative Proteomic Analysis: Isobaric-Labeling Using TMT Multiplex
3.7 Mass Spectrometry Data Analysis: Protein Identification
3.8 Databases Selection, Including Proteogenomic Approaches
3.9 Mass Spectrometry Data Analysis: Protein Quantification
3.10 Statistical Analysis: Differentially Expressed Proteins
4 Notes
References
Part III: Clinical Proteomics
Chapter 7: SWATH-MS Protocols in Human Diseases
1 Introduction
1.1 SWATH-MS Application in Proteomics
2 Materials
2.1 Methods for Protein Extraction
2.1.1 Protein Extractions from Different Body Fluids
Saliva Samples
Tear Samples
Urine Samples
Blood, Serum, or Plasma Samples
2.2 Major Protein Removal
2.2.1 Dithiothreitol (DTT)
2.2.2 Nanoparticles for Protein Enrichment
Nanoparticles in Blood Serum Plasma Samples
Au, Ag, and Pt Nanoparticles
Fe Nanoparticles
Nanoparticles in Urine Samples
2.3 SDS Gels
2.4 Colloidal Coomassie Blue Staining
2.5 In-Gel Protein Digestion
2.6 SWATH-MS Methods
3 Methods
3.1 Methods for Protein Extraction
3.1.1 Protein Extractions from Different Body Fluids (See Note 4)
Saliva Samples
Saliva Collection
Saliva Sample Preparation
Tear Samples
Tear Sample Preparation
Urine Samples
Urine Sample Preparation (Fig. 6)
Blood, Serum, or Plasma Samples
Sample Collection
Serum Sample Preparation
3.2 Major Protein Removal
3.2.1 DTT
3.2.2 Nanoparticles for Protein Enrichment
Nanoparticles in Blood Serum Plasma Samples
Au, Ag, and Pt Nanoparticles
Fe Nanoparticles
Nanoparticles in Urine Samples
Au Nanoparticles
3.3 SDS Gels
3.4 Colloidal Coomassie Blue Staining
3.5 In-Gel Protein Digestion
3.6 SWATH-MS Methods
3.6.1 LC-MS Data Acquisition
Instrument Quality Control Considerations
3.6.2 Spectral Library Building and Data Generation
3.6.3 DDA (IDA) Analysis of Sample Proteolytic Digests to Build a Spectral Ion Library
TOF MS (MS1)
Product Ion (MS2)
SWATH-MS Data Acquisition (DIA-MS Acquisition)
3.6.4 SWATH-MS Data Analysis Using PeakView 2.2 and SWATH-MS Microapp 2.0 (See Note 10)
Introduction to SWATH-MS Data Analysis Procedure
Creation of Spectral Ion Library Using Protein Pilot Paragon Method
Importing Ion Libraries into the SWATH-MS Microapp and Analyzing SWATH-MS Data
4 Notes
References
Chapter 8: Serum Proteomic Profiling in Rheumatoid Arthritis by Antibody Suspension Bead Arrays
1 Introduction
2 Materials
2.1 Bead Coupling
2.2 Sample Labeling
2.3 Assay Procedure
3 Methods
3.1 Coupling of Antibodies on Beads
3.2 Bead Array Preparation and Coupling Efficiency Test
3.3 Sample Labeling
3.4 Assay Procedure
4 Notes
References
Chapter 9: Human Blood Plasma Investigation Employing 2D UPLC-UDMSE Data-Independent Acquisition Proteomics
1 Introduction
2 Materials
2.1 Liquid Chromatography Equipment and Software
2.2 Mass Spectrometry Equipment and Software
2.3 Sample Preparation and Digestion
3 Methods
3.1 Human Plasma or Serum Protein Depletion
3.2 Desalting and Buffer Exchange
3.3 Tryptic Digestion Using RapiGest SF Surfactant
3.4 HDMSE Spectra Acquisition and Scouting Runs
3.5 Selection Rules and UDMSE Acquisition
3.6 Data Processing, Quantification, and Identification
3.7 In Silico Analysis: Available Tools
4 Notes
References
Chapter 10: Metaproteomics Analysis of Host-Microbiota Interfaces
1 Introduction
2 Materials
2.1 Collection of Intestinal Mucus
2.2 Sample Lysis
2.3 Filter-Aided Sample Preparation (FASP)
2.4 C18-StageTip Cleanup
2.5 LC-MS
2.6 Computational Resources and Required Software
3 Methods
3.1 Collection of Mucus Sample
3.2 Sample Lysis
3.3 Filter-Aided Sample Preparation (FASP)
3.4 C18-StageTip Cleanup
3.5 Peptide Analysis by Liquid Chromatography-Mass Spectrometry
3.6 De Novo Peptide Identification
3.7 Species Annotation of De Novo Peptide Identifications
3.8 Database Generation for Targeted Protein Search
3.9 Targeted Peptide Identification
4 Notes
References
Chapter 11: Mass Spectrometry-Based Analysis of Mycobacterial Single-Colony Proteome
1 Introduction
2 Materials
2.1 Mycobacterium Smegmatis
2.2 7H9 Liquid Media (See Note 1)
2.3 7H10 Solid Media (See Note 2)
2.4 Sterile Inoculation Loops
2.5 Cell Lysis
2.6 Acetone-Methanol Protein Precipitation and Extraction
2.7 Reduction
2.8 Alkylation
2.9 Predigestion
2.10 Tryptic Digestion
2.11 Desalting
3 Methods
3.1 Mycobacterial Strain
3.2 Cell Lysis
3.3 Acetone-Methanol Protein Precipitation and Extraction
3.4 Reduction, Alkylation, Predigestion (See Note 5)
3.5 Tryptic Digestion
3.6 StageTip Desalting
3.7 Preparing Peptides for MS Analysis
3.8 LC-MS/MS Measurement
3.9 Protein Identification and Data Handling
4 Results
5 Notes
6 Summary
References
Chapter 12: Proteogenomic Approach for Mycobacterium tuberculosis Investigation
1 Introduction
2 Materials
2.1 Purification of Mycobacterial Cells
2.2 Total Protein Extraction
2.3 Gel Electrophoresis
2.4 In-Gel Trypsin Digestion
2.5 Tandem Mass Spectrometric Analysis Combined with Nano-Liquid Chromatography
2.6 Identification of Mass Spectrometric Data
2.7 Proteogenomic Analysis
3 Methods
3.1 Purification of Mycobacterial Cells
3.2 Total Protein Extraction
3.3 Gel Electrophoresis
3.4 In-Gel Trypsin Digestion
3.5 Tandem Mass Spectrometric Analysis Combined with Nano-Liquid Chromatography
3.6 Identification of Mass Spectrometry Data
3.7 Proteogenomic Analysis
4 Notes
References
Part IV: Food Proteomics
Chapter 13: Shotgun Proteomics for Food Microorganism Detection
1 Introduction
2 Materials
2.1 Strain Culture
2.2 Protein Extraction
2.3 Peptide Preparation
2.4 Shotgun LC-MS/MS Analysis
2.5 LC-MS/MS Mass Spectrometry Databases and Software
3 Method
3.1 Strain Culture
3.2 Protein Extraction
3.3 Soluble Protein Quantification
3.4 Protein Denaturalization, Reduction, Alkylation, and Tryptic Digestion
3.5 Peptide Desalting and Purification
3.6 Shotgun LC-MS/MS Analysis
3.7 LC-MS/MS Mass Spectrometry Data Processing
3.8 Selection of Potential Peptide Biomarkers
4 Notes
References
Chapter 14: Shotgun Proteomics and Protein-Based Bioinformatics for the Characterization of Food-Derived Bioactive Peptides
1 Introduction
2 Materials
2.1 Protein Extraction
2.2 Protein Concentration
2.3 Protein Digestion with Trypsin
2.4 Shotgun LC-MS/MS Analysis
2.5 MS Data Processing
2.6 Bioactive Peptides Prediction
3 Methods
3.1 Discovery Phase: Sarcoplasmic Protein Extraction of the Selected Seafood
3.2 Trypsin Protein Digestion
3.3 LC-MS/MS Analysis
3.4 MS Data Processing
3.5 Bioinformatic Phase: In Silico Human Gastrointestinal Digestion
3.6 Evaluation of the Final List of Potential Bioactive Peptides
3.7 Potential Bioactive Peptides
4 Notes
References
Part V: Analysis of Postranslational Modifications and Protein Complexes
Chapter 15: FTSC-Labeling Coupled with 2DE-LC-MS/MS Analysis of Complex Protein Mixtures for Identification and Relative Quant...
1 Introduction
2 Materials
2.1 Preparation of Protein Extracts
2.2 FTSC-Labeling of Protein-Bound Carbonyls
2.3 First Dimension: Isoelectrofocusing (IEF)
2.4 Second Dimension: SDS Polyacrylamide Gel
2.5 In-Gel Protein Digestion
2.6 LC-MS/MS Analysis of Carbonylome
3 Methods
3.1 Preparation of Protein Extracts
3.2 FTSC-Labeling of Protein-Bound Carbonyls
3.3 First Dimension: Isoelectrofocusing (IEF)
3.4 Second Dimension: SDS Polyacrylamide Gel
3.5 In-Gel Protein Digestion
3.6 LC-MS/MS Analysis of Carbonylome
4 Notes
References
Chapter 16: Mass Spectrometry-Based Proteomics for Analysis of Hydrophilic Phosphopeptides
1 Introduction
2 Materials
2.1 Cell Lysis
2.2 Protein Digestion
2.3 TMT Labeling
2.4 Peptide Desalting
2.5 Phosphopeptide Enrichment
2.6 Liquid Chromatography and Electrospray Tandem Mass Spectrometry
3 Methods
3.1 Cell Lysis
3.2 Protein Digestion
3.3 Digestion of Immunoprecipitated Proteins (Phosphorylated CXCR3 in This Example)
3.4 TMTzero (TMT0) Labeling
3.5 Peptide Desalting by C18 Cartridge for Large Amounts of Samples
3.6 Phosphopeptide Enrichment by IMAC
3.7 Peptide Desalting by C18 StageTip for Small Amounts of Samples
3.8 LC-MS Setup
3.9 Data Analysis
4 Notes
References
Chapter 17: Rapid Shotgun Phosphoproteomics Analysis
1 Introduction
2 Materials
2.1 Cell Culture and Cell Lysis
2.2 Cell Lysis
2.3 Protein Concentration
2.4 Accelerated Trypsin Protein Digestion Using HIFU
2.5 Phosphopeptide Enrichment Using TiO2
2.6 Phosphopeptide Fractionation Using SCX
2.7 LC-MS/MS Analysis
2.8 MS Data Processing
3 Methods
3.1 Cell Culture and Cell Lysis
3.2 Trypsin Protein Digestion Using HIFU
3.3 Phosphopeptide Enrichment Using TiO2
3.4 Phosphopeptide Fractionation Using SCX
3.5 LC-MS/MS Analysis
3.6 MS Data Processing
4 Notes
References
Chapter 18: System-Wide Profiling of Protein Complexes Via Size Exclusion Chromatography-Mass Spectrometry (SEC-MS)
1 Introduction
2 Material
2.1 Cell Culture and Tissue Samples
2.2 Lysis Buffer
2.3 Ultracentrifugation and Sample Concentration
2.4 HPLC Standard Operation Procedure
2.5 High Throughput Filter-Aided Sample Preparation Protocol of SEC-Fraction for MS-Analysis (FASP Protocol)
2.6 MS Setup and Material
2.7 Data Analysis Workflow
2.8 Protein Co-Elution Profiling Tools
3 Methods
3.1 Tissue Culture
3.2 Native Lysis
3.3 SEC Sample Preparation
3.4 HPLC Standard Operation Procedure
3.5 Filter-Aided Sample Preparation Protocol of SEC Fraction for MS Analysis (FASP Protocol)
3.6 MS Acquisition in SWATH Mode
3.7 Data Analysis Pipeline
3.8 Protein Complex Analysis
4 Expected Outcome and Applications
5 Notes
References
Part VI: Shotgun Proteomics Data Processing and Storage
Chapter 19: Qualitative and Quantitative Shotgun Proteomics Data Analysis from Data-Dependent Acquisition Mass Spectrometry
1 Introduction
2 Materials
2.1 Peptide Identification by Database Search
2.2 Quantification of Peptides and Detection of Protein Changes
2.3 Gene Ontology Term Enrichment Analysis and Network Visualization
3 Methods
3.1 Peptide Identification by Database Search
3.2 Quantification of Peptides and Detection of Protein Changes
3.3 Term Enrichment Analysis of Protein Changes
4 Notes
References
Chapter 20: The jPOST Repository as a Public Data Repository for Shotgun Proteomics
1 Introduction
2 Materials
3 Methods
3.1 Types of Registered Data
3.2 Presets and Projects
3.3 Registration of Presets
3.3.1 Sample
3.3.2 Fractionation
3.3.3 Enzyme/Mod
3.3.4 MS Mode
3.4 Creation of a Project
3.5 Input of Registered Files
3.5.1 Raw Data
3.5.2 Peak List
3.5.3 Search Results
3.5.4 Others
3.6 Submission of the Files
3.7 Condition for Submission
3.8 Selection of Submission Types
3.9 Registration of Correspondence Between Files
3.10 Complete Submission
4 Notes
References
Index