Quantitative Methods in Proteomics

Preface
Contents
Contributors
Chapter 1: Important Issues in Planning a Proteomics Experiment: Statistical Considerations of Quantitative Proteomic Data
1 Introduction
2 Planning a Proteomic Experiment
2.1 Experimental Design for Proteomic Experiments
2.2 Design Considerations in Labeled Mass Spectrometry Experiments
3 Data Preprocessing
3.1 Missing Values
3.2 Normalization
4 Basic Statistical Concepts for Difference Detection
4.1 Statistical Hypothesis Tests
4.2 Power and Sample Size
4.3 Multiple Testing
4.4 Statistical Significance and Biological Relevance
5 Statistical Tests in Proteomic Experiments
5.1 Comparing Two Sample Groups
5.2 Analysis of Multiple Sample Groups and Additional Factors
6 Notes
6.1 Normalization in Case of Many Changing Proteins
6.2 Fold Change
6.3 Expected Effect Size and Variance for Choosing the Sample Size
6.4 Adjusted p-Values
6.5 Fold Change Cutoff
6.6 Euclidean Distance Measure in Volcano Plots
References
Chapter 2: Good Old-Fashioned Protein Concentration Determination by Amino Acid Analysis
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 Acid Hydrolysis
2.3 Derivatization of Samples
2.4 External Amino Acid Standard for System Calibration/Data Analysis
2.5 HPLC Measurement
2.6 Generation of Calibration Curves for Sample Analysis Using the External Amino Acid Standard and Data Analysis
3 Methods
3.1 Sample Preparation
3.2 Acid Hydrolysis
3.3 Derivatization of Samples
3.4 External Amino Acid Standard for System Calibration/Data Analysis
3.5 HPLC Measurement of the Samples and External Amino Acid Standard
3.6 Generation of Calibration Curves for Sample Analysis Using the External Amino Acid Standard
3.7 Data Analysis of Samples
4 Notes
References
Chapter 3: Protein Quantification Using the ``Rapid Western Blot´´ Approach
1 Introduction
2 Materials
2.1 Gel Casting
2.2 SDS Gel Electrophoresis
2.3 Blotting
2.4 Immunostaining
2.5 Imaging and Data Analysis
3 Methods
3.1 Gel Casting
3.2 SDS Gel Electrophoresis
3.3 Blotting
3.4 Immunostaining
3.5 Imaging an Data Analysis with LabImage1D SPL Software
4 Notes
References
Chapter 4: The Whereabouts of 2D Gels in Quantitative Proteomics
1 Introduction
2 The Protein Detection Methods
2.1 Protein Detection Via Radioisotopes
2.2 Protein Detection Via Organic Dyes
2.3 Protein Detection Via Silver Staining
2.4 Protein Detection Via Fluorescence
3 The Data Analysis Issues
4 The Protein Unicity Issue
5 Conclusions
References
Chapter 5: Proteome Analysis with Classical 2D-PAGE
1 Introduction
2 Materials
2.1 Technical Equipment (See Note 1)
2.1.1 IPG-Based IEF
2.1.2 CA-Based IEF
2.1.3 SDS-PAGE
2.2 Buffers and Solutions
2.2.1 IPG-Based IEF
2.2.2 CA-Based IEF
2.2.3 SDS-PAGE
3 Methods
3.1 IPG-Based IEF
3.2 CA-Based IEF
3.3 SDS-PAGE
4 Notes
References
Chapter 6: Silver Staining of 2D Electrophoresis Gels
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents and Protocols
2.2.1 Reagents
2.2.2 Fast Silver Staining
2.2.3 Silver Staining with Aldose-Based Developer
2.2.4 Spot Destaining Prior to Mass Spectrometry
3 Methods
3.1 General Practice
3.2 Fast Silver Staining
3.3 Silver Staining with Aldose-Based Developer
3.4 Spot Destaining Prior to Mass Spectrometry
3.4.1 Procedure
4 Notes
References
Chapter 7: Differential Proteome Analysis Using 2D-DIGE
1 Introduction
2 Materials
2.1 Technical Equipment
2.1.1 CyDye Minimal Labeling
2.1.2 Refraction-2D Labeling
2.1.3 CyDye Saturation Labeling
2.1.4 Saturn-2D Labeling
2.1.5 CA- and IPG-Based 2D-PAGE
2.2 Buffers and Solutions
2.2.1 CyDye Minimal Labeling
2.2.2 Refraction-2D Labeling
2.2.3 CyDye Saturation Labeling
2.2.4 Saturn-2D Labeling
2.2.5 CA- and IPG-Based 2D-PAGE
3 Methods
3.1 CyDye Minimal Labeling/Refraction-2D Labeling
3.1.1 CyDye Saturation Labeling/Saturn-2D Labeling
3.2 CA- and IPG-Based 2D-PAGE
4 Notes
References
Chapter 8: Quantitative Mass Spectrometry-Based Proteomics: An Overview
1 Introduction
2 General Principles for MS-Based Quantification
2.1 Global and Targeted Quantification
2.2 Peptide or Protein-Centric Approach
2.3 Label-Free and Label-Based Quantification by Mass Spectrometry
2.4 Quantitative Cross-Linking/Mass Spectrometry
3 Methods for Protein/Peptide Quantification
3.1 Relative Quantification
3.1.1 Stable Isotope Labeling-Based Methods
Chemical Labeling
Metabolic Labeling
Enzymatic Labeling
3.1.2 Label-Free Quantification (LFQ)
Spectral Counting-Based LFQ
Intensity-Based LFQ
3.2 Absolute Quantification
3.2.1 Label-Based Absolute Quantification
3.2.2 Label-Free Absolute Quantification
4 Notes
References
Chapter 9: Tandem Mass Tags for Comparative and Discovery Proteomics
Abbreviations
1 Introduction
2 Materials
2.1 Cell Lysis of Biological Samples and Protein Concentration Estimation
2.2 Reduction and Alkylation (Carbamidomethylation)
2.3 Sample Cleanup and on-Filter Enzymatic Digestion
2.4 Digestion Control by Monolithic Reversed-Phase Chromatography (RPC)
2.5 Solid-Phase Extraction Cartridges (SPEC) for Sample Cleanup
2.6 TMT Labeling
2.7 Sample Fractionation Using High-pH C18 RP Chromatography
2.8 NanoLC-NanoESI MS and MS/MS
2.9 Data Analysis
3 Methods
3.1 Sample Lysis and Determination of Protein Concentration
3.2 Carbamidomethylation, On-Filter Sample Cleanup, and Proteolytic Digestion
3.3 Digestion Control with RP Monolithic Chromatography
3.4 SPEC Sample Cleanup (Desalting)
3.5 TMT Labeling
3.6 Data Analysis, Evaluation of Labeling Efficiency, and Multiplexing
3.7 Off-Line High-pH C18 RP Fractionation
3.8 LC-MS/MS Analysis
3.8.1 LC Conditions
3.8.2 MS Conditions
4 Notes
References
Chapter 10: An Approach for Triplex-IPTL
1 Introduction
2 Materials
2.1 Protein Digestion with Endoproteinase Lys-C
2.2 Dimethylation of Alpha-N-Termini
2.3 Dimethylation of Lysine Residues
2.4 MALDI-MS Analysis
2.5 LC-ESI-MS Analysis
2.6 Data Analysis
3 Methods
3.1 General Practice
3.2 Protein Digestion with Endoproteinase Lys-C
3.3 Dimethylation of Peptides
3.3.1 Dimethylation of Peptide N-Termini
3.3.2 Dimethylation of Lysine Residues
3.4 MALDI-MS
3.5 LC-ESI Mass Spectrometry
3.6 Protein Identification Using Mascot and Quantification Using IsobariQ
4 Notes
References
Chapter 11: Targeted Protein Quantification Using Parallel Reaction Monitoring (PRM)
1 Introduction
2 Materials
2.1 Stable Isotope-Labeled Synthetic (SIS) Peptides
2.2 LC-MS/MS Analysis
2.3 Data Analysis
3 Methods
3.1 Selection of Stable Isotope-Labeled Synthetic (SIS) Peptides
3.2 SIS Peptide Concentration Correction
3.3 PRM Analysis of SIS Peptides
3.4 Calibration Curve of SIS Peptides
3.5 Measurement of SIS Peptides in a Complex Matrix
3.6 Peptide-Specific Optimization of Normalized Collision Energies (NCEs)
3.7 Calibration Curves of SIS Peptides in a Complex Matrix
3.8 Use Optimized PRM Assay to Measure Real Samples
4 Notes
References
Chapter 12: Quantitative Approach Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-ToF) Mass Spectromet...
1 Introduction
2 Materials
2.1 MALDI-ToF MS Calibrants and Matrix
2.2 PFOS Standards
2.3 MALDI-ToF MS
2.4 Solid-Phase Extraction (SPE)
3 Methods
3.1 Minimization of Contamination
3.2 Calibration Preparation
3.3 MALDI-ToF MS Acquisition and Analysis
3.4 Extraction and Analysis of Contaminated Samples
3.5 Quantification of PFAS
4 Notes
References
Chapter 13: Application of SILAC Labeling in Phosphoproteomics Analysis
1 Introduction
2 Materials
2.1 SILAC Medium and Cell Culture Experiment
2.2 Cell Lysis and Protein Extraction
2.3 Protein Digestion and Peptide Purification
2.4 Phosphopeptide Enrichment
2.5 Stage Tip Purification
2.6 LC-MS/MS Analysis
2.7 Raw Data Processing (Peptide Identification and Quantification) and Data Analysis
3 Methods
3.1 General Considerations and Remarks
3.2 SILAC Labeling and Cell Culture Experiment
3.3 Cell Lysis and Protein Extraction
3.4 Mixing of SILAC Samples
3.5 Protein in-Solution Digestion
3.6 Peptide Purification on Oasis HLB Cartridges
3.7 Phosphopeptide Enrichment
3.7.1 Preparation of TiO2 Removal Tips
3.7.2 Preparation of TiO2 Beads
3.7.3 Phosphopeptide Enrichment
3.8 Stage Tip Purification
3.9 LC-MS/MS Analysis
3.10 Raw Data Processing (Peptide Identification and Quantification) and Data Analysis
4 Notes
References
Chapter 14: Relative Quantification of Phosphorylated and Glycosylated Peptides from the Same Sample Using Isobaric Chemical L...
1 Introduction
2 Materials
2.1 Laboratory Equipment
2.2 Cell Lysis and Protein Extraction and Digestion
2.3 TMT Labelling Reaction
2.4 Glycopeptide Enrichment
2.5 Phosphopeptide Enrichment
2.6 Peptide Desalting Using C18 Spin Columns
2.7 High-pH Reversed-Phase Fractionation
2.8 LC/MS/MS Analysis
3 Methods
3.1 Cell Lysis and Protein Extraction and Digestion
3.2 TMT Labelling Reaction
3.3 Glycopeptide Enrichment
3.4 Phosphopeptide Enrichment
3.5 Peptide Desalting Using C18 Spin Columns (See Notes 19 and 20)
3.6 Basic Reversed-Phase Fractionation
3.7 LC/MS/MS Analysis
3.8 Data Processing
4 Notes
References
Chapter 15: High-Throughput Profiling of Proteome and Posttranslational Modifications by 16-Plex TMT Labeling and Mass Spectro...
1 Introduction
2 Materials
2.1 Protein Extraction from Cells and Tissues
2.2 Protein In-Solution Digestion
2.3 Peptide Desalting
2.4 Enrichment of Di-GG Peptides and Di-GG Peptides Depletion Test
2.5 TMT16 Labeling of Peptides and Desalting
2.6 Offline Basic pH Reverse-Phase Liquid Chromatography Fractionation
2.7 Acidic pH RPLC-MS/MS Analysis
2.8 MS Data Analysis
3 Methods
3.1 Protein Extraction from Cells and Tissues
3.1.1 Whole Proteome
3.1.2 Ubiquitinome
3.2 Protein In-Solution Digestion
3.3 Peptide Desalting
3.3.1 Whole Proteome
3.3.2 Ubiquitinome
3.4 Enrichment of Di-GG Peptides
3.5 TMT16 Labeling of Peptides and Desalting
3.5.1 Whole Proteome
3.5.2 Ubiquitinome
3.6 Offline Basic pH RPLC Fractionation
3.7 Acidic pH RPLC-MS/MS Analysis
3.8 MS Data Analysis
3.8.1 Database Search
3.8.2 Peptide-Spectrum Match (PSM) Filtering
3.8.3 TMT-Based Quantification
4 Notes
References
Chapter 16: Quantification and Identification of Post-Translational Modifications Using Modern Proteomics Approaches
1 Introduction
2 Materials
2.1 Tissue Lysis and Tryptic Protein Digestion
2.2 Desalting of Proteolytic/Tryptic Peptides After Digestion (Oasis/HLB)
2.3 Anti-Acetyl Immunoaffinity Enrichment
2.4 Small-Scale Acetyl-Peptide Desalting Prior to MS Analysis
2.5 Chromatography and Mass Spectrometry: Nanoflow HPLC-MS/MS
3 Methods
3.1 Tissue Lysis
3.2 Tryptic Digestion
3.3 Desalt Tryptic Peptides with Oasis HLB Cartridges
3.4 Anti-Acetyl Immunoaffinity Enrichment
3.5 Small-Scale Acetyl-Peptide Desalting with C18 StageTips
3.6 Nanoflow LC-MS/MS Analysis
3.7 Identification and Quantification of Acetylation Sites Using DDA and DIA
3.8 Anticipated Results
4 Notes
References
Chapter 17: Affinity Enrichment Chemoproteomics for Target Deconvolution and Selectivity Profiling
1 Introduction
2 Materials
2.1 Whole Cell Lysis and Tissue Lysis
2.2 Preparation of Affinity Matrix
2.3 Rebinding and Competition Binding
2.4 Protein Digestion
2.5 Labeling of Tryptic Digests with Isobaric Mass Tags
2.6 Peptide Cleanup
2.7 Mass Spectrometry
2.8 Data Analysis
3 Methods
3.1 Cell and Tissue Lysis
3.2 Preparation of Affinity Matrix
3.3 Rebinding and Competition Binding Experiment
3.4 Protein Digestion
3.5 Labeling of Tryptic Digests with Isobaric Mass Tags
3.6 Peptide Cleanup
3.7 Mass Spectrometry
3.8 Data Analysis
4 Notes
References
Chapter 18: 2nSILAC for Quantitative Proteomics of Prototrophic Baker´s Yeast
1 Introduction
2 Materials
2.1 Metabolic Labeling of Prototrophic Yeast Using 2nSILAC
2.1.1 Yeast Strains
2.1.2 Culture Media
2.2 Preparation of Whole Cell Lysates
2.3 Small-Scale Preparation of Mitochondria-Enriched Fractions
2.4 LC-MS Sample Preparation
2.4.1 Acetone Precipitation
2.4.2 Proteolytic In-Solution Digest
2.4.3 Desalting of Peptides
2.5 LC-MS/MS and Data Analysis
3 Methods
3.1 Cultivation and Metabolic Labeling of Yeast Cells
3.2 Preparation of Whole Cell Lysates
3.3 Small-Scale Preparation of Mitochondria-Enriched Fractions
3.4 LC-MS Sample Preparation
3.4.1 Acetone Precipitation
3.4.2 Proteolytic In-Solution Digest
3.4.3 Desalting of Peptides
3.5 LC-MS/MS and Data Analysis
4 Notes
References
Chapter 19: Metabolic Labeling of Clostridioides difficile Proteins
1 Introduction
2 Materials
2.1 Buffer, Media, and Solutions for Preparatory Operations
2.2 Equipment
3 Methods
3.1 Preparatory Operation
3.2 Metabolic Labeling Procedure
4 Notes
References
Chapter 20: Application of Label-Free Proteomics for Quantitative Analysis of Urothelial Carcinoma and Cystitis Tissue
1 Introduction
2 Materials
2.1 FTIR Imaging and Laser Microdissection
2.2 Sample Preparation and Digestion
2.3 Liquid Chromatography
2.4 Mass Spectrometry
2.5 Data Analysis
3 Methods
3.1 General Practice
3.2 Automatic Annotation Via FTIR Imaging and Laser Microdissection
3.3 Sample Preparation and Enzymatic Digestion
3.4 Peptide Separation with Reversed Phase High-Performance Liquid Chromatography
3.5 Detection of Separated Peptides with Mass Spectrometry
3.6 Identification of Measured Proteins
3.7 Quantitative Proteome Analysis
3.8 Statistical Analysis
4 Notes
References
Chapter 21: Quantitative MS Workflow for a High-Quality Secretome Analysis by a Quantitative Secretome-Proteome Comparison
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Collection of Supernatant and Cell Harvest
2.2.1 Cell Lysis
2.2.2 Precipitation of Proteins from Cell Supernatant
2.3 Determination of Protein Concentration
2.4 In-Gel Digestion
2.5 LC-MS/MS
2.6 Database Search and Quantification
2.7 Processing of Quantitative Data
2.8 Supplementary Data Annotation
3 Methods
3.1 Cell Culture
3.2 Collecting Supernatant and Cell Harvest
3.2.1 Cell Lysis
3.2.2 Precipitation of Secreted Proteins
3.3 Determination of the Protein Concentration
3.4 In-Gel Digestion
3.5 LC-MS/MS
3.6 Database Search and Quantification
3.7 Analysis of Quantitative Protein Data
3.8 Data Supplementation
4 Notes
References
Chapter 22: Establishing a Custom-Fit Data-Independent Acquisition Method for Label-Free Proteomics
1 Introduction
2 Materials
2.1 Liquid Chromatography
2.2 Mass Spectrometry
2.3 Data Analysis
3 Methods
3.1 LC Optimization
3.2 MS Optimization
3.3 Calculation of Fixed Window Sizes
3.4 Variable DIA Windows
3.5 Creation of a Spectral Library
3.6 DIA Method Analysis and Validation
3.7 Workflows for Data Analysis, Visualization, and Interpretation
3.7.1 Workflow to Determine Peak Lengths
3.7.2 Workflow for Variable DIA Window Calculation
4 Notes
References
Chapter 23: Label-Free Proteomics of Quantity-Limited Samples Using Ion Mobility-Assisted Data-Independent Acquisition Mass Sp...
1 Introduction
2 Materials
2.1 Common Consumables and Lab Equipment
2.2 Cell Lysis
2.3 Single Pot Solid-Phase-Enhanced Sample Preparation
2.4 LC-MS Analysis
2.5 Data Analysis
3 Methods
3.1 Cell Lysis
3.2 Single Pot Solid-Phase-Enhanced Sample Preparation
3.3 LC-MS Analysis
3.4 Data Analysis and Label-Free Quantification
4 Notes
References
Chapter 24: DIA-MSE to Study Microglial Function in Schizophrenia
1 Introduction
2 Materials
2.1 Cellular Processes
2.2 In-Gel Digestions
2.3 Mass Spectrometry Analysis (LC-MS/MS)
2.4 UDMSE Analysis
2.5 Data Processing
3 Methods
3.1 Cell Thawing
3.2 Cell Growth
3.3 Freezing
3.4 Collecting the Cells
3.5 Cell Lysis and Protein Extraction (See Note 5)
3.6 In-Gel Digestion (See Fig. 2)
3.7 Mass Spectrometry Analysis (LC-MS/MS) (See Note 10)
3.8 UDMSE Analysis (Fig. 3)
3.9 Data Processing (See Note 12)
4 Notes
References
Chapter 25: Detailed Method for Performing the ExSTA Approach in Quantitative Bottom-Up Plasma Proteomics
1 Introduction
2 Materials
2.1 Sample and Solution Preparation Supplies
2.2 Control and Experimental Sample Preparations
2.3 LC-MS Equipment
2.4 Data Collection and Analysis Software
3 Methods
3.1 Digest Preparations (for Control and Experimental Samples)
3.2 Standard Solution Preparations (for the Control Samples)
3.3 Standard Spiking to Control/Experimental Digests
3.4 Extraction and Reconstitution for LC-MS/MS
3.5 LC System Setup
3.6 MRM/MS System Setup
3.7 PRM/MS System Setup
3.8 LC-MS/MS Platform Performance Test
3.9 Sample Injection
3.10 Data Analysis
4 Notes
References
Chapter 26: Quantitative Cross-Linking of Proteins and Protein Complexes
1 Introduction
1.1 Cross-Linking
1.2 Sample Preparation and LC-MS/MS Analysis
1.3 Data Analysis
1.4 Quantitative Cross-Linking
1.5 Quantitative Cross-Linking Workflow and Example Data
2 Materials
2.1 Chemical Cross-Linking
2.2 In-Solution Digestion
2.2.1 Ethanol Precipitation
2.2.2 In-Solution Digestion in the Presence of RapiGest
2.3 Enrichment of Cross-Links
2.4 LC-MS/MS Analysis
2.5 Identification of Cross-Linked Peptides
2.6 Quantification of Identified Cross-Links
3 Methods
3.1 Chemical Cross-Linking
3.1.1 Identification of Optimal Cross-Linker Concentration
3.1.2 Quantitative Cross-Linking
3.2 Sample Preparation for LC-MS/MS Analysis
3.2.1 Ethanol Precipitation and in-Solution Digestion
3.2.2 Enrichment of Cross-Linked Di-Peptides
3.3 LC-MS/MS Analysis
3.4 Identification of Cross-Linked Peptides
3.5 Quantification of Identified Cross-Links
4 Notes
References
Chapter 27: Missing Value Monitoring to Address Missing Values in Quantitative Proteomics
1 Introduction
2 Materials
2.1 Stage Tip Preparation
2.2 Protein Digestion
2.3 Materials for LC-MS/MS.
2.4 Software
3 Methods
3.1 Protein Digestion
3.2 Mass Spectrometry Analysis
3.3 MvM Workflow
4 Notes
References
Chapter 28: Quantitative Proteome Data Analysis of Tandem Mass Tags Labeled Samples
Abbreviations
1 Introduction
2 Materials
2.1 Raw MS and MS/MS Data
2.2 Database Analysis Software
2.3 Data Processing
2.4 Data Interpretation and Visualization
3 Methods
3.1 Database Search
3.2 Data Visualization, Filtering and Export from PD
3.3 Data Normalization and Scaling Using MS Excel
3.4 Data Consolidation and Ratio Calculation
3.5 Threshold Definition for Regulations
3.6 Data Evaluation According to Ratios and p-Value
4 Notes
References
Chapter 29: Mining Protein Expression Databases Using Network Meta-Analysis
1 Introduction
1.1 Public Data and Reproducibility of Research
1.2 Research Synthesis and Meta-Analyses of High-Throughput Omics Data
1.3 Network Meta-Analysis
2 Materials
3 Methods
3.1 Normalization and Differential Expression Analysis
3.2 Analysis of Merged Data
3.3 Network Meta-Analysis Based on Individual Study Results
4 Results and Discussion
4.1 Network Meta-Analysis Versus Analysis of Merged Data
4.2 Biological Interpretation
4.2.1 Differences Regarding the Method of Analysis
4.2.2 Role of Significant Proteins in Breast Cancer
5 Discussion
6 Notes
References
Chapter 30: A Tutorial for Variance-Sensitive Clustering and the Quantitative Analysis of Protein Complexes
1 Introduction
2 Materials
2.1 Resources
2.2 Software
3 Methods
3.1 Data Preparation
3.2 Statistics and Clustering Using VSClust
3.3 Quantitative Behavior of Protein Complexes
4 Notes
References
Chapter 31: Automated Workflow for Peptide-Level Quantitation from DIA/SWATH-MS Data
1 Introduction
2 Materials
3 Methods
4 Notes
References
Index