Clinical Proteomics: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Bile Processing Protocol for Improved Proteomic Analysis
1 Introduction
2 Materials
2.1 Bile
2.2 S-TRAP Digestion
2.3 Cleaning-Up and Desalting Peptides with C18 Stage-Tips
2.4 Qubit Peptide Quantitation Assay
2.5 Liquid Chromatography
2.6 Mass Spectrometry
3 Methods
3.1 S-TRAP Digestion
3.2 Cleaning-up and Desalting Peptides with C18 Stage-Tips (Fig. 2)
3.3 Qubit Peptide Quantitation Assay
3.4 Liquid Chromatography and Mass Spectrometry Analysis
3.5 Data Analysis and Quantification
4 Notes
References
Chapter 2: MS-Based Extracellular Vesicle (EVs) Analysis: An Application to Helminth-Secreted EVs
1 Introduction
2 Materials
2.1 Equipment Needed
2.2 Reagents for the Isolation of EVs
2.3 Reagents for the Isolation of Proteins and in-Gel Digestion
3 Methods
3.1 Isolation of Helminth-Secreted EVs
3.2 Isolation of the Proteins from the Different Compartments of EVs
3.3 In-Gel Protein Digestion and Preparation for Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
4 Notes
References
Chapter 3: Proteomic Profiling of Cerebrospinal Fluid by 16-Plex TMT-Based Mass Spectrometry
1 Introduction
2 Materials
2.1 CSF Protein Extraction and In-Solution Digestion
2.2 Peptide Desalting
2.3 TMT Labeling of Peptides and Desalting
2.4 Offline Basic pH Reverse Phase Liquid Chromatography (RPLC) Fractionation
2.5 Acidic pH RPLC-MS/MS Analysis
2.6 MS Data Analysis
3 Methods
3.1 Cerebrospinal Fluid
3.2 Protein Extraction, Quality Control, and In-Solution Digestion
3.2.1 Protein Extraction and Quality Control
3.2.2 In-Solution Protein Digestion, Peptide Reduction and Alkylation, Digestion Efficiency Test, and Peptide Desalting
3.3 TMT16 Labeling of Peptides, Label Efficiency Test, Sample Pooling, and Labeled Peptide Desalting
3.4 Offline Basic pH RPLC Fractionation
3.5 Acidic pH RPLC-MS/MS Analysis
3.6 MS Data Analysis
3.6.1 Database Search
3.6.2 Peptide-Spectrum Match Filtering
3.6.3 TMT-Based Protein Quantification
4 Notes
References
Chapter 4: Data-Independent Acquisition Mass Spectrometry-Based Deep Proteome Analysis for Hydrophobic Proteins from Dried Blo...
1 Introduction
2 Materials
2.1 DBS Samples
2.2 Protein Extraction Solutions and Other Reagents
2.3 Trypsin Digestion
2.4 LC-MS
2.5 Data Analysis
3 Methods
3.1 DBS Preparation
3.2 Preparation of the Insoluble Fraction Using 100 mM Sodium Carbonate (Fig. 1)
3.3 Trypsin Digestion
3.4 Purification
3.5 DIA-MS by LC-MSMS
3.6 oDIA-MS Data Analysis by Scaffold DIA
4 Notes
References
Chapter 5: Sample Processing for Metaproteomic Analysis of Human Gut Microbiota
1 Introduction
2 Materials
2.1 Microbial Enrichment and Protein Extraction
2.2 Precipitation and Protein Digestion
2.3 Purification and Sample Preparation for LC-MS/MS
3 Methods
3.1 Microbial Collection (Fig. 1a)
3.2 Protein Extraction (Fig. 1b)
3.3 Precipitation, Quantification, and Trypsin Digestion
3.4 Purification and MS/MS Analysis
4 Notes
References
Chapter 6: Mass Spectrometry-Based Analytical Strategy for Single-Cell Proteomics
1 Introduction
2 Materials
2.1 Reagents
2.2 Buffers and Solutions to Prepare
2.3 Instrumentation
2.4 Software for Data Analysis
3 Methods
3.1 Culture and Isolation of Cells
3.2 Protein Extraction, Denaturation, and Digestion
3.3 TMT Labeling
3.4 Pooling of TMT-Labeled Peptides
3.5 LC-MS/MS Analysis
3.6 Data Analysis
3.6.1 Protein Search and Identification
3.6.2 Statistical Analysis
4 Notes
References
Chapter 7: Lysine Acetylation Stoichiometry Analysis at the Proteome Level
1 Introduction
2 Materials
2.1 Synthesis of N-Acetoxysuccinimide-d3 (NAS-d3)
2.2 Sample Preparation in Solution
2.2.1 Instruments
2.2.2 Stock Solutions
2.3 Sample Preparation in S-Trap
2.3.1 Instruments
2.3.2 Stock Solutions
2.4 LC-MS/MS Analysis
2.4.1 Instruments
2.4.2 Chromatographic Solutions
3 Methods
3.1 Synthesis of N-Acetoxysuccinimide-d3 (NAS-d3)
3.2 Sample Preparation in Solution
3.2.1 Cell Lysis: Protein Extraction
3.2.2 Protein Lysine Acetylation
3.2.3 Protein Digestion with Trypsin
3.3 Sample Preparation in S-Trap
3.3.1 Cell Lysis: Protein Extraction
3.3.2 Protein Lysine Acetylation
3.3.3 Protein Digestion with Trypsin
3.3.4 Peptide Elution
3.4 LC-MS/MS Analysis
3.5 Data Processing for Identifying Lysine Acetylation Sites
4 Notes
References
Chapter 8: Implementation of Clinical Phosphoproteomics and Proteomics for Personalized Medicine
1 Introduction
2 Materials and Software
2.1 Sample Lysis
2.2 Cysteine Reduction and Alkylation
2.3 Desalting
2.4 Phosphoenrichment
2.5 Peptide Reconstitution
2.6 Mass Spectrometry Analysis
3 Methods
3.1 Sample Lysis
3.1.1 Adherent Cells
3.1.2 Suspension Cells
3.1.3 Solid Tissue Samples
3.1.4 Common Steps
3.2 Cysteine Reduction and Alkylation
3.3 Tryptic Digestion
3.4 Desalting
3.4.1 Desalting for Phosphoproteomics
3.4.2 Desalting for Proteomics
3.5 Phosphoenrichment
3.6 Peptide Reconstitution
3.6.1 Reconstitution for Phosphoproteomics
3.6.2 Reconstitution for Proteomics
3.7 Mass Spectrometry Analysis
3.7.1 Liquid Chromatography Separation
3.7.2 Mass Spectrometry Analysis
3.8 Phosphopeptide and Protein Identification
3.9 Phosphopeptides and Protein Quantification
3.10 Estimation of Kinase Activity
3.11 Statistical Learning
4 Notes
References
Chapter 9: pH/Acetonitrile-Gradient Reversed-Phase Fractionation of Enriched Hyper-Citrullinated Library in Combination with L...
1 Introduction
2 Materials
2.1 Cell/Tissue Lysis
2.2 Protein Quantification
2.3 Filter-Aided Sample Preparation (FASP) Reagents
2.4 Deamidation Reaction
2.5 Desalting
2.6 High-pH Reversed-Phase Fractionation
2.7 Mass Spectrometry Sample Preparation
2.8 Data Analysis Software and Setup
3 Methods
3.1 Cell/Tissue Lysis
3.2 Protein Quantification
3.3 FASP Digestion with Lys-C and Deimidation Reaction on Filters
3.4 Desalting
3.5 High-pH Reversed-Phase Fractionation and Mass Spectrometry Sample Preparation
3.6 Liquid Chromatography
3.7 Mass Spectrometry
3.8 Data Preparation
3.9 CitFinder: Identification of Citrullinated Peptides
3.10 CitFinder: Validation of Citrullinated Peptides
3.11 Anticipated Results
4 Notes
References
Chapter 10: Purification of HLA Immunopeptidomes from Human Thymus
1 Introduction
2 Materials
2.1 Materials
2.2 Reagents, Buffers, and Solutions
2.3 Human Thymus Samples
3 Methods
3.1 Collection and Processing of Thymus Samples
3.2 Genomic DNA (gDNA) Extraction and HLA Typing
3.3 Homogenization of Thymus Samples and Cell Lysis
3.4 Precolumn and Antibody Column
3.5 Purification of pHLA Complexes
3.6 LC-MS/MS Analysis
3.7 MS/MS Ion Search and Peptide Identification
4 Notes
References
Chapter 11: Generation of HLA Allele-Specific Spectral Libraries to Identify and Quantify Immunopeptidomes by SWATH/DIA-MS
1 Introduction
2 Materials
2.1 Hardware
2.2 Software
2.2.1 Trans Proteomic Pipeline
2.2.2 NetMHCpan-4.1
2.2.3 Python 3.5 (or Greater)
2.2.4 OpenSWATH (Only Required If Generating TraML Files)
2.2.5 Msproteomicstools Package for Python
2.2.6 Additional Python Scripts and Files
2.3 Data
3 Methods
3.1 Organization of Data
3.2 Add iRT Peptides to FASTA
3.3 Generate Decoy Database
3.4 Prepare Search Parameters
3.5 Run Database Searches
3.6 Convert X! Tandem Output Files
3.7 Validation with PeptideProphet
3.8 Integration with iProphet
3.9 iProphet pepXML Extraction and False Discovery Rate Filtering
3.10 HLA Allele Annotation and Building SpectraST Inclusion Lists
3.11 Generation of HLA Allele-Specific Peptide Spectral Libraries
3.12 Conversion to SWATH Assay Libraries
4 Notes
References
Chapter 12: Immunopeptidomic Analysis of the Phosphopeptidome Displayed by HLA Class I Molecules
1 Introduction
2 Materials
2.1 Preparation of the Affinity Column and Precolumn
2.2 Cell Lysis and Immunopurification of MHC-I Complexes
2.3 Phosphopeptide Enrichment
2.4 LC-MS/MS Analysis
2.5 Database Search
3 Methods
3.1 Preparation of the Affinity Column and Precolumn
3.2 Cell Lysis and Affinity Purification of MHC-I Complexes
3.3 Phosphopeptide Enrichment
3.4 LC-MS/MS Analysis
3.5 Database Search
4 Notes
References
Chapter 13: Development of a Standardized MRM Method for the Quantification of One Carbon Metabolism Enzymes
1 Introduction
2 Materials
2.1 Database for Peptide Selection and Validation (See Note 1)
2.2 Recombinant Proteins for Peptide Library Generation
2.3 Software for Data Analysis
2.4 Peptide Synthesis
2.5 Synthetic Peptide Purification
2.6 Synthetic Peptide Analytic Chromatography
2.7 Synthetic Peptide Purity Assay
2.8 Collision Energy Optimization
2.9 Scheduling/Selectivity/Interference Screening/Validation
2.10 Shotgun Proteomics Analysis
2.11 Targeted Analysis
2.12 Lysis of Frozen (-80 C) Liver Tissue Sample
2.13 Protein Concentration Measurement
2.14 Protein Precipitation
2.15 Protein Digestion
2.16 Sample Desalting
2.17 Peptide Concentration Measurement
3 Methods
3.1 Protein and Peptide Selection for Method Development and Peptide Library Generation (See Note 4)
3.2 Synthetic Peptides
3.2.1 Peptide Synthesis
3.2.2 Peptide Purification and Quantification
3.3 Method Optimization
3.3.1 Targeted Proteomics MRM Method Test
3.3.2 Collision Energy Optimization
3.3.3 Development of an MRM Scheduled Method (See Note 8)
3.4 Selectivity/Interference Screening
3.5 Concentration-Signal Correlation Assessment
3.6 Routine Application
3.6.1 Lysis
3.6.2 Protein Quantification
3.6.3 Protein Precipitation
3.6.4 Protein Digestion (See Subheading 3.1, step 1)
3.6.5 Sample Desalting (see Note 12)
3.6.6 Peptide Concentration Measurement
3.6.7 LC-MS Analysis
4 Notes
References
Chapter 14: Molecular Histology Analysis of Cryopreserved Tissue Using Peptide/Protein MALDI-TOF Imaging Mass Spectrometry (MA...
1 Introduction
2 Materials
2.1 Tissue Sampling, Sectioning, and Storage
2.2 Matrix Deposition and Spectra Collection
2.3 Histological Analysis
3 Methods
3.1 Tissue Sampling, Sectioning, and Storage
3.2 MALDI IMS
3.3 Histological Analysis
4 Notes
References
Chapter 15: The Human Protein Atlas and Antibody-Based Tissue Profiling in Clinical Proteomics
1 Introduction
2 Materials
2.1 Tissue Microarray
2.2 Tissue Sectioning
2.3 Immunohistochemistry
3 Method
3.1 Selection of Antibodies and Tissues
3.2 Tissue Microarray (TMA)
3.3 Tissue Sectioning
3.4 Immunohistochemistry (IHC)
3.5 Annotation
3.6 Antibody Validation
4 Notes
References
Chapter 16: Microbial Identification in the Clinical Microbiology Laboratory Using MALDI-TOF-MS
1 Introduction
2 Materials
2.1 General Purpose
2.2 For Identification Directly from Blood Cultures
2.3 For Filamentous Fungi (Molds) and Mycobacteria
2.4 Matrix and BTS Preparation
3 Methods
3.1 Identification of Bacterial and Yeast Species from Agar Plate Colonies
3.2 Ethanol/Formic Acid Extraction
3.3 Identification of Bacterial and Yeast Species Directly from Positive Blood Culture Bottles
3.3.1 Fast Protocol Using Triton X-100
3.3.2 Alternative Protocol (Not for Yeast Species)
3.4 Identification of Mycobacteria and Other Difficult Bacterial Groups
3.5 Identification of Filamentous Fungi from Agar Plate Colonies
4 Notes
References
Chapter 17: Combining Electron Microscopy (EM) and Cross-Linking Mass Spectrometry (XL-MS) for Structural Characterization of ...
1 Introduction
2 Materials
2.1 Biological Samples
2.2 Grafix (Gradient Fixation)
2.3 Negative Staining EM
2.4 Chemical Cross-Linking and Trypsin Digestion
2.5 LC-MS/MS Analysis and Peptide Identification
3 Methods
3.1 GraFix
3.2 Negative Staining EM
3.3 Image Processing and 3D Reconstruction
3.4 Docking of Atomic Structures
3.5 Chemical Cross-Linking and Trypsin Digestion
3.6 LC-MS/MS Analysis and Peptide Identification
3.7 Model Validation
4 Notes
References
Chapter 18: Accurate Prediction of Protein Sequences for Proteogenomics Data Integration
1 Introduction
1.1 Multi-omics Profiles
1.2 Proteogenomics
1.3 Highly Similar, Yet Different, Individual Genomes
2 Different Types of Variants
2.1 Single-Nucleotide Variants
2.2 Multi-nucleotide Variants
2.3 Insertions and Deletions
2.4 Structural Variants
2.5 Haplotypes
2.6 Epigenetic Regulation: Inherited, Dynamic, Regulated, and Conditional
2.7 Genomic Features
2.8 Gene Architecture
2.9 Alternative Splicing
2.10 From Transcription to Mature Transcript
2.11 RNA Regulation
2.12 Translation Gatekeeping
3 DNA/RNA Sequencing Methods
3.1 Basis of (Nearly) All Sequencing Approaches
3.2 Whole-Genome Sequencing
3.3 Whole-Exome Sequencing
3.4 RNA Sequencing
3.5 Sequencing Actively Translated Transcripts
3.6 Sequencing Actively Translated Transcripts, Fractionated by Number of Attached Ribosomes
3.7 Cell Sorting and Laser Capture Microdissection
3.8 Unique Molecular Identifier
3.9 Single-Cell and Single-Nuclei Sequencing
4 DNA/RNA Sequencing Technologies
4.1 Next-Generation Sequencing
4.2 Next-Generation Sequencing Library Types
4.3 Long-Read Sequencing
4.4 Implications of Long Reads for Transcriptomics
4.5 Implications of Long Reads for Genomics
4.6 Striking a Balance Between Quality and Read Length
5 Sequencing Data Analysis
5.1 From Reads to Sample-Specific Protein Database
5.2 Quality Control
5.3 Read Mapping
5.4 Variant Calling
5.5 Enumerating Protein Variants
6 Conclusion
References
Index