Serum/Plasma Proteomics: Methods and Protocols

Preface
Contents
Contributors
Part I: Blood Processing and Handling Strategies
Chapter 1: Protocols for the Isolation of Platelets for Research and Contrast to Production of Platelet Concentrates for Trans...
1 Introduction
2 Materials
2.1 Blood Collection and Platelet Sample Preparation
3 Methods
3.1 Blood Collection
3.2 Platelet-Rich Plasma (PRP) Preparation Method
3.3 Buffy Coat (BC) Platelet Preparation Method
3.4 How Blood Processing Facilities Prepare PCs for Transfusion
4 Notes
References
Chapter 2: Collection of Plasma Samples in Areas with Limited Healthcare Access
1 Introduction
2 Materials
2.1 Plasma Collection and Storage
2.2 Extraction of Intact Proteins: General Procedure
2.3 Extraction of Intact Proteins: SARS-CoV-2 Total Ab Assay
2.4 Extraction and Digestion of Proteins for Peptide-Based LC-MS Proteomics: Direct Digestion
2.5 Extraction and Digestion of Proteins for Peptide-Based LC-MS Proteomics: SP3 Digestion
2.6 Extraction of Metabolites for Global Profiling
3 Methods
3.1 Plasma Collection and Storage
3.2 Extraction of Intact Proteins: General Procedure
3.3 Extraction of Intact Proteins: SARS-CoV-2 Total Ab Assay
3.4 Extraction and Digestion of Proteins for Peptide-Based LC-MS Proteomics: Direct Digestion
3.5 Extraction and Digestion of Proteins for Peptide-Based LC-MS Proteomics: SP3 Digestion (See Note 16)
3.6 Extraction of Metabolites for Global Profiling (See Note 17)
4 Notes
References
Chapter 3: Blood Collection Processing and Handling for Plasma and Serum Proteomics
1 Introduction
2 Materials
2.1 Blood Collection
2.2 Plasma/Serum Generation
3 Methods
3.1 Participant Recruitment and Study Design
3.2 Venous Blood Collection for Plasma
3.3 Venous Blood Collection for Serum
3.4 Sample Preparation
3.5 Sample Thawing
4 Notes
Checklist for Addressing Pre-analytical Variables in a Proteomic Study
References
Chapter 4: Preparation of Cryoprecipitate and Cryo-depleted Plasma for Proteomic Research Analysis
1 Introduction
2 Materials
2.1 Whole Blood Collection, Plasma Preparation, and Frozen Storage
2.2 Cryoprecipitate/Cryo-depleted Plasma Preparation
3 Methods
3.1 Blood Collection/Phlebotomy
3.2 Cryoprecipitation
3.3 Thawing of Cryoprecipitate or Cryo-depleted Plasma
4 Notes
References
Part II: Discovery-Based Mass Spectrometry
Chapter 5: High-Throughput Proteome Profiling of Plasma and Native Plasma Complexes Using Native Chromatography
1 Introduction
1.1 Native Profiling of Protein Complexes
1.2 Native Separation of Plasma
1.3 HT Label-Free SEC-AutoP3 Approach
1.4 HT Isobaric Labeling AutoMP3 Approach
2 Materials
2.1 Native Fractionation Using SEC
2.2 Sample Preparation for AutoP3/HT AutoMP3 SEC Fractions
2.3 Sample Preparation for AutoMP3 for SEC Fractions
2.4 LC-MS Acquisition
2.5 LC-MS Data Analysis: Software Tools
3 Methods
3.1 Native Fractionation Using SEC
3.2 Sample Preparation Using SEC-AutoP3
3.3 Sample Preparation Using HT AutoMP3
3.4 LC-MS/MS Acquisition
3.4.1 LC-MS/MS Acquisition of SEC-AutoP3 Label-Free Samples
3.4.2 LC-MS Acquisition of TMT-Labeled Samples
3.5 Data Analysis
4 Notes
References
Chapter 6: High-Throughput and In-Depth Proteomic Profiling of 5 μL Plasma and Serum Using TMTpro 16-Plex
1 Introduction
2 Materials
2.1 Depletion of Top-Abundance Proteins in Plasma/Serum
2.2 Protein Digestion
2.3 TMT Labeling
2.4 Basic pH Reversed Phase Liquid Chromatography (bRPLC)
2.5 LC-MS/MS
2.6 Software for MS Data Analysis
3 Methods
3.1 Depletion of High-Abundance Proteins
3.2 Protein Digestion and Peptide Desalting
3.3 TMT Labeling (See Note 11)
3.4 High pH Fractionation by Basic pH Reversed Phase Liquid Chromatography
3.5 LC-MS/MS
3.6 MS Data Analysis
4 Notes
References
Chapter 7: An Optimized Data-Independent Acquisition Strategy for Comprehensive Analysis of Human Plasma Proteome
1 Introduction
2 Materials
2.1 Plasma Collection and Abundant Plasma Protein Depletion
2.2 Single-Pot Solid-Phase-Enhanced Sample Preparation and Enzymatic Digestion
2.3 Proteomic Profiling Using NanoLC-MS/MS
2.4 MS Data Processing and Analysis
2.5 DIA Method Development and Optimization (Optional)
3 Methods
3.1 Plasma Collection and Abundant Plasma Protein Depletion
3.2 Single-Pot Solid-Phase-Enhanced Sample Preparation and Enzymatic Digestion
3.3 Proteomic Profiling Using NanoLC-MS/MS
3.4 MS Data Processing and Analysis
3.5 DIA Method Development and Optimization (Optional)
4 Notes
References
Chapter 8: In-Depth Blood Proteome Profiling by Extensive Fractionation and Multiplexed Quantitative Mass Spectrometry
1 Introduction
2 Materials
2.1 Plasma Protein Precipitation
2.2 In-Solution Digesting
2.3 Peptide Desalting
2.4 TMT Labeling and Desalting of Peptides
2.5 Offline Basic pH RPLC
2.6 Acidic pH RPLC MS/MS Analysis
2.7 MS Data Analysis
3 Methods
3.1 Plasma/Serum Collection
3.2 Protein Precipitation and In-Solution Digestion
3.2.1 Protein Precipitation
3.2.2 In-Solution Digestion and Peptide Desalting
3.3 TMTpro 18-Plex Labeling, Pooling, and Desalting
3.4 Basic pH RPLC Extensive Fractionation
3.5 Acidic pH RPLC-MS/MS Analysis
3.6 Computational Analysis
3.6.1 Database Search of MS Raw Data
3.6.2 Peptide Identification After Data Filtering
3.6.3 Quantification of Peptides/Proteins
4 Notes
References
Chapter 9: Early Cancer Biomarker Discovery Using DIA-MS Proteomic Analysis of EVs from Peripheral Blood
1 Introduction
2 Materials
2.1 Serum Sample Preparation
2.2 Extracellular Vesicle (EV) Purification
2.3 EV Protein Extraction for LC-MS/MS
2.4 Protein Quantitation (EZQ Protein Quantitation Kit) (See Note 8)
2.5 Sample Preparation (Reduction and Alkylation) for LC-MS/MS
2.6 Single-Pot Solid-Phase-Enhanced Sample Preparation (SP3) Protocol (See Note 11)
2.7 Enzymatic Digestion of Protein
2.8 Peptide Sample Desalting Using ZipTips and Spin Columns
2.9 Off-Line Peptide Fractionation by Microflow High pH RP-HPLC
2.10 LC-MS/MS Analysis of Peptide Samples
2.11 LC-MS/MS Data Processing
2.12 Statistical Analysis
3 Methods
3.1 Serum Sample Preparation
3.2 Extracellular Vesicle Extraction
3.3 EV Protein Extraction for LC-MS/MS
3.4 Protein Quantitation (EZQ Protein Quantitation Kit) (See Note 8)
3.5 Sample Preparation (Reduction and Alkylation) for LC-MS/MS
3.6 Single-Pot, Solid-Phase-Enhanced Sample Preparation (SP3) Protocol (See Note 27)
3.7 Enzymatic Digestion of Proteins and Peptide Desalting (See Note 41)
3.8 Off-Line Peptide Separation by High pH Reversed Phase HPLC
3.9 DDA-MS Data Processing and Spectral Library Generation
3.10 DIA-MS Data Processing
3.11 Statistical Analysis
4 Notes
References
Part III: Developments in Technologies for Plasma Proteomics
Chapter 10: Strategies to Enrich, Identify, and Characterize Glycoproteome in Blood Plasma Using Liquid Chromatography High-Re...
1 Introduction
2 Materials
2.1 Plasma Sample Collection and Storage
2.2 Enrichment of Glycoproteins Using Lectin Affinity Chromatography (LAC)
2.3 Glycoprotein Digestion and Peptide Preparation
2.4 Selective Enrichment of Glycopeptides by LAC and HILIC, Deglycosylation, and O18 Labeling
2.5 MS Acquisition (Nano-LC-HR-MS/MS)
2.6 LC-HRMS Data Analysis: Software Tools
3 Methods
3.1 Blood Collection, Isolation of Plasma, and Storage
3.1.1 Isolation of Plasma from Blood Samples
3.2 Glycoprotein Enrichment from Plasma Samples
3.2.1 Selective Enrichment of Plasma Glycoproteins through Lectin Affinity Chromatography (LAC)
3.3 Glycoprotein Digestion
3.4 Glycopeptide Enrichment, Deglycosylation, and O18 Labeling
3.4.1 Glycopeptide Enrichment Using LAC-HILIC, PNGase-Mediated Deglycosylation, and O18 Labeling
3.5 HR-MS Acquisition (Nano-LC-HR Orbitrap MS)
3.6 Software-Based Data Analysis
4 Notes
References
Chapter 11: Proteome Analysis of Whole Blood Collected by Volumetric Absorptive Microsampling
1 Introduction
2 Materials
2.1 Blood Collection
2.2 VAMS Processing
2.3 Tryptic Digestion and Peptide Purification
2.4 Mass Spectrometry
3 Methods
3.1 Blood Collection
3.2 VAMS Processing
3.3 Tryptic Digestion
3.4 Mass Spectrometry Acquisition
3.5 Peptide Identification
4 Notes
References
Chapter 12: Proteomic Applications and Considerations: From Research to Patient Care
1 Introduction
1.1 Recent Advancements in Proteomics
2 Analytical Strategies in Proteomics
2.1 Label-Based Proteomics
2.2 Label-Free-Based Proteomics
3 Proteomic Biomarker Studies
4 Blood
4.1 Plasma- and Serum-Derived Extracellular Vesicles
5 Blood-Based Proteomic Biomarkers
5.1 Cancer
5.2 Stroke and Venous Thromboembolism (VTE)
5.3 Mild Traumatic Brain Injury (mTBI)
6 The Future of Proteomics
6.1 Machine Learning
6.2 Proteogenomics
7 Considerations for Biomarker Studies
References
Part IV: Enrichment & Detection Strategies
Chapter 13: Immunoaffinity Mass Spectrometry Diagnostic Tests for Multi-Biomarker Assays
1 Introduction
2 Materials
2.1 Antibody-Bead Production
2.2 Calibrator Preparation
2.3 Immunoaffinity Bead Method
2.4 MS Acquisition (Microflow-LC-MS/MS)
2.5 MS Data Evaluation: Software Tools
3 Methods
3.1 Antibody-Bead Preparation
3.2 Calibrator Preparation
3.3 Synthetic Label Preparation
3.4 IA-MS Method
3.5 LCMS Acquisition
3.6 MS Data Analysis
4 Notes
References
Chapter 14: Secretome Profile of Leukocyte-Platelet-Rich Fibrin (L-PRF) Membranes
1 Introduction
2 Materials
2.1 Blood Collection
2.2 L-PRF Membrane Culture and Secretome Collection
2.3 Protein Precipitation
2.4 Protein Quantitation
2.5 Gel-Based Proteomics: Qualitative Proteomics
2.5.1 Trypsin in-Gel Digestion
2.6 Gel-Based Proteomics: MS Analysis
2.7 Differential Secretome Protein Quantitation by SWATH-MS
3 Methods
3.1 Blood Collection: L-PRF Generation
3.2 L-PRF Membrane Culture and Secretome Collection
3.3 Protein Precipitation
3.4 Protein Quantitation
3.5 Qualitative Proteomics Analysis
3.5.1 Gel-Based Protein Separation
3.5.2 Trypsin in-Gel Digestion (See Note 9)
3.5.3 MS Analysis
3.6 Differential Quantitative SWATH-MS-Based Proteomic Analysis of L-PRF Secretomes
3.7 Systems Biology
4 Notes
References
Chapter 15: Quantification of Proteins in Blood by Absorptive Microtiter Plate-Based Affinity Purification Coupled to Liquid C...
1 Introduction
2 Materials
2.1 Antibody-Based Immunocapture
2.2 Affimer-Based Immunocapture
2.3 Direct (in-Well) Tryptic Digestion
2.4 Indirect Tryptic Digestion
2.5 LC-SRM/MS Analysis
3 Methods
3.1 Antibody-Based Immunocapture
3.2 Affimer-Based Immunocapture
3.3 Direct (in-Well) Tryptic Digestion
3.4 Indirect Tryptic Digestion
3.5 μLC-SRM/MS Analysis
4 Notes
References
Chapter 16: Glycomics-Assisted Glycoproteomics Enables Deep and Unbiased N-Glycoproteome Profiling of Complex Biological Speci...
1 Introduction
2 Materials
2.1 Initial Protein Handling
2.2 N-Glycomics Workflow
2.2.1 Protein Immobilization on PVDF Membrane
2.2.2 Release of N-Glycans
2.2.3 Reduction of N-Glycans
2.2.4 Desalting of N-Glycans Using SCX-C18-SPE
2.2.5 Desalting of N-Glycans Using PGC-C18-SPE
2.2.6 PGC-LC-MS/MS
2.2.7 Data Analysis (Manual- and Software-Aided Annotation): Software
2.3 N-Glycoproteomics Workflows
2.3.1 Tryptic Digestion and Peptide Desalting Using Oligo R3-C18-SPE
2.3.2 TMT-Labeling of Peptides (Optional)
2.3.3 N-Glycopeptide Enrichment Using ZIC-HILIC-C8-SPE
2.3.4 Peptide De-N-glycosylation
2.3.5 High-pH Prefractionation Using R2-C18-SPE (Optional)
2.3.6 Reversed-Phased LC-MS/MS of Intact N-Glycopeptides, De-N-glycopeptides, and Non-modified Peptides
2.3.7 Data Analysis of LC-MS/MS Data of Intact N-Glycopeptides: Software
2.3.8 Data Analysis of LC-MS/MS Data of De-N-glycopeptides and Non-modified Peptides: Software
3 Methods
3.1 Initial Protein Handling
3.2 N-Glycomics Workflow
3.2.1 Protein Immobilization on PVDF Membrane
3.2.2 Release of N-Glycans
3.2.3 Reduction of N-Glycans
3.2.4 Desalting of N-Glycans Using SCX-C18-SPE
3.2.5 Desalting of N-Glycans Using PGC-C18-SPE (See Note 4.1.7)
3.2.6 PGC-LC-MS/MS
3.2.7 Data Analysis (Manual- and Software-Aided Annotation)
3.3 N-Glycoproteomics Workflows
3.3.1 Tryptic Digestion and Peptide Desalting Using Oligo R3-C18-SPE
3.3.2 TMT-Labeling (Optional)
3.3.3 N-Glycopeptide Enrichment Using ZIC-HILIC-C8-SPE
3.3.4 Peptide De-N-glycosylation
3.3.5 High-pH Prefractionation Using R2-C18-SPE (Optional) (See Note 4.2.5)
3.3.6 Reversed-Phased LC-MS/MS of Intact N-Glycopeptides, De-N-glycopeptides, and Non-modified Peptides
3.3.7 Data Analysis of LC-MS/MS Data of Intact N-Glycopeptides
3.3.8 Data Analysis of LC-MS/MS Data of Non-modified and De-N-glycosylated Peptides
4 Notes
4.1 N-Glycomics Workflow
4.2 N-Glycoproteomics Workflows
References
Chapter 17: The Small-Protein Enrichment Assay (SPEA) for Analysis of Low Abundance Peptide Hormones in Plasma
1 Introduction
2 Materials
2.1 Plasma Precipitation
2.2 Chloroform/Ethanol Delipidation
2.3 SEC Liquid Chromatography Separation
2.4 Sample Preparation for Mass Spectrometry
2.5 DDA Mass Spectrometry Analysis
2.6 DIA Mass Spectrometry Analysis
3 Methods
3.1 Plasma Precipitation
3.2 Chloroform/Ethanol Delipidation
3.3 SEC Liquid Chromatography Separation
3.4 Sample Preparation for Mass Spectrometry
3.5 DDA Mass Spectrometry Analysis
3.6 DIA Mass Spectrometry Analysis
3.7 DDA Data Analysis
3.8 DIA Data Analysis
4 Notes
References
Part V: Analysis of Extracellular Vesicles from Blood
Chapter 18: In-Depth Proteomic Analysis of Blood Circulating Small Extracellular Vesicles
1 Introduction
2 Materials
2.1 Plasma Preparation
2.2 Exosome Isolation and Lysis
2.3 Protein Precipitation and Resuspension
2.4 Immunodepletion of Plasma Contaminants
2.5 Protein Digestion and Peptide Purification
2.6 MS Acquisition (Nano-LC-MS/MS)
2.7 MS Data Analysis and Bioinformatic Tools
3 Methods
3.1 Collection of Blood and Preparation of Plasma
3.2 sEVs Isolation and Lysis
3.3 Immunodepletion of Most Abundant Plasma Proteins
3.4 Protein Digestion and Peptide Purification
3.5 MS Acquisition (Nano-LC-MS/MS)
3.6 MS Data Analysis
3.7 Bioinformatic Analysis
4 Notes
References
Chapter 19: Protocol for Plasma Extracellular Vesicle and Particle Isolation and Mass Spectrometry-Based Proteomic Identificat...
1 Introduction
2 Materials
2.1 Blood Collection and Plasma EVPs Isolation
2.2 LC-MS/MS
2.3 Quantification and Bioinformatic Analysis
3 Methods
3.1 EVPs Isolation
3.2 Preparation of EVPs Samples for LC-MS/MS Processing
3.3 LC-MS/MS Analysis of EVPs Peptides
3.4 Bioinformatic Analysis of Proteomic Data
4 Notes
References
Chapter 20: Analysis of Extracellular Vesicle and Contaminant Markers in Blood Derivatives Using Multiple Reaction Monitoring
1 Introduction
2 Materials
2.1 Blood Serum/Plasma Collection
2.2 Isolation of Extracellular Vesicles
2.3 Isolation of Platelets and Red Blood Cells (RBCs) from Whole Blood
2.4 EV Protein Quantification, Extraction, and Digestion
2.5 Targeted Peptide Analysis by LC-MRM-MS
3 Methods
3.1 Collection and Storage of Blood Serum/Plasma
3.2 Isolation of Extracellular Vesicles
3.3 Isolation of Platelet and Red Blood Cells from Whole Blood
3.4 Protein Digestion
3.5 SIL Peptides
3.6 Analytical Method
4 Notes
References
Chapter 21: Generation of Red Blood Cell Nanovesicles as a Delivery Tool
1 Introduction
2 Materials
2.1 Isolation of Erythrocytes from Whole Blood
2.2 RBC Ghost Generation by Hypotonic Hemolysis
2.3 Erythrocyte-Derived Nanovesicle (edNV) Generation and Purification
2.4 Biophysical Characterization
2.4.1 Nanoparticle Tracking Analysis (NTA) Using ZetaView Particle Metrix
2.4.2 Cryoelectron Microscopy
2.5 Drug Loading and Detection
2.5.1 Active Curcumin Loading by Extrusion
2.5.2 Passive Curcumin Loading by Diffusion
2.5.3 Drug Detection by Spectrophotometry
2.6 MS Sample Preparation
2.6.1 Protein Reduction and Alkylation
2.6.2 Single-Pot, Solid-Phase-Enhanced Sample Preparation
2.6.3 Nano-LC MS/MS
2.7 Data Analysis
3 Methods
3.1 Isolation of Erythrocytes from Whole Blood
3.2 Erythrocyte Ghost Generation by Hypotonic Hemolysis
3.3 Erythrocyte-Derived Nanovesicle Generation and Purification (See Note 3)
3.4 Biophysical Characterization (See Note 9)
3.4.1 Nanoparticle Tracking Analysis (NTA) Using Zetaview Particle Metrix
3.4.2 Cryoelectron Microscopy (See Note 12)
3.5 Drug Loading and Detection (See Note 13)
3.5.1 Active Loading by Extrusion
3.5.2 Passive Loading by Diffusion
3.5.3 Drug Detection by UV-Visible Spectrophotometry
3.6 Proteomic Profiling of Erythrocyte-Derived Nanovesicles
3.6.1 Single-Pot, Solid-Phase-Enhanced Sample Preparation
3.6.2 Nano-LC MS/MS
3.7 Data Analysis
4 Notes
References
Part VI: Targeted-Based Mass Spectrometry
Chapter 22: Progress in Targeted Mass Spectrometry (Parallel Accumulation-Serial Fragmentation) and Its Application in Plasma/...
1 Introduction
2 Materials
2.1 Protein Digestion from Human Serum/Plasma
2.2 prmPASEF Assay Generation and Targeted MS Analysis
2.3 MRM Assay Generation and Targeted MS Analysis
2.4 Websites and Software
3 Methods
3.1 Protein Digestion from Human Serum/Plasma
3.2 Generation of the Secreted Protein Library
3.3 Generation of the Peptide Library for prmPASEF
3.4 Generation of prmPASEF Assays
3.5 Validation and Optimization of prmPASEF Assays
3.6 Generation of MRM Assays
3.7 Verification and Optimization of MRM Assays
3.8 Query MRM or PRM Assays from Cancer Serum Atlas
3.9 Perform prmPASEF or MRM Assays in Serum or Plasma Samples
3.10 Quantitative Analysis of Target Proteins in Serum or Plasma
4 Notes
References
Chapter 23: Offline Peptide Fractionation and Parallel Reaction Monitoring MS for the Quantitation of Low-Abundance Plasma Pro...
1 Introduction
2 Materials
2.1 Enzymatic Digestion
2.2 Standard Curve Preparation and Sample Spiking
2.3 Solid-Phase Extraction (SPE)
2.4 High-pH Reversed-Phase Fractionation
2.5 Determine Plasma Digest Protein Concentration
2.6 Nano-LC-MS/MS
2.7 MS Data Analysis: Software Tools
3 Methods
3.1 Human Plasma
3.2 Enzymatic Digestion of Human Plasma and BSA
3.3 Preparation of NAT and SIS Peptide Dilutions
3.4 Spiking Plasma and BSA Digests with Synthetic Peptides
3.5 SPE of Standard Curve Samples and Plasma Samples
3.6 Reversed-Phase High-pH Peptide Fractionation
3.7 Quantitation of Fractionated Plasma Peptide Concentration
3.8 Nano-LC-MS/MS Analysis by PRM on Q Exactive Plus
3.9 MS Data Analysis
4 Notes
References
Chapter 24: Profiling Serum Intact N-Glycopeptides Using Data-Independent Acquisition Mass Spectrometry
1 Introduction
2 Materials
2.1 Example Data
2.2 Software Tools
3 Methods
3.1 Spectral Library Building
3.1.1 DDA Database Searching
3.1.2 Preparation for Library Building
3.1.3 Library Building from DDA Results
3.1.4 Library Generation for OpenSWATH
3.2 DIA Data Analysis
3.2.1 Raw Data Conversion to mzML
3.2.2 Targeted Data Extraction
3.2.3 Statistical Control
3.2.4 Multi-run Alignment
3.2.5 Post-analysis Data Visualization
4 Notes
References
Part VII: Assay Development in Biomarker Discovery and Translational Proteomics
Chapter 25: Semi-Automated Lectin Magnetic Bead Array (LeMBA) for Translational Serum Glycoprotein Biomarker Discovery and Val...
1 Introduction
2 Materials
2.1 Lectin Conjugation to Magnetic Beads
2.2 Serum Preparation
2.3 Lectin Pulldown
3 Method
3.1 Lectin Conjugation to Tosyl-Activated Dynabeads MyOne
3.1.1 Day 1: Lectin Coupling to Dynabeads
3.1.2 Day 2: Blocking the Conjugated Beads
3.1.3 Day 3: Lectin-Bead Conjugate Storage
3.2 Serum Sample Preparation
3.2.1 Plate Layout and Controls
3.2.2 Serum Protein Denaturation (See Note 5)
3.3 Lectin Magnetic Bead Array (LeMBA) Pulldown
3.3.1 AssayMAP Bravo System Start-Up
AssayMAP Bravo Deck Layout
Serum Glycoprotein Capture with Lectin-Conjugated Beads
On-Bead Trypsin Digestion
Collect Digested Peptides
AssayMAP Bravo System Shutdown
3.3.2 Overview of the Integra VIAFLO 96 GripTip and Tecan HydroFlex Microplate Washer
Programming of the Tecan HydroFlex Microplate Washer
Programming of the Integra VIAFLO 96 GripTip
Procedures to Work with the Tecan HydroFlex Microplate Washer and the Integra VIAFLO 96 GripTip Systems for LeMBA
3.4 Mass Spectrometry
3.5 Data Quality Control Analysis
4 Notes
References
Chapter 26: Accessing Antibody Reactivities in Serum or Plasma to (Auto-)antigens Using Multiplexed Bead-Based Protein Immunoa...
Abbreviations
1 Introduction
2 Materials
2.1 Coupling of Proteins to MagPlex Beads
2.2 Coupling Confirmation Assay
2.3 Multiplexed Bead-Based Protein Immunoassay
3 Methods
3.1 Preparations for Protein Coupling to MagPlex Beads
3.1.1 Bead Preparation
Selection of Bead Types
Calculation of Required Bead Amounts
Coupling Plate Preparation
3.1.2 Protein Preparation
3.1.3 Control Antigens
3.2 Coupling of Proteins to MagPlex Beads
3.2.1 Bead Activation
3.2.2 Protein Immobilization on Activated MagPlex Beads
3.3 Preparing the Flexmap 3D Instrument
3.4 Coupling Confirmation Assay
3.4.1 Preparations of the Multiplexed Bead Mix
3.4.2 Detection of Tagged Proteins with Penta-HIS-Specific Antibody
3.4.3 Analysis and Interpretation of Results
3.5 Multiplexed Bead-Based Protein Immunoassay for the Determination of Antibody Reactivities
3.5.1 Selection of Sample Matrix
Serum or Plasma Dilution Without IgG Purification
Purification and Dilution of IgG Samples from Serum or Plasma
3.5.2 Preparation of the Bead Mix
3.5.3 Sample Processing and Detection
3.5.4 Data Export and Analysis
4 Notes
References
Chapter 27: Absolute Quantitative Targeted Proteomics Assays for Plasma Proteins
1 Introduction
2 Materials
2.1 Samples
2.2 Sample Preparation Chemicals and Reagents
2.3 Peptide Synthesis
2.4 Equipment and Liquid Chromatography-Mass Spectrometry Instrumentation
2.5 Software Tools and Data Analysis
3 Methods
3.1 Protein Assay Design
3.2 Peptide Synthesis
3.3 Sample Preparation
3.4 LC Separation and MRM-MS Acquisition
3.5 Data Evaluation
4 Notes
References
Part VIII: Plasma-Based Peptide, Lipid, and Metabolite Assays
Chapter 28: Rapid and Quantitative Enrichment of Peptides from Plasma for Mass Spectrometric Analysis
1 Introduction
2 Materials
2.1 Peptide Extraction from Human Plasma
2.1.1 Protein Precipitation
2.1.2 Solid Phase Extraction (SPE)
2.1.3 Reduction and Alkylation
2.2 Peptidomics Characterization
3 Methods
3.1 Peptide Extraction from Human Plasma
3.1.1 Protein Precipitation
3.1.2 Solid Phase Extraction (SPE)
3.1.3 Reduction and Alkylation
3.2 Peptidomics Characterization
3.3 PEAKS Peptidomics Data Analysis
4 Notes
References
Chapter 29: Comprehensive Targeted Lipidomic Profiling for Research and Clinical Applications
1 Introduction
2 Materials
2.1 General
2.2 Chromatography and Mass Spectrometry Analysis
3 Method
3.1 Lipid Extraction
3.2 Mass Spectrometry Setup
3.2.1 Instrument Setup (Chromatography, Agilent 6495C)
3.2.2 Instrument Setup (Mass Spectrometry, Agilent 6495C)
3.2.3 Retention Time Adjustment
3.2.4 Sample Thawing and Preparation
3.2.5 Data Acquisition, Quality Control Monitoring, and Sample Changeover
3.3 Data Integration and Reporting (Agilent)
4 Notes
References
Chapter 30: Multiplexed Bead-Based Peptide Immunoassays for the Detection of Antibody Reactivities
1 Introduction
2 Materials
2.1 Coupling of Peptides to MagPlex Beads
2.2 Multiplexed Immunoassay and Coupling Confirmation Assay with Peptide-Coupled Microspheres
3 Methods
3.1 Peptide Design and Modifications for Coupling
3.2 Coupling of Peptides to MagPlex Beads
3.2.1 Preparation of MagPlex Beads
3.2.2 Preparation of the Antigenic Peptides
3.2.3 Preparation of Control Antigens
3.2.4 Preparation of 2-Azidoethan-1-Amine Hydrochloride
3.2.5 Coupling of 2-Azidoethan-1-Amine Hydrochloride to MagPlex Beads
3.2.6 Coupling of Propargylglycine-Tagged Peptides to Azide-Coupled MagPlex Beads
3.3 FLEXMAP 3D Instrument Preparation
3.4 Coupling Confirmation Assay
3.4.1 Preparation of the Bead Mix
3.4.2 Detection of Tagged Peptides with FLAG-Tag-Specific Antibody
3.4.3 Data Export and Analysis
3.5 Multiplexed Bead-Based Peptide Immunoassay for the Detection of Antibody Reactivities
3.5.1 Preparation of Purified IgG Samples from Serum or Plasma
3.5.2 Preparation of the Bead Mix
3.5.3 Processing of IgG Samples
3.5.4 Data Export and Analysis
4 Notes
References
Chapter 31: Array-Based Multiplex and High-Throughput Serology Assays
1 Introduction
2 Materials
2.1 Bead Coupling and Coupling Efficiency Test
2.2 Serology Assay
2.3 Data Analysis
3 Methods
3.1 Bead Coupling and Coupling Efficiency Test
3.2 Serology Assay
3.3 Data Analysis
4 Notes
References
Part IX: Software and Bioinformatics for the Plasma Proteome
Chapter 32: Bioinformatics Tools and Knowledgebases to Assist Generating Targeted Assays for Plasma Proteomics
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 33: Use of Longitudinal Serum Analysis and Machine Learning to Develop a Classifier for Cancer Early Detection
1 Introduction
2 Materials
2.1 Immunoaffinity Depletion
2.2 Protein Digestion and Sample Cleanup
2.3 LC-SRM Data Acquisition
2.4 SRM Data Analysis and Longitudinal Data Analysis: Software Tools
3 Methods
3.1 Randomization and Batching
3.2 Immunoaffinity Depletion
3.3 Protein Digestion and Sample Cleanup
3.4 LC-SRM Data Acquisition
3.5 LC-SRM Data Analysis and Processing
3.6 Classifier Development and Evaluation Using Random Forest
4 Notes
References
Index