Vascular Proteomics: Methods and Protocols (Methods in Molecular Biology, 1000)

✍ Scribed by Fernando Vivanco (editor)

• Publisher: Humana
• Year: 2013
• Tongue: English
• Leaves: 234
• Category: Library

✦ Synopsis

Proteomics is a rapidly expanding investigation platform in cardiovascular medicine. Driven by major improvements in mass spectrometry (MS) instrumentation and data analysis, the proteomics field has flourished in recent years particularly in the study of complex diseases. These recent advances are characterized by the development of quantitative MS-based methods that promoted the field from primarily identifying proteins to also providing measurements of relative changes in protein levels between different cell states. This progress is reflected in the application of proteomic techniques to vascular pathology. Vascular Proteomics: Methods and Protocols provides up-to-date methods and protocols for the analysis of arteries, cells, lipoproteins, body fluids, and metabolites, with a particular focus on MS-based methods of protein and peptide quantification. Written in the successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.

 

Authoritative and easily accessible, Vascular Proteomics: Methods and Protocols is a representative selection of methods that can be a useful resource for experienced proteomics practitioners as well as newcomers interested in becoming acquainted with the practice of proteomic techniques for cardiovascular research.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Vascular Proteomics
1 Introduction
1.1 Atherosclerosis Development and Clinical Needs
2 Biomarkers of Disease
3 A Step Forward: “Omics” Multi-target
4 Sample Sources: The Advantages of Sub-proteomes
4.1 Biological Fluids
4.2 Tissue
4.2.1 Cells
4.2.2 Secretome
5 Proteomics in Cardiovascular Disease
5.1 Proteins as Diagnostic Biomarkers
5.2 Classical Markers of Vascular Disease
5.3 Proteomic Approaches for the Study of Atherosclerosis
5.4 Differential Expression of Proteins by Atherosclerotic Lesions
5.4.1 Biological Fluids
5.4.2 Tissue
5.4.3 Secretome
6 Metabolomics in Cardiovascular Disease
7 Emerging Mass Spectrometry Imaging: High Potential in CVD
8 Systems Biology
References
Chapter 2: Laser Microdissection and Saturation Labeling DIGE Method for the Analysis of Human Arteries
1 Introduction
2 Materials
2.1 Histology
2.2 Tissue Processing and LMD Isolation
2.3 Saturation Labeling DIGE Buffers
3 Methods
3.1 Arterial Sample Collection and Histology (Fig.  1)
3.2 Processing for LMD Isolation
3.3 Laser Microdissection (Fig.  2)
3.4 Protein Extraction
3.5 Saturation Labeling DIGE (Fig.  3)
3.6 Preparative Gels and MS/MS Identification
4 Notes
References
Chapter 3: Use of TOF-SIMS in Vascular Biology
1 Introduction
1.1 SIMS Principles
1.2 TOF-SIMS Instrumentation
1.3 TOF-SIMS as a Tool in Pathology
2 Materials
2.1 Sample Preparation
2.2 TOF-SIMS Analysis
3 Methods
3.1 Sample Preparation
3.1.1 Sample Preservation
3.1.2 Cutting
3.1.3 Surface Washing
3.1.4 Dehydration
3.2 TOF-SIMS Measurements
3.3 Data Analysis
3.3.1 Calibration
3.3.2 Chemical Identification
3.3.3 Imaging
4 Notes
References
Chapter 4: Proteomic Analysis of Vascular Smooth Muscle Cells with S- and R-Enantiomers of Atenolol by iTRAQ and LC-MS/MS
1 Introduction
2 Materials
3 Methods
3.1 Cell Culture
3.2 Cell Lysis, Protein Digestion, and Labeling with iTRAQ Reagents
3.3 Online 2D Nano-LC-MS/MS Analysis
3.4 Data Analysis and Interpretation
4 Notes
References
Chapter 5: Phosphoproteomic Analysis of Aortic Endothelial Cells Activated by Oxidized Phospholipids
1 Introduction
2 Materials
2.1 Components for Treatment of Bovine Aortic Endothelial Cells with Oxidized Phospholipids and Lysis
2.2 Components for Alkylation, Digestion, and Reverse Phase Extraction
2.3 Components for Immunoprecipitation of Tyrosine-Phosphorylated Peptides and Enrichment with Iron Metal Affinity Media
2.4 Components for Affinity Enrichment of Serine/Threonine-Phosphorylated Peptides
2.5 Components for Sample Clean-Up for Mass Spectrometry Analysis
2.6 Mass Spectrometry Analysis
3 Methods
3.1 Treatment of Primary Aortic Endothelial Cells with Oxidized Phospholipids
3.2 Alkylation, Digestion, and Reverse Phase Extraction
3.3 Immuno
3.4 Affinity Enrichment of Serine/Threonine-Phosphorylated Peptides
3.5 Sample Clean-Up for Mass Spectrometry Analysis
3.6 Mass Spectrometry and Phosphopeptide Identification
3.7 Chromatography Profile Alignment and Alignment-Based Peak Identification
4 Notes
References
Chapter 6: Characterization of Membrane and Cytosolic Proteins of Erythrocytes
1 Introduction
2 Materials
2.1 Sample Collection and Fractions Isolation
2.2 Membrane and Cytosolic Fractions Pretreatment
2.3 Two-Dimensional Electrophoresis
2.4 Proteins Digestion and Peptide Analysis by LC-MS/MS Analysis
3 Methods
3.1 Sample Collection and Red Blood Cells Isolation
3.2 Fractionation of Erythrocyte Membrane and Cytosol
3.3 Cytosolic Hemoglobin Depletion and Sample Pretreatment Prior to 2-DE
3.4 Membrane Fraction Pretreatment Prior to Analysis by 2-DE
3.5 Two-Dimensional Electrophoresis of Erythrocyte Membrane and Cytosolic Proteins
3.6 nLC-MS/MS Membrane Proteins Identification
3.7 nLC-MS/MS Cytosolic Proteins Identification
4 Notes
References
Chapter 7: Characterization and Analysis of Human Arterial Tissue Secretome by 2-DE and nLC-MS/MS
1 Introduction
2 Materials
2.1 Tissue Culture
2.2 Histology
2.3 Gel Electrophoresis (2-DE and SDS-PAGE)
2.4 Silver Staining
2.5 Proteins Digestion and Peptide Analysis by LC-MS/MS Analysis
3 Methods
3.1 Arterial Tissue Collection and Culture
3.2 Arterial Tissue Histology (Fig.  1)
3.3 Secretome Analysis by 2-DE
3.4 Secretome Analysis by nLC-MS/MS
3.4.1 Proteins Digestion and Peptides Extraction
3.4.2 Analysis by nLC-MS/MS
4 Notes
References
Chapter 8: Identification of Novel Biomarkers of Abdominal Aortic Aneurysms by 2D-DIGE and MALDI-MS from AAA- Thrombus-Cond...
1 Introduction
2 Materials
2.1 AAA-Tissue-Conditioned Media Preparation
2.2 Sample Preparation for 2D-DIGE
2.3 Protein Labeling for 2D-DIGE
2.4 Sample Preparation and Isoelectric Focusing
2.5 Second Dimension (SDS-PAGE)
2.6 Staining of Gels and Analysis of Gel Images
2.7 Spot Digestion
2.8 Sample Preparation for MALDI-MS
3 Methods
3.1 Patient Recruitment and Samples
3.2 AAA-Thrombus-Conditioned Media Isolation
3.3 Experimental Design and DIGE Protein Labeling
3.4 Two-Dimensional Electrophoresis and Image Acquisition
3.5 DIGE Image Analysis
3.6 Gel Silver Staining
3.7 In-Gel Tryptic Digestion
3.8 MALDI Mass Spectrometry
3.9 MALDI-MS Database Searching
4 Notes
References
Chapter 9: Metabolites Secreted by Human Atherothrombotic Aneurysm
1 Introduction
2 Materials
2.1 Chemicals
2.2 Samples
2.3 Equipment
2.4 LC-MS Equipment
2.5 Software
2.6 Others
3 Methods
3.1 Secretomes Preparation ( See Note 4)
3.2 Sample Preparation for LC-MS Fingerprinting
3.3 Metabolite Fingerprinting with LC-MS
3.4 Data Analysis
3.5 Metabolites Identification
4 Notes
References
Chapter 10: Quantitative Analysis of Apolipoproteins in Human HDL by Top-Down Differential Mass Spectrometry
1 Introduction
2 Materials
2.1 HDL Preparation
2.2 LC-MS Reagents
3 Methods
3.1 Preparation of Human High-Density Lipoprotein Samples
3.2 Preparation for LC-MS Analysis
3.3 LC-MS Analysis of Prepared HDL 3 Samples
3.4 dMS Analysis of HDL 3 Samples
3.5 Targeted Analysis and Online LC-MS/MS of Features of Interest
3.6 Protein Identification of Targeted Features by ProSightPC v2.0
4 Notes
References
Chapter 11: Quantitative Proteomics Analysis of High-Density Lipoproteins by Stable 18 O-Isotope Labeling
1 Introduction
2 Materials
2.1 Equipment
2.2 Buffers and Reagents
3 Methods
3.1 FPLC Affinity Isolation of HDL
3.2 Concentrating Gel Electrophoresis and In-Gel Digestion
3.3 18 O Labeling
3.4 Peptide Fractionation by Isoelectric Point (p I)
3.5 Mass Spectrometric Analysis by LC–ESI–LIT
3.6 Protein Identification
3.7 Protein Quantification
3.8 Statistical Analysis
4 Notes
References
Chapter 12: Unraveling Biomarkers of Abdominal Aortic Aneurisms by iTRAQ Analysis of Depleted Plasma
1 Introduction
2 Materials
2.1 Plasma Depletion and Quantitation
2.2 Trypsin Digestion and iTRAQ Labelling
2.3 LC–MS/MS Analysis
2.4 Specialized Equipment
3 Methods
3.1 Plasma
3.2 High-Abundant Protein Depletion from Plasma
3.3 Trypsin Digestion
3.4 iTRAQ Labelling
3.5 Reversed-Phase Liquid Chromatography
3.6 Mass Spectrometry
3.7 Database Searching and iTRAQ Labelling Analysis
4 Notes
References
Chapter 13: Absolute Quantitation of Proteins in Human Blood by Multiplexed Multiple Reaction Monitoring Mass Spectrometry
1 Introduction
2 Materials
2.1 Biological Samples
2.2 Dried Blood Spot Processing
2.3 Sample Preparation
2.4 LC/MRM-MS Systems
2.5 Data Analysis Software
3 Methods
3.1 Dried Blood Spot Processing
3.2 Sample Preparation
3.3 LC Separation Parameters
3.4 MRM-MS Acquisition Parameters
3.5 Preliminary Experiments
3.5.1 Empirical Optimization of Peptide Collision Energies
3.5.2 LC Gradient Optimization
3.5.3 Optimization of Sample Loading Amount
3.5.4 Interference Determination
3.6 Protein Quantitation
3.7 Conclusions and Significance
4 Notes
References
Chapter 14: Multiple Reaction Monitoring (MRM) of Plasma Proteins in Cardiovascular Proteomics
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
2.3 Solutions
3 Methods
3.1 Tryptic Digestion
3.1.1 Sample Denaturation, Reduction, and Alkylation
3.1.2 Digestion Reaction
3.1.3 Sample Digests Cleaning
3.2 MRM Analysis
3.2.1 MRM Assay Development Without Any Synthetic Standard Molecules
3.2.2 Quantitative Data Acquisition
4 Notes
References
Chapter 15: Proteomic Analysis of Plasma of Patients with Left Ventricular Remodeling After Myocardial Infarction: Usefulne...
1 Introduction
2 Materials
2.1 Combinatorial Peptide Ligand Library Components
2.2 SELDI-TOF Components
2.3 Liquid-Phase Isoelectric Focusing Components
3 Methods
3.1 Sample Preparation
3.2 Sample Treatment with CPLL
3.3 SELDI-TOF Analysis
3.4 Purification of Proteins
4 Notes
References
Chapter 16: Proteomic Analysis of Urinary Exosomes in Cardiovascular and Associated Kidney Diseases by Two-Dimensional Elec...
1 Introduction
2 Materials
2.1 Urine Collection
2.2 Exosome Isolation
2.3 Depletion of Urine Major Proteins
2.4 Exosome Characterization
2.5 Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE)
2.6 Protein Digestion and Peptide Analysis by nLC–MS/MS Analysis
3 Methods
3.1 Urine Collection
3.2 Exosome Isolation
3.3 THP and Albumin Depletion (Optional Step)
3.4 Electron Microscopy and Western Blotting (Figs.  1 and 2)
3.5 Two-Dimensional Electrophoresis (Fig.  3)
3.6 LC–MS/MS Label Free Analysis (Fig.  4)
4 Notes
References
Index