Proteins and Proteomics

  1. Core Technologies in Proteomics
    1. Sample Preparation and Protein Extraction
      1. Sample Collection and Storage
        1. Tissue Sampling
          1. Cell Culture Preparation
            1. Biofluid Collection
              1. Sample Preservation
              2. Cell Lysis and Tissue Homogenization
                1. Mechanical Disruption
                  1. Sonication
                    1. Bead Beating
                      1. French Press
                      2. Chemical Lysis
                        1. Detergent-Based Lysis
                          1. Chaotropic Agents
                            1. Buffer Systems
                            2. Enzymatic Digestion
                              1. Protease Treatment
                                1. Cell Wall Degradation
                              2. Protein Solubilization and Denaturation
                                1. Detergents and Chaotropes
                                  1. SDS and Other Ionic Detergents
                                    1. Non-Ionic Detergents
                                      1. Urea and Guanidinium
                                      2. Reducing Agents
                                        1. DTT and TCEP
                                          1. Beta-Mercaptoethanol
                                        2. Protein Concentration Assays
                                          1. Bradford Assay
                                            1. Principle and Protocol
                                              1. Advantages and Limitations
                                              2. BCA Assay
                                                1. Copper Reduction Method
                                                  1. Compatibility Issues
                                                  2. Lowry Assay
                                                    1. Classical Method
                                                      1. Interference Factors
                                                      2. UV Absorbance Methods
                                                      3. Protein Cleanup and Desalting
                                                        1. Precipitation Methods
                                                          1. Dialysis and Ultrafiltration
                                                            1. Spin Columns
                                                              1. Buffer Exchange
                                                            2. Protein and Peptide Separation
                                                              1. Gel-Based Electrophoresis
                                                                1. SDS-PAGE
                                                                  1. Principle and Protocol
                                                                    1. Gel Preparation
                                                                      1. Molecular Weight Estimation
                                                                        1. Staining Methods
                                                                          1. Limitations and Artifacts
                                                                          2. Native PAGE
                                                                            1. Blue Native PAGE
                                                                              1. Clear Native PAGE
                                                                              2. 2D-Gel Electrophoresis (2-DE)
                                                                                1. Isoelectric Focusing
                                                                                  1. IPG Strips
                                                                                    1. pH Gradients
                                                                                    2. Second Dimension SDS-PAGE
                                                                                      1. Spot Detection and Analysis
                                                                                        1. Image Analysis Software
                                                                                          1. Reproducibility Challenges
                                                                                          2. Urea-PAGE
                                                                                            1. Tricine-PAGE
                                                                                            2. Liquid Chromatography (LC)
                                                                                              1. Reversed-Phase LC (RP-LC)
                                                                                                1. C18 and Other Stationary Phases
                                                                                                  1. Gradient Elution
                                                                                                    1. Mobile Phase Optimization
                                                                                                    2. Ion-Exchange Chromatography (IEX)
                                                                                                      1. Cation Exchange
                                                                                                        1. Anion Exchange
                                                                                                          1. Salt Gradients
                                                                                                          2. Size-Exclusion Chromatography (SEC)
                                                                                                            1. Molecular Weight Separation
                                                                                                              1. Buffer Considerations
                                                                                                              2. Hydrophobic Interaction Chromatography
                                                                                                                1. Affinity Chromatography
                                                                                                                  1. Immunoaffinity
                                                                                                                    1. Antibody Selection
                                                                                                                      1. Elution Conditions
                                                                                                                      2. Metal Affinity
                                                                                                                        1. IMAC Principles
                                                                                                                          1. His-Tag Purification
                                                                                                                          2. Lectin Affinity
                                                                                                                            1. Heparin Affinity
                                                                                                                            2. Multidimensional Chromatography
                                                                                                                              1. 2D-LC Systems
                                                                                                                                1. Online vs Offline Coupling
                                                                                                                            3. Mass Spectrometry (MS) for Protein Analysis
                                                                                                                              1. Fundamental Principles
                                                                                                                                1. Ionization Process
                                                                                                                                  1. Mass-to-Charge Ratio (m/z)
                                                                                                                                    1. Resolution and Accuracy
                                                                                                                                      1. Sensitivity and Dynamic Range
                                                                                                                                      2. Key Components of a Mass Spectrometer
                                                                                                                                        1. Ion Source
                                                                                                                                          1. Electrospray Ionization (ESI)
                                                                                                                                            1. Mechanism and Conditions
                                                                                                                                              1. Multiple Charging
                                                                                                                                                1. Nano-ESI
                                                                                                                                                2. Matrix-Assisted Laser Desorption/Ionization (MALDI)
                                                                                                                                                  1. Matrix Selection
                                                                                                                                                    1. Sample Preparation
                                                                                                                                                      1. Imaging Applications
                                                                                                                                                      2. Atmospheric Pressure Chemical Ionization (APCI)
                                                                                                                                                        1. Electron Impact (EI)
                                                                                                                                                        2. Mass Analyzer
                                                                                                                                                          1. Time-of-Flight (TOF)
                                                                                                                                                            1. Linear and Reflectron Modes
                                                                                                                                                              1. Resolution Enhancement
                                                                                                                                                              2. Quadrupole
                                                                                                                                                                1. Mass Filtering
                                                                                                                                                                  1. Scanning Modes
                                                                                                                                                                  2. Ion Trap
                                                                                                                                                                    1. 3D and Linear Traps
                                                                                                                                                                      1. MSn Capabilities
                                                                                                                                                                      2. Orbitrap
                                                                                                                                                                        1. High Resolution and Accuracy
                                                                                                                                                                          1. Fourier Transform Detection
                                                                                                                                                                          2. Fourier Transform Ion Cyclotron Resonance (FT-ICR)
                                                                                                                                                                            1. Hybrid Instruments
                                                                                                                                                                            2. Detector
                                                                                                                                                                              1. Electron Multipliers
                                                                                                                                                                                1. Microchannel Plates
                                                                                                                                                                                  1. Image Current Detection
                                                                                                                                                                                2. Tandem Mass Spectrometry (MS/MS)
                                                                                                                                                                                  1. Peptide Fragmentation
                                                                                                                                                                                    1. Collision-Induced Dissociation (CID)
                                                                                                                                                                                      1. Higher-Energy Collisional Dissociation (HCD)
                                                                                                                                                                                        1. Electron Transfer Dissociation (ETD)
                                                                                                                                                                                          1. Electron Capture Dissociation (ECD)
                                                                                                                                                                                          2. Fragmentation Patterns
                                                                                                                                                                                            1. b and y Ion Series
                                                                                                                                                                                              1. a, c, x, z Ions
                                                                                                                                                                                                1. Neutral Losses
                                                                                                                                                                                                2. Data Acquisition Modes
                                                                                                                                                                                                  1. Data-Dependent Acquisition (DDA)
                                                                                                                                                                                                    1. Data-Independent Acquisition (DIA)
                                                                                                                                                                                                      1. Targeted Acquisition
                                                                                                                                                                                                    2. Ionization Efficiency and Suppression
                                                                                                                                                                                                      1. Matrix Effects
                                                                                                                                                                                                        1. Salt Effects
                                                                                                                                                                                                          1. Peptide Properties
                                                                                                                                                                                                        2. Major Proteomic Strategies
                                                                                                                                                                                                          1. Bottom-Up Proteomics (Shotgun Proteomics)
                                                                                                                                                                                                            1. Enzymatic Digestion
                                                                                                                                                                                                              1. Trypsin Specificity
                                                                                                                                                                                                                1. Alternative Proteases
                                                                                                                                                                                                                  1. Digestion Optimization
                                                                                                                                                                                                                  2. Peptide Separation
                                                                                                                                                                                                                    1. LC-MS/MS Workflows
                                                                                                                                                                                                                      1. Gradient Optimization
                                                                                                                                                                                                                      2. Database Searching
                                                                                                                                                                                                                        1. Peptide Identification
                                                                                                                                                                                                                          1. Protein Inference
                                                                                                                                                                                                                        2. Top-Down Proteomics
                                                                                                                                                                                                                          1. Analysis of Intact Proteins
                                                                                                                                                                                                                            1. Ionization Challenges
                                                                                                                                                                                                                              1. Fragmentation Methods
                                                                                                                                                                                                                              2. Advantages and Challenges
                                                                                                                                                                                                                                1. PTM Characterization
                                                                                                                                                                                                                                  1. Isoform Identification
                                                                                                                                                                                                                                    1. Technical Limitations
                                                                                                                                                                                                                                    2. Middle-Down Approaches
                                                                                                                                                                                                                                    3. Targeted Proteomics
                                                                                                                                                                                                                                      1. Selected Reaction Monitoring (SRM)
                                                                                                                                                                                                                                        1. Triple Quadrupole MS
                                                                                                                                                                                                                                          1. Transition Selection
                                                                                                                                                                                                                                            1. Method Development
                                                                                                                                                                                                                                            2. Parallel Reaction Monitoring (PRM)
                                                                                                                                                                                                                                              1. High-Resolution MS
                                                                                                                                                                                                                                                1. Spectral Libraries
                                                                                                                                                                                                                                                2. Applications in Quantitative Proteomics
                                                                                                                                                                                                                                                  1. Biomarker Validation
                                                                                                                                                                                                                                                    1. Pathway Analysis
                                                                                                                                                                                                                                                  2. Cross-Linking Proteomics
                                                                                                                                                                                                                                                    1. Chemical Cross-Linkers
                                                                                                                                                                                                                                                      1. Structural Information
                                                                                                                                                                                                                                                        1. Protein Complex Analysis

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                                                                                                                                                                                                                                                    7. Introduction to Proteomics

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                                                                                                                                                                                                                                                    9. Quantitative Proteomics