Proteomics Data Analysis
Mass Spectrometry, Quantitative Proteomics, and Protein-Protein
Interactions
Instructor : Ajay Samuel
Introduction to Proteomics
Definition:
• Proteomics is the large-scale study of proteins, particularly their
structures and functions.
Importance:
• Cellular Functions: Proteins are key players in most biological
processes.
• Disease Mechanisms: Altered protein expressions can indicate
diseases.
• Drug Development: Identifying potential therapeutic targets.
Mass Spectrometry in Proteomics
Overview:
• An analytical technique to measure the mass-to-charge ratio of ions.
Components:
• Ion Source: Converts molecules into ions (e.g., ESI, MALDI).
• Mass Analyzer: Separates ions based on their mass-to-charge ratio (e.g., TOF,
Orbitrap).
• Detector: Records the number of ions at each mass-to-charge ratio.
Workflow:
• Sample Preparation: Protein extraction, digestion (trypsin).
• Ionization: Methods like ESI (Electrospray Ionization) or MALDI (Matrix-Assisted
Laser Desorption/Ionization).
• Mass Analysis: Separation and detection of ions.
• Data Acquisition: Generating spectra for analysis.
Mass Spectrometry Data Preprocessing
• Raw Data: High-throughput spectral data.
• Noise Reduction: Techniques like wavelet transform, smoothing
algorithms.
• Baseline Correction: Adjustments to correct for variations in the
baseline signal.
• Peak Detection: Algorithms for identifying true peaks from the
spectra (e.g., centroiding).
Peptide Identification
Database Searching:
• Matching: Matching observed spectra to theoretical spectra.
• Tools: Mascot, Sequest, Andromeda.
• Steps: Peptide mass fingerprinting, tandem mass spectrometry.
• De Novo Sequencing:
• Direct interpretation of MS/MS spectra to derive peptide sequences.
Validation:
• False Discovery Rate (FDR): Estimating the proportion of false
positives.
• Peptide Prophet: A tool for evaluating peptide identification
confidence.
Quantitative Proteomics Methods
Label-Free Quantification:
• Ion Intensity: Comparing the intensity of peptide ions across samples.
• Spectral Counting: Counting the number of spectra matching a
peptide/protein.
• Advantages/Disadvantages: Cost-effective, but can be less precise.
Isobaric Labeling:
• Tandem Mass Tags (TMT): Tags that produce reporter ions in MS/MS.
• iTRAQ (Isobaric Tags for Relative and Absolute Quantitation): Similar to TMT
but different chemistry.
• Advantages/Disadvantages: High multiplexing capability, expensive, potential
for ratio compression.
Label-Free Quantification Workflow
Steps:
• Sample Preparation: Protein extraction, digestion, desalting.
• LC-MS/MS Analysis: Liquid chromatography coupled with tandem
mass spectrometry.
• Data Preprocessing: Noise reduction, peak detection.
• Quantification: Using tools like MaxQuant, Progenesis.
• Statistical Analysis: Normalization, significance testing.
Isobaric Labeling Workflow
Steps:
• Sample Labeling: Covalently attaching isobaric tags to peptides.
• Sample Mixing: Combining labeled samples.
• LC-MS/MS Analysis: Separation and detection.
• Reporter Ion Quantification: Extracting and comparing reporter ion
intensities.
• Normalization and Statistical Analysis: Adjusting for technical
variability.
Protein-Protein Interaction (PPI) Analysis
Definition: Investigating the physical contacts between proteins within
a cell.
Methods:
• Yeast Two-Hybrid: Detecting binary interactions in yeast.
• Co-immunoprecipitation: Pulling down protein complexes with
antibodies.
• Cross-Linking Mass Spectrometry: Stabilizing interactions with cross-
linkers and analyzing with MS.
• Bioinformatics: Predictive models and databases like STRING.
Software Tools for Proteomics Data Analysis
MaxQuant:
• Functionality: Comprehensive software for MS data analysis.
• Features: Label-free quantification, isobaric labeling support, PTM analysis,
FDR estimation.
• Example: Quantification of differential protein expression.
Skyline:
• Functionality: Targeted proteomics, method refinement, and quantification.
• Features: Support for various MS instruments, robust analysis pipelines.
• Example: Targeted quantification of specific peptides.
Software Tools for Proteomics Data Analysis
Cytoscape:
Functionality: Visualization and analysis of molecular interaction
networks.
Features: Integration with proteomics data, various plugins for network
analysis.
Example: Visualizing protein interaction networks and pathways.
Links
1. https://www.maxquant.org/
2. https://skyline.ms/project/home/software/Skyline/begin.view
3. https://cytoscape.org/
4. https://string-db.org/
