Proteomics Study Notes MCB3025F (3b)

OMICS AND LARGE DATASETS IN BIOLOGY MCB3025F: PROTEOMICS

PART 3a: Proteomics Overview

  • Proteomics: The large-scale study of proteins, particularly their functions and structures.

  • Key Methods in Proteomics:

    • 1D-PAGE (One-Dimensional Polyacrylamide Gel Electrophoresis):

    • Performs separation of proteins based on their mass and charge.

    • Useful for initial separation and visualization.

    • 2D-PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis):

    • Combines isoelectric focusing and 1D-PAGE to separate proteins based on isoelectric point and molecular weight.

    • Proteome Mining:

    • The process of identifying and quantifying proteins in a sample.

    • Protein Expression Profiling:

    • Assessment of the expression levels of thousands of proteins simultaneously.

    • Protein-Network Mapping:

    • Analysis of interactions between different proteins within the proteome.

    • Mapping of Protein Modifications:

    • Understanding how post-translational modifications impact protein function.

    • Structural Proteomics:

    • Focuses on the 3D structure of proteins to understand their function and interactions.

PART 3b: Advanced Proteomics Techniques

  • Liquid Chromatography (LC):

    • Technique for separating mixtures with a liquid mobile phase. Can be coupled with mass spectrometry.

    • HPLC (High-Performance Liquid Chromatography): A type of LC used for separating peptides before mass spectrometry.

    • Mobile Phase Gradient: Refers to varying conditions of the solvent during LC to optimize separation.

Mass Spectrometry (MS) Overview

  • Mass Spectrometer (MS) Components:

    • Ionization Source: Converts sample into ions. Achieved through:

    • Addition of protons (+).

    • Electrospray Ionization (ESI):

      • Peptides introduced via liquid flow into an electromagnetic field.

      • Heated needle creates a mist of charged droplets, where solvent evaporates to form smaller droplets until charged peptides are produced.

      • Coulombic Explosion: When the electric field becomes too strong, leading to smaller charged droplets.

      • Most peptides contain 2 or more protons in ESI, important for understanding ionization.

  • Mass Analyser:

    • Detects ions based on their mass-to-charge ratio (m/z).

  • Detector: Constructs a mass spectrum, which represents the mass distribution of ions.

Peptide Sequence Determination

  • Process of determining peptide sequences through mass spectrometry.

  • Coupled high-performance liquid chromatography (HPLC) allows for separation before sequencing via MS.

Peptide Identification Techniques

  • Peptide Mass Fingerprinting (PMF):

    • Involves using online databases to search peptide m/z for identification.

    • Challenges include handling posttranslational modifications (PTMs) that affect peptide mass.

    • Example: Phosphorylation at different serine residues can yield similar m/z values.

  • Example of PMF:

    • Trypsin digestion of human hemoglobin α yields specific peptides:

    • Peptide example: VGAHAGEYGAEALER - mass: 1528.7348 Da.

    • One proton results in m/z 1529.7348.

    • Search Parameters:

    • Utilizing the MS-FIT program for searching with variable mass tolerance.

    • Identifies unique peptides from protein sequences based on their mass and allows for comparing similar m/z values from different peptides.

Tandem Mass Spectrometry (MS/MS)

  • Concept:

    • Two mass spectrometers linked with a collision cell for further analysis.

  • Collision Cell:

    • Precursor ions (parent ions) selected for fragmentation.

    • Fragmentation occurs in collision cell using inert gas.

    • Results in daughter ions (newly fragmented ions) in the second mass spectrum.

    • Neutral loss can occur if only one daughter ion retains a charge when the precursor ion is singly charged. Techniques like ESI help reduce neutral loss.

Online Data Acquisition in Proteomics

  • Utilization of amino acid composition and sequences derived from mass spectra to match with existing protein databases.

  • Data-reduction Algorithms & Software Tools:

    • High-throughput capabilities of HPLC-MS/MS create thousands of spectra.

    • Database comparison for peptide sequence identification.

Implications and Applications of Proteomics

  • Shotgun Proteomics: Utilizes ultra-high-performance liquid chromatography (UHPLC) to enhance sensitivity and reduce sample volume.

    • Faster and more effective compared to traditional methods.

Comparing LC-MS and GC-MS

  • Gas Chromatography (GC) vs. Liquid Chromatography (LC):

    • GC is more suited for volatile compounds with a gas mobile phase while LC handles a broader range of compounds including larger molecules.

    • GC often requires additional steps like derivatization.

Case Study: Early Diagnosis of Hepatocellular Carcinoma (HCC)

  • Xing et al. (2021) explored noninvasive proteomics-based screening for early HCC detection:

    • HCC ranks 4th in cancer mortality due to late-stage diagnosis.

    • Developed experimental procedures using HPLC-MS/MS to identify biomarkers differentiating between HCC and other liver diseases.

    • A predictive model was constructed based on protein expression data and validated with patient samples.

    • Findings:

    • 11 candidate proteins were identified, with further validation indicating only 5 retained statistical significance.

    • The model shows the potential to predict HCC emergence approximately 11 months in advance.

Additional Notes

  • Focus on recent advancements in peptide mass identification techniques, spectra data comparisons, and the implications of these developments in diagnostics and biological research, particularly related to liver disease.

  • Graphical Data Representation: Utilizes gene network clusters, protein-protein interaction (PPI) networks, and heatmaps to visually represent proteomics data and findings.