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Introduction to Comparative Proteomics and SDS-PAGE

Introduction to Comparative Proteomics and SDS-PAGE

  • Proteomics

    • Study of proteins, focusing on structures and functions.

  • Proteome

    • The complete set of proteins present in a system, whether throughout its lifecycle or within specific cell types under certain stimuli.

  • Human Proteome Organization (HUPO)

    • An organization dedicated to cataloging human proteins, their functions, and their interactions.

  • Central Dogma of Molecular Biology

    • Concept stating DNA → RNA → Protein.

Key Processes in Protein Biology

  • Transcription

    • Creation of an RNA molecule based on a DNA template by RNA polymerase.

  • Translation

    • Synthesis of a polypeptide based on the mRNA sequence by ribosomes.

  • RNA Editing

    • Modification of RNA sequences by substituting, inserting, or deleting bases, impacting amino acid sequence and protein translation.

  • Alternative Splicing

    • Involves spliceosomes that can change mRNA by removing introns and retaining exons depending on environmental conditions.

  • mRNA Degradation

    • Processes that affect the stability of mRNA, thus regulating protein production (e.g., protective 7-methyl guanosine cap and poly A tail).

Protein Modifications and Interactions

  • Proteolytic Cleavage

    • The removal of initiator methionine and activation/inactivation of proteins through specific cleavages.

  • Protein Degradation

    • Mechanisms like ubiquitination regulate protein levels by tagging them for destruction.

  • Protein-Protein Interaction

    • Many proteins require interaction with other proteins to function effectively.

  • Glycosylation

    • The attachment of carbohydrates to proteins/lipids impacting their function and interaction.

  • Phosphorylation

    • Addition of a phosphoryl (PO₃) group, altering enzyme activity.

  • Phenotypic Diversity

    • One gene can lead to multiple protein forms, providing evolutionary adaptability without changing DNA.

Muscle Proteins Fortifying Structure and Function

  • Muscle Proteins

    • Myofibrils form the fundamental contractile units, bundled into muscle fibers.
    • Sarcomeres consist of actin and myosin filaments, crucial for muscle contraction.

  • Key Proteins

    • Actin: Thin filament protein that aligns with myosin.
    • Myosin: Thick filament protein that interacts with actin for contraction.
    • Titin: Contributes to muscle elasticity and stability.
    • Dystrophin: Anchors muscle cell membranes.
    • Filamin: Crosslinks actin filaments forming a network.
    • Nebulin: Regulates the length of actin filaments.

Evolution and Adaptation

  • Evolutionary Processes

    • Natural selection influences adaptations in muscle proteins based on environmental demands.

  • Genotypes and Phenotypes

    • Genotype: Genetic constitution influencing physical traits (phenotypes).

  • Evolutionary Trees

    • Diagrams representing lineages and evolutionary changes.

Methods in Proteomics: SDS-PAGE

  • Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-PAGE)

    • A key method for protein analysis, separating proteins based on molecular weight.

  • Electrophoresis

    • Movement of charged molecules in an electric field; protein separation relies on their charge and mass.

  • Protein Molecular Weight

    • Measured in Daltons (Da) and kilodaltons (kD), most proteins range from 10 kD to 220 kD.

  • Buffers and Gels

    • Discontinuous System: Allows proteins to separate concurrently.
    • Stacking Buffer: Compresses proteins at gel interface.
    • Resolving Gel: Separates proteins based on higher acrylamide concentration.

  • Migration Speed

    • Varies; for instance, chloride ions migrate faster than proteins during electrophoresis.

  • Denaturation

    • Structural disruption of protein complexes through SDS, heat, or reducing agents, allowing analysis of the protein's primary structure.

  • Reducing Agents

    • BME (Beta mercaptoethanol) or DTT (Dithiothreitol) break disulfide bridges ensuring complete denaturation.

Applications and Limitations of SDS-PAGE

  • Identifying Proteins

    • Cannot definitively identify unknown proteins without mass spectrometry or other techniques.

  • Western Blotting

    • Employs electric current to transfer proteins to membranes for antibody recognition.

Plotting and Analysis

  • Creating Standard Curves

    • Measure migration distances of known protein bands to estimate sizes of unknown proteins.

  • Cladistic Analysis

    • Use protein data to identify genetic relationships among fish, establishing which fish share characteristics.

  • Phylogenetic Comparison

    • Compare derived cladograms with published phylogenetic data to validate or challenge existing theories on species evolution.