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.