Proteomics

Native vs. Denatured Protein Molecular Weights:

Understanding the difference between the native and denatured state of a protein is fundamental to determining its quaternary structure and biological assembly.

  • Native Molecular Weight: This refers to the molecular weight of a protein in its biologically active, folded state.

    • Proteins in their native state often exist as oligomers (e.g., dimers, tetramers), where multiple polypeptide subunits are held together by non-covalent interactions or disulphide bridges.

    • Determining the native molecular weight allows researchers to identify the oligomeric state of the protein.

  • Denatured Molecular Weight: * In the denatured state, proteins are unfolded into linear polypeptide chains, and any non-covalent quaternary associations are disrupted.

    • Denaturation is typically achieved using detergents like Sodium Dodecyl Sulphate (SDS) and reducing agents such as β-mercaptoethanol to break disulphide bonds.

    • Comparing the native molecular weight to the denatured molecular weight reveals whether a protein is a monomer, a homomultimer (identical subunits), or a heteromultimer (different subunits).

Methods for Determining Protein Molecular Weights:

To fully characterise a protein, multiple techniques are employed to measure its mass under different conditions.

  • Gel Filtration (Size Exclusion Chromatography):

    • Principle: This technique separates proteins in their native state based on their hydrodynamic volume.

    • The stationary phase consists of porous beads. Large proteins are excluded from the pores and elute first (the void volume), while smaller proteins enter the pores, resulting in a longer path and later elution.

    • Calibration: By running a set of standard proteins of known molecular weight, a calibration curve can be generated to estimate the native mass of an unknown sample.

  • SDS-PAGE (Polyacrylamide Gel Electrophoresis):

    • Principle: This method determines the molecular weight of denatured polypeptide subunits.

    • SDS coats proteins with a uniform negative charge, ensuring the charge-to-mass ratio is constant. Consequently, separation in the polyacrylamide gel is strictly dependent on the size (length) of the chain.

    • Comparison: If a protein appears as a 100 kDa peak in gel filtration but shows a single 25 kDa band on SDS-PAGE, it is likely a homotetramer.

Primary Structure and Internal Cleavage:

Determining the primary sequence (the linear order of amino acids) is essential for identifying the protein and understanding its function.

  • Enzymatic Proteolysis: Large proteins are often too complex for direct sequencing. Instead, they are cleaved into smaller, manageable peptides using specific proteases.

    • Trypsin: A widely used serine protease that highly specifically cleaves the peptide bond on the carboxyl side (C-terminus) of Lysine (K) and Arginine (R) residues.

    • This predictable cleavage pattern produces a unique set of peptides known as a "peptide map" or "fingerprint".

  • Peptide Sequencing: * Once cleaved, these peptides can be sequenced to reconstruct the full primary structure of the protein.

    • Historically, Edman degradation was the standard, but modern proteomics relies almost exclusively on mass spectrometry.

Mass Spectrometry and Proteomics:

Mass Spectrometry (MS) has revolutionised biochemistry by providing extremely accurate mass measurements and rapid protein identification.

  • Ionisation Techniques: Proteins and peptides must be converted into gas-phase ions.

    • Two primary methods are used:

      • ESI (Electrospray Ionisation):

        • The sample is ionised from a liquid stream, allowing for direct coupling with high-performance liquid chromatography (LC-MS).

      • MALDI (Matrix-Assisted Laser Desorption/Ionisation):

        • The sample is co-crystallised with a matrix and ionised by a laser pulse. It is typically coupled with a TOF (Time of Flight) analyser.

  • Peptide Mass Fingerprinting (PMF) and Database Searching: The experimental masses of trypsin-cleaved peptides are measured and compared against theoretical masses derived from genomic databases.

    • Bioinformatics Tools: Programmes such as BLAST are used to interrogate databases like SWISS-PROT to identify the protein based on these matching sequences.

    • Successful identification is achieved when multiple peptide sequences from the sample align perfectly with a database entry