Important Techniques for the Detection of Post-Translation Modification of Proteins

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1. Introduction to PTMs:

  • PTMs refer to the covalent and enzymatic modification of proteins following protein biosynthesis.
  • PTMs can alter protein function, stability, location, and interactions.
  • Examples of PTMs: phosphorylation, acetylation, ubiquitination, methylation, glycosylation, etc.

2. Mass Spectrometry (MS):

  • Principle: Detects protein mass changes due to PTMs.
  • Procedure: a. Proteins are digested into peptides. b. Analyze peptide masses using mass spectrometers. c. Changes in peptide mass indicate potential PTMs.
  • Advantages: Highly sensitive and can detect multiple PTMs simultaneously.

3. Western Blotting with PTM-specific Antibodies:

  • Principle: Detection of protein bands with antibodies specific to particular PTMs.
  • Procedure: a. Separate proteins via SDS-PAGE. b. Transfer proteins to a membrane. c. Probe with PTM-specific antibodies.
  • Advantages: Specific and can be quantitative when coupled with imaging systems.

4. Phos-tag SDS-PAGE:

  • Principle: Uses a specific compound, Phos-tag, to retard the migration of phosphorylated proteins.
  • Procedure: Similar to regular SDS-PAGE but includes Phos-tag in the gel.
  • Advantages: Direct visualization of phosphorylated vs. non-phosphorylated proteins.

5. Edman Degradation:

  • Principle: Sequential removal of N-terminal residues of peptides.
  • Procedure: a. Attach peptide to solid support. b. Sequentially remove and identify the N-terminal residues.
  • Advantages: Can locate exact site of PTM, but mostly phased out due to newer, more efficient methods.

6. Immunoprecipitation (IP):

  • Principle: Uses antibodies to isolate specific proteins or PTM-modified proteins from a complex mixture.
  • Procedure: a. Incubate cell lysate with PTM-specific antibody. b. Capture antibody-protein complexes using protein A/G beads. c. Analyze captured proteins by western blotting or MS.
  • Advantages: Specific isolation of PTM proteins for detailed studies.

7. Proximity Ligation Assay (PLA):

  • Principle: Detects proteins in close proximity (usually <40 nm) by using PTM-specific antibodies linked to DNA oligonucleotides.
  • Procedure: a. Incubate cells with two PTM-specific antibodies. b. Add oligonucleotide-conjugated secondary antibodies. c. Produce circular DNA if proteins are in proximity. d. Amplify and detect DNA.
  • Advantages: High sensitivity and specificity; allows for visualization of PTMs in situ.

8. Protein Microarrays:

  • Principle: High-throughput screening of protein interactions or detection of PTMs.
  • Procedure: a. Spot proteins/peptides on a solid support. b. Probe with PTM-specific antibodies or enzymes. c. Detect bound antibodies/enzymes.
  • Advantages: Rapid screening of multiple PTMs or proteins.

9. Enzymatic Assays:

  • Principle: Use enzymes specific for a certain PTM (e.g., kinases for phosphorylation) to modify substrates.
  • Procedure: a. Mix protein substrates with specific enzymes in presence of co-factors. b. Monitor reaction progress (e.g., using radiolabeled ATP for kinase reactions).
  • Advantages: Functional assays that can provide mechanistic insights.

10. Peptide Mapping:

  • Principle: Correlates the structural changes due to PTMs with alterations in peptide profiles.
  • Procedure: Digest proteins and compare peptide fragments before and after PTM.
  • Advantages: Can identify specific sites of modification.

Conclusion: Detection of PTMs is crucial for understanding protein functions in biological processes. A combination of techniques can provide comprehensive insights into the type, site, and consequence of PTMs on proteins.

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