Proteomics and Biomedical Research .pptx

An overview of proteomic methods and biomedical research concerns.

1. Proteomics and Biomedical Research

2. Objectives Define proteomics and describe its importance in molecular medicine. Explain the principles of Two-Dimensional Gel Electrophoresis and Mass Spectrometry. Describe the process and uses of Shotgun Proteomics. Recognize techniques used in elucidating protein structure. Discuss the role of animal models in biomedical research. Identify alternatives to animal testing and their importance in ethical research.

3. What is Proteomics ? Proteome: refers to the entire set of proteins and includes their posttranslational modification in any cell type, tissue, or organism. The proteome varies with time in response to environmental influences or extracellular signals. Proteomics = large-scale study of all proteins expressed in a cell, tissue, or organism.

4. Introduction to Proteomics Proteins are the 'workers' of the cell. Proteomic and genomic studies are complementary approaches to understanding biological and pathological processes. Proteomics helps to overcome some of the limitations of genomics. Applications: disease biomarkers, drug targets, personalized medicine.

5. Proteomics

6. Gel Electrophoresis Uses an electric current to separate DNA, RNA, or proteins by size and charge through a porous gel matrix.  DNA and RNA are loaded into wells, and an electric current is applied, causing the negatively charged molecules to migrate toward the positive electrode.  Smaller molecules move faster through the gel's pores, resulting in separation by size A DNA ladder of known sizes is run simultaneously to estimate fragment lengths.  After electrophoresis, a DNA-binding dye allows the separated DNA to be visualized as fluorescent bands under UV light. 

7. Two-dimensional Gel electrophoresis Separates complex protein mixtures by: first separating proteins by their isoelectric point (charge) in a pH gradient via isoelectric focusing (IEF) separating these same proteins by their molecular weight (size) using SDS-PAGE in the second dimension.  2D map of protein spots, with proteins resolving into distinct spots based on these two properties, allowing for detailed proteomic analysis.  

8. Two-Dimensional Gel Electrophoresis (2D-GE) Step 1: Separation by isoelectric point ( pI ). Step 2: Separation by molecular weight. Produces a protein map (spots). Gel is stained to visualize the spots: Spots excised → proteins digested → analyzed.

9. Benefits and Applications High-Resolution Separation: 2DE offers greater resolution than one-dimensional gels, allowing for the separation of thousands of proteins from complex biological samples.  Proteomic Analysis: It is used to analyze protein expression, quantify protein abundance, and study protein modifications like isoforms and post-translational modifications.  Comparative Studies: 2DE helps researchers to compare protein profiles between different biological samples, such as healthy versus diseased tissues. 

10. Limitations Labor-intensive and time-consuming Poor reproducibility, Difficulty in resolving low-abundance, hydrophobic, or extremely acidic/basic proteins, Limited detection dynamic range.

11. Proteoforms Any of the different molecular forms that can be generated from a single gene, including different amino acid sequences (due to genetic variations or splice variants) and various post-translational modifications (PTMs) (like phosphorylation, glycosylation, or ubiquitination). Understanding the diversity of proteoforms is crucial for comprehending cell function and disease, but requires specialized top-down proteomics methods that analyze intact proteins rather than just peptides.

12. Mass Spectrometry (MS) Measures mass-to-charge ratio (m/z) of ionized peptides. Produces peptide fingerprints. Types: MALDI-TOF, ESI-MS. Applications: biomarker discovery, drug development.

13. Mass Spectrometry

14. Pros and Cons Pros: Very accurate and sensitive. Works with small samples. Cons: Expensive equipment. Needs trained experts.

15. Shotgun Proteomics Proteins are digested into peptides. Peptides analyzed directly by MS. High-throughput: thousands of proteins at once. Application: cancer proteomics.

16. Pros and Cons Pros: Very high-throughput (analyzes thousands of proteins). Great for complex samples. Cons: Data analysis is complex. Can miss rare proteins.

17. Elucidation of Protein Structure Levels of structure: Primary = sequence of amino acids. Secondary = local folding (like coils and sheets). Tertiary = full 3D shape. Quaternary = multiple proteins working together.

18. Elucidation of Protein Structure Techniques: X-ray crystallography → high detail, needs crystals. NMR → proteins in solution, good for smaller proteins. Cryo-EM → studies big complexes, very powerful. Medical relevance: drug design, misfolding diseases.

19. Elucidation of protein Structure Pros: Precise 3D structures. Cons: Some methods are expensive and slow.

20. Animal Models in Biomedical Research Used to mimic human physiology & disease. Used to test new drugs Common models: mice, zebrafish, fruit flies, primates. Example: Mouse models in diabetes & cancer.

21. Pros and Cons Pros: Similar physiology to humans in many ways. Controlled experiments possible. Cons: Not always identical to humans. Ethical concerns.

22. Alternatives to Animal Testing New approaches: In vitro = human cell cultures in a dish. Organoids = mini 3D organs from stem cells. In silico = computer models and simulations. 3Rs principle : Replacement, Reduction, Refinement .

23. Pros and Cons Pros: Reduce animal use. Sometimes more human-relevant. Cons: Still developing, not always replacing animals fully yet.

24. Conclusion Proteomics provides insight into cell function & disease. 2D-GE, MS, Shotgun = complementary methods. Protein structure knowledge aids drug design. Animal models important, but alternatives are rising. Future: Personalized medicine with ethical testing.

25. Quiz Questions 1. What are two pros and cons of 2D-GE? 2. What is the main advantage of mass spectrometry? 3. What makes shotgun proteomics 'high-throughput'? 4. Name one alternative to animal testing.

26. Quiz: 5. Which protein structure method would you use for a large protein complex? 6. A doctor wants to compare proteins in cancer vs normal cells. Which method is best? 7. Compare one advantage and one disadvantage of 2D-GE vs mass spectrometry. 8. What ethical issue do animal models raise? 9. How do organoids help address this ethical issue?

27. This or That Scenario 1 A patient with HIV needs viral protein levels measured to monitor treatment. Which method would you use? 👉 2D-GE OR Mass Spectrometry?

28. This or That Scenario 2 A researcher wants to identify thousands of proteins in a cancer biopsy sample. Which method would you use? 👉 Shotgun Proteomics OR 2D-GE?

29. This or That Scenario 3 You need to determine the 3D shape of a protein involved in Alzheimer’s disease. Which method would you use? 👉 X-ray Crystallography OR Cryo-EM?

30. Class Debate Activity Debate Topic: Should animal models still be used in biomedical research? Team A: Argue FOR animal models. Team B: Argue FOR alternatives (in vitro, organoids, in silico). Rules: 3 minutes prep time for each side. 3 minutes to present arguments. 2 minutes for rebuttal.

31. References Kurreck J, Stein C. Molecular Medicine. Peer-reviewed sources on proteomics and bioethics.