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생명과학부 석사 1 년차 김 원

Proteome analysis and its impact on the discovery of serological tumor markers Terence C.W.Poon, Philip J. Johnson Clinica Chimica Acta 313 (2001) 231-239. 생명과학부 석사 1 년차 김 원. 1. Introduction 2. Concept of proteome and proteomics 3. Advances in proteome analysis

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생명과학부 석사 1 년차 김 원

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  1. Proteome analysis and its impact on the discovery of serological tumor markersTerence C.W.Poon, Philip J. JohnsonClinica Chimica Acta 313 (2001) 231-239 생명과학부 석사 1년차 김 원

  2. 1. Introduction • 2. Concept of proteome and proteomics • 3. Advances in proteome analysis • 4. What are tumor markers? • 5. Hepatocellular carcinoma as a model tumor for tumor marker discovery 5.1 Current clinical tools for diagnosis and for monitoring treatment response 5.2 Discovery of novel tumor markers by proteome analysis of sera • 6. Future Prospects • 7. Conclusion

  3. 1. Introduction • Tumor marker – screening, diagnosis, staging, prognosis, monitoring • There is little consensus on their clinical use -> We need better methods for discovering Sensitive and specific markers

  4. 2. Concept of proteome and proteomics • Proteome : Entire protein complement • Proteome is dynamic : change with time and according to physiological • Proteomics:‘the study of protein properties’ (expression level, post-translational modification, interaction) • Proteomic study 1)structure : protein-protein interaction 2)quantitative regulation : expression level

  5. 3. Advances in proteome analysis

  6. History of 2D PAGE • 1970s (by O’Farrel) -> 2D PAGE • 1980s -> Immobilized pH gradients -> Consistent,reproducible • Recent -> High sensitivity stain, narrow pI range

  7. Merits of 2D PAGE • Separation of a large number of proteins • Quantitative information(by intensity of the spots) • Information on post-translational modifications, glycosylation, phosphorylation

  8. Use of 2D PAGE • Identify with MALDI-TOF • Comparison under different experimental conditions -> especially useful in finding disease-associated proteins • We have not databases of full-length sequences of a large percentage of human proteins -> unidentified proteins -> Q-TOF MS for amino acid sequencing

  9. 4. What are tumor markers? • Biomolecules that can be measured quantitatively in body tissues or fluids • Can be clinically useful in patients with malignancies • Need for screening, diagnosis, staging, prognosis, monitoring treatment response, detection of tumor recurrence • Highly specific for a single type of tumor -> rare->specific to tumor cells but, cancer type Ex) AFP (alpha-fetoprotein) : HCC(mainly), testicular cancer, ovarian cancer(frequently) • Detect in blood or tissue section

  10. 5. Hepatocellular carcinoma as a model tumor for tumor marker discovery

  11. 5.1 Current clinical tools for diagnosis and for monitoring treatment response • AFP is the conventional marker for HCC * level up : 80% of patients with HCC • Most HCCs arise in patients with coexisting chronic liver disease -> limits the use of AFP for early detection of HCC • Current standard method -> Imaging Technique -> difficult ( < 2cm nodule) We need general method to detect novel marker for the diagnosis and monitoring

  12. 5.2 Discovery of novel tumor markers by proteome analysis of sera • By analysing and comparing the proteomes of cancer and normal tissues or cells, We found the disease-associated proteins (2D PAGE) ->They can also be detected in biological fluids (plasma or serum) • Direct analysis on biological fluids such as urine or serum -> compare samples from cancer patients and healthy controls

  13. Typical example HCC Normal About 20 spots detected

  14. Disappearance of this AFP strongly indicates that it is a protein specifically released by the HCC Studies of more patient and control specimens ㅡ> This will be potential tumor marker

  15. Antibody Detection Method

  16. SCC Normal

  17. Cystectomy

  18. 6. Future prospects( Other separation technique ) • A. HPLC 1D : size-exclusion chromatography 2D : reverse-phase HPLC 1D : size-exclusion HPLC 2D : capillary isoelectric focusing faster sample separation, higher throughput, automation • B. Direct analysis of unseparated protein mixture : protease digestion -> LC -> tandem MS -> limited by the current MS technology (only simple protein mixture) ->next generation

  19. 7. Conclusion • The combination of 2D PAGE and MS is the most widely used strategy for proteome analysis • Proteome analysis on sera from cancer patients and control subjects has been shown to be capable of identifying novel tumor markers -> not sufficient • Automatic high-throughput analysis and large dynamic range -> can find high sensitivity & specificity novel tumor marker

  20. Problems • Heterogeneity of biopsy materials • Technical problems Separation Focusing of some proteins • Large dynamic range of protein expression • Lack of suitable procedures for quantitating protein changes • Better image analysis system • Databases

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