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  1. Proteomik

  2. Proteomics Proteomics (PROTEOME Analysis) – analysis of the entire PROTEin complement expressed by a genOME (Wilkins et al., 1996). Proteomics (PROTEOME Analysis) – analysis of the entire PROTEin complement expressed by a genOME(Wilkins et al., 1996). Professor Maxey C.M. Chung

  3. TIME Magazine July 3, 2000

  4. Ctr Ctr 12 h 12 h 24 h 24 h 72 h 72 h Proteomics Proteomics refers to the large-scale study of the structure & functions of proteins. A) Normal Liver A) B) B) Hepatocellular carcinoma Traditional Western Blot (single candidate analysis) Hsp 27 Colorectal cancer cell lines treated with Butyrate, a HDAC inhibitor Cathepsin D

  5. Proteomics versus traditional approach

  6. Normal Lesion In Situ Invasive Cancer Metastatic Cancer The Proteome: A snapshot of proteins in the cell

  7. Why Proteomics? • The genome is a rather constant entity whereas the proteome is dynamic. (differs from cell to cell, is constantly changing through its interactions with the genome and the environment) • Proteins, not genes, are the functional workhorses of a cell. • In addition, there is a poor correlation between mRNA levels and protein levels in the cell. • Proteomics also allows for the identification of post-translational modifications (eg phosphorylations) which directly regulate activity by switching on/off proteins. • Biofluids (eg blood, GI fluids, urine, CSF, amniotic fluid) do not have corresponding genomes or transcriptomes from which gene expression can be analysed.

  8. NUMBER OF PROTEINS IN A PROTEOME • 20,000 – 40,000 genes in human • Each gene produces 3-6 final modified proteins • Post-translational modifications (PTMs) • Alternate splicing • Number of proteins in higher organism is therefore between 60,000 to 250,000. • 3. A single genome can give rise to different proteomes depending on such variables such as cell cycle, stress response, pathological conditions

  9. PROTEOMICS • The proteome is a dynamic entity • Protein changes resulting from drug treatment, diseases etc • This could be a result of differences in expression and/or post-translational modifications • Note that the GENOME is static

  10. Protein Profiling and separation Protein Identification by Mass Spectrometry Sample Collection Sample Preparation Bioinformatics Pipeline for Clinical Proteomics Clinical Validation Biomarkers / Drug targets / Mechanisms Functional Validation

  11. Protein Elektroforesis Gel Poliakrilamid • SDS-PAGE • Pergerakan molekul bermuatan dalam medan listrik • Sodium lauryl sulfate membentuk kompleks bermuatan negatif dengan protein • 2-merkaptoetanol untuk memutus ikatan disulfida • Pemisahan berdasarkan massa molekul

  12. Elektroforesis 2-D: Pemisahan protein (10.000 jenis) berdasarkan muatan dan ukuran

  13. Western blot: identifikasi protein menggunakan antibodi yang spesifik

  14. Clinical Proteomics in O&G Proteomics. 2006 Jun;6(11):3445-54 Detection of candidate biomarkers in follicular fluid of controls and RSA patients by western blots Spots differentially expressed in patients with RSA Fibrinogen chains, a, b and g Antithrombin

  15. HPLC: Pemisahan Campuran Protein

  16. Protease: hidrolisis ikatan peptida

  17. Spektrofotometri Massa: Penentuan massa molekul • Pembentukan ion. Konversi molekul dalam bentuk padat atau larutan menjadi ion dalam fasa gas • Pemisahan ion dalam fasa gas berdasarkan m/z. • Detektor. Penentuan m/z setiap ion

  18. Matrix-Assisted Desorption Ionization (MALDI) • Protein dicampur dengan komponen matriks (Asam dihidroksi benzoat) • Sinar 337 nmmengeksitasi matriks dan protein melalui mekanisme transfer energi dari matriks ke sampel protein • MALDI menghasilkan ion [M+H]+

  19. Electrospray Ionization (ESI) Tegangan listrik tinggi (3-5 kv) pada larutan Pengupan pelarut (dengan gas hidrogen yang dipanaskan) Menghasilkan ion bermuatan lebih dari satu [M+nH]+n

  20. Surface enhaced laser desorption ionizaiton (SELDI). Profil protein dari cairan tubuh (darah)

  21. Spektrofotometri Peptida

  22. Tandem Mass SpectroscopyPeptida DNA polymerase

  23. Spektrofotometri massa:Kuantitatif Peptida

  24. Diagnosa Pola Proteom

  25. Protein Tagging System:Isolasi dan Pemurnian Protein

  26. Interaksi Protein-ProteinSistem Dua Hibrida Ragi

  27. Interaksi Protein dengan Koimunopresipitasi