2 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).Indeed with the completion of human genome sequencing, functional genomics which is the high-throughput expression analysis of genes and gene products is of utmost importance. Proteomics is the analysis of proteome which is defined as the entire protein complement expressed by a genome. The study of proteome is essential as there is poor correlation between mRNA level and protein abundance. Moreover, proteins are the functional molecules in cells and important pharmaceutical targets. Proteomics has the advantage of unbiased search for novel relationship at global level, and hence could delineate unexpected ways in which proteins regulate cellular responses. Hence, proteomics is widely used in the identification of disease biomarkers or drug targets esp in oncology.Professor Maxey C.M. Chung
4 Traditional Western Blot (single candidate analysis) Ctr12 h24 h72 hHsp 27Colorectal cancer cell lines treated with Butyrate, a HDAC inhibitorCathepsin DA)B)Proteomics refers to the large-scale study of thestructure & functions of proteins.ProteomicsA) Normal LiverB) Hepatocellularcarcinoma
6 A snapshot of proteins in the cell The Proteome:A snapshot of proteins in the cellNormalLesionIn SituInvasiveCancerMetastatic
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 humanEach gene produces 3-6 final modified proteinsPost-translational modifications (PTMs)Alternate splicingNumber 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
10 PROTEOMICSThe 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
11 Pipeline for Clinical Proteomics ValidationProtein Identification by Mass SpectrometryProtein Profiling and separationBiomarkers /Drug targets / MechanismsSample CollectionSample PreparationBioinformaticsFunctionalValidation
12 Protein Elektroforesis Gel Poliakrilamid SDS-PAGEPergerakan molekul bermuatan dalam medan listrikSodium lauryl sulfate membentuk kompleks bermuatan negatif dengan protein2-merkaptoetanol untuk memutus ikatan disulfidaPemisahan berdasarkan massa molekul
13 Elektroforesis 2-D: Pemisahan protein (10 Elektroforesis 2-D: Pemisahan protein ( jenis) berdasarkan muatan dan ukuran
14 Western blot: identifikasi protein menggunakan antibodi yang spesifik
16 Clinical Proteomics in O&G Proteomics Jun;6(11):Detection of candidate biomarkers in follicular fluid ofcontrols and RSA patients by western blotsSpots differentially expressed in patients with RSAFibrinogen chains, a, b and gAntithrombinRecurrent spontaneous abortion (RSA) is defined as the loss of 3 or more consecutive pregnancies prior to 20th week of gestation affects 5% of population. To investigate the proteins associated with RSA, protein expression in follicular fluidn was analyzed by 2DE. Follicular fluid contains a variety of biologically important proteins for oocyte fertilization and follicle maturation in the mammalian reproductive system. Depletion of six most abundant proteins (i.e. albumin, transferrin, IgG, IgA, haptoglobin, and antitrypsin) in follicular fluid was carried out using a multiple affinity removal column (MARC) (Agilent, Wilmington, DE, USA). A 4.6 mm650 mm MARC with binding capacity for 20 mL of follicular fluid was used.The results showed a decreased expression in fibrinogen gamma and antithrombin – coagulation factors- suggesting the role played by these proteins in maintaining normal pregnancy.
20 Spektrofotometri Massa: Penentuan massa molekul Pembentukan ion. Konversi molekul dalam bentuk padat atau larutan menjadi ion dalam fasa gasPemisahan ion dalam fasa gas berdasarkan m/z.Detektor. Penentuan m/z setiap ion
21 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 proteinMALDI menghasilkan ion [M+H]+
22 Electrospray Ionization (ESI) Tegangan listrik tinggi (3-5 kv) pada larutanPengupan pelarut (dengan gas hidrogen yang dipanaskan)Menghasilkan ion bermuatan lebih dari satu [M+nH]+n
23 Surface enhaced laser desorption ionizaiton (SELDI) Surface enhaced laser desorption ionizaiton (SELDI). Profil protein dari cairan tubuh (darah)