Proteomik

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

TIME Magazine July 3, 2000

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

Proteomics versus traditional approach

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

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.

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

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

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

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

Elektroforesis 2-D: Pemisahan protein (10 Elektroforesis 2-D: Pemisahan protein (10.000 jenis) berdasarkan muatan dan ukuran

Western blot: identifikasi protein menggunakan antibodi yang spesifik

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 Recurrent 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.

HPLC: Pemisahan Campuran Protein

Protease: hidrolisis ikatan peptida

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

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]+

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

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

Spektrofotometri Peptida

Tandem Mass Spectroscopy Peptida DNA polymerase

Spektrofotometri massa: Kuantitatif Peptida

Diagnosa Pola Proteom

Protein Tagging System: Isolasi dan Pemurnian Protein

Interaksi Protein-Protein Sistem Dua Hibrida Ragi

Interaksi Protein dengan Koimunopresipitasi