Lecture-8 Introduction to Proteomics Huseyin Tombuloglu, Phd GBE423 Genomics & Proteomics.

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Presentation transcript:

Lecture-8 Introduction to Proteomics Huseyin Tombuloglu, Phd GBE423 Genomics & Proteomics

What is Proteomics? Defined as “the analysis of the entire protein complement in a given cell, tissue, or organism.” Proteomics “also assesses activities, modifications, localization, and interactions of proteins in complexes.” Proteomes of organisms share intrinsic differences across species and growth conditions.

The proteome Organisms have one genome But multiple proteomes Proteomics is the study of the full complement of proteins at a given time Humans - ~20,000 genes - >500,000 proteins

Quantitation Experimental Paradigm - Labelling Label samples in such a way as to not affect subsequent processing but allow differentiation in final analysis. Examples: –Fluorescent dyes (2DGE) –SILAC amino acid labels (MS) –Isobaric mass tags (MS/ MS)

2D-GE Separate proteins by isoelectric point, then by mass Visualise with silver staining or coomassie Use CyDyes to label samples so they can be run together on the same gel Appl Microbiol Biotechnol October; 76(6): 1223–1243.

The isoelectric point (pI) is the pH at which a particular molecule or surface carries no net electrical charge.

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Two Dimensional Gel Electrophoresis (2-DE) - Sample preparation Protein precipitation…… TCA/Acetone -First dimension: Isoelectric focusing (IEF) -Second dimension: SDS-PAGE -Detection of protein spots: Staining Collaidal Comassie G-250 Silver Staining Florescent dyes (Cy3- Cy5) - Imaging analysis & 2D Gel databases - Spot handling: excision, in gel digestion - MS (Mass Spectrometry)

IPG strip ranges IPG strips (3 mm x 18 cm x 0.5 mm)  Narrow range  Medium range  Broad range Identification of B tolerance mechanisms

Program power supply Number of gels: (1-12) Max voltage: V Vhold: 125 V Duration: 24 hrs Max current: 50  A/strip Volt hours: 80,000 Vh pI pH 4 pH 7 Cathode Anode IEF run Place wet wicks (  H 2 O) under each end of strip Set chiller temperature for 20°C (setting ~2.5)

After IEF run Remove IPG strip from tray Let oil drip off the strip Place IPG strip gel facing up in equilibration tray + - Add 10 ml equilibration buffer 1 per tray

Lipids (detergents) Proteases (inhibitor cocktails) Nucleic acids (ultracentrifugation, nucleases) Polysaccharides (ultracentrifugation) Salts (dialyse;  less than 20 mM) Interfering substancesLimit sample degradation Fresh cells/tissue Protease inhibitors Keep sample cold Long term storage at –80°C Limit sample contamination Gloves (keratin)