Medical Biotechnology lecture 3.pptx

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1. Proteomics Global analysis of proteins Proteome is the entire protein complement of an organism Translatome is the complement of proteins under specific circumstances. It is dynamic and changes when the environmental conditions change. The relationship among the genome, proteome, and translatome are not linear

2. Proteomics The proteome correlates highly with the genome for proteins are the products of the majority of genes. Some genes code for non-translated RNA and so do not contribute to the proteome. The translatome is highly dynamic, changing from minute to minute depending on many different stimuli microRNA and siRNA control the expression of proteins

3. Proteomics The translatome and proteome are also affected by post-translational modifications Proteins also undergo proteolytic cleavage and as such, the composition of the translatome is affected by the rate of protein degradation, and protein stability has a major influence.

4. Gel Electrophoresis of Proteins Done on polyacrylamide gels (PAGE) to separate proteins by size Polyacrylamide has smaller pores than agarose and is thus suitable for proteins since proteins are smaller than DNA molecules. Most proteins do not have a negative charge. Some are positively charged. SDS comes in. Amount of SDS and therefore the amount of charge correlates with the length (molecular weight) of the protein

5. SDS PAGE

6. SDS PAGE Use Coomassie Blue or Silver Stain (more sensitive) Suitable for samples with only a few proteins of different sizes. With many proteins and of similar sizes, individual bands may not appear. Two dimensional PAGE comes in. Here, Isoelectric focusing is done to first separate proteins by their native charge Then SDS-PAGE is done for in the second dimension.

7. Two Dimensional PAGE

8. Two Dimensional PAGE 2-D PAGE allows for quantification of the relative amounts of different proteins. This is done by looking at the size of each spot and quantifying by scanning with a laser. Computer analysis then determines the density of the spot and relative abundance. Disadvantages of 2D PAGE is that Certain classes (hydrophobic, extremely large/small, rare) of proteins are under represented on a gel due to their inability to migrate through acrylamide Must isolate from the acrylamide for analysis by mass spectrometry

9. Western Blotting of Proteins Used to identify proteins Relies on having an antibody to the protein First separate proteins by size using SDS-PAGE or 2D-PAGE Then transfer to nitrocellulose membrane Membrane must have a positive charge Electricity used to transfer Blocking is done then primary antibody added followed by washing and adding the secondary antibody then washing and visualizing.

10. Western Blotting of Proteins

11. High-Pressure Liquid Chromatography (HPLC) Seperates protein mixtures Chromatography is a general term for separation techniques where a sample of molecules, the analyte , is dissolved in a mobile phase and then forced through a stationary phase. In HPLC, the sample is dissolved in a mobile phase and forced through a chromatography column (a narrow tube packed with the stationary phase under high pressure) in a process called elution

12. HPLC HPLC can be used to Separate proteins Identify proteins Purify proteins Quantify proteins The separated proteins are in a liquid state thereby making further analysis easier.

13. HPLC B: seperation of a mixture of dyes into Tartrazine Amaranth Indigo Carrine New Coccine Sunset Yello Fast Green Brilliant Blue Erythosine

14. Types of HPLC Size exclusion chromatography columns contain porous beads that separate mixtures of proteins by size Reverse phase HPLC uses columns packed with hydrophobic alkyl chains attached to silica-based material. Hydrophilic molecules elute faster Ion-exchange HPLC uses a stationary phase with charged functional groups that bind oppositely charged molecules in the sample Affinity HPLC contains molecules that specifically bind the target proteins.

15. HPLC Detection Refractive index detectors shine a light beam through the sample to monitor refraction of light by the elutes Ultraviolet detectors have a UV light to determine UV light absorption Fluorescence detectors detect compounds that fluoresce Radiochemical detectors detect radioactively labeled compounds Electrochemical detectors measure compounds that undergo redox reactions

16. HPLC with Mass Spectrometry Mass spectrometry can be combined with HPLC – an approach that is increasingly used in proteomics Many experimental conditions can be adjusted to get high resolution

17. Mass Spectrometry Used to determine the mass of molecules The molecule is fragmented into different ions whose masses are accurately measured Two different ionization techniques have made proteins manageable Matrix-associated laser desorption-ionization – proteins are embedded in a matererial that absorbs laser light and time of flight (TOF) detector records the intensity and mass Electroscopy ionization – sample dissolved in liquid and small droplets a narrow capillary tube

18. Mass Spectrometry

19. Peptide Sequencing using Mass Spectrometry Currently only for short peptides Obtain a pure sample of the protein A protease is used to digest it into fragments thereby reducing undesirable characteristics of the entire protein. Two rounds os mass spectroscopy are used in what is refered to as tandem mass spectroscopy. One ion produced in round 1.

20. Peptide Sequencing using Mass Spectrometry Fragmented by collision Seperated (by mass to charge ratio (m/z)) Each peak varies by one AA, and the difference between the peak determines the AA sequence Sometimes results maybe ambiguous in which case databases of peptide ion spectra are used for comparison

21. Protein Quantification using Mass Spectrometry

22. Protein Tagging Systems Target protein genetically fused to a DNA that codes for a tag (short and long tags) Short tags Polyhistidine / His6 tag – binds to nickel ions Flag tag – binds to anti-flag Strep tag – similar to biotin, binds to streptavidin

23. Protein Tagging Systems Long tags Protein A Glutathione-S- transferase (GST) Maltose-binding protein (MBP)

24. Protein Tagging Systems

25. Digestion of Proteins by Proteases Proteases (aka proteinases or peptidases) hydrolyze the peptide bond between AA residues in a polypeptide chain. Proteases are used to cleave fusion proteins or remove single AAs for protein sequencing During apoptosis, they digest cellular components for recycling Plants use proteases to protect themselves from fungal or bacterial invaders In biotechnology industries, they are used as additives to detergents to digest proteins in ketchup, blood, or grass stains

26. Digestion of Proteins by Proteases Classification criteria of proteases: Reaction catalyzed Chemical nature of the catalytic site Evolutionary relationships Types: Endopeptidases – cleave the target protein internally Exopeptidases – remove single AA from terminals (can either be carboxy or amino-peptidases)

27. Digestion of Proteins by Proteases Serine proteases – contain a serine in their active sites (include chymotrypsin , trypsin , elastase ) Cysteine proteases contain cysteine instead e.g. papain , and bromelain Aspartic proteases have two essential aspartic acid residues close together in the active site and include digestive enzymes pepsin and chymosin Metalloproteases use metal ion co-factors eg thermolysin Threonine proteases have threonine in the active sites

28. Digestion of Proteins by Proteases The degradome is the complete set of proteases expressed at one specific time by a cell, tissue, or organism