Chapter 9 Proteomics. From genomics to proteomics

Transcription

1 Chapter 9 Proteomics From genomics to proteomics

2 From structural genomics, functional genomics to proteomics The Proteome: Discovering the Structure and Function of Proteins

3 Why proteomics? The proteome is neither as uniform nor as static as the genome

4 The strategies in genomics and proteomics: from past to present Present Study single gene or protein and expand to others related Static Monitor the whole changes- for example, by microarrays and find the related info Dynamic Past

5 From gene to protein Hemoglobin beta-subunit GAG mutated to GTG Questions? 1. How phenotype changed? 2. How hemoglobin changed in its structure? 3. How hemoglobin lost its function as a carrier to O 2?

6 Multidimensional proteome analysis of cells and tissues Nature Reviews Molecular Cell Biology 16, (2015)

7 What people do in proteomics? Proteinprotein interaction Complex proteins Metabolic cascade Organelle composition Localization of organelle proteins Function of transport intermediates Protein profiles Diseases Environmental factors Protein activity patterns Proteomic signatures in diseases From genomics to proteomics, Nature, 2003

8 Some skills we adapt to determine protein behaviors Separation PAGE.Centrifugation LC.Affinity Quantification To quantify protein itself To quantify visualization or identification Localization To localize visualization and identification Visualization In vivo: Reporter genes In vitro: Staining or labeling Detection and Identification Western Blot Mass spectrometry Protein array Interaction.Array.Pull-down.Y2H

9 Separation PAGE Liquid chromatography Centrifugation

10 PAGE (Polyacrylamide gel electrophoresis) SDS-PAGE (sodium dodecyl sulfate PAGE) Principle: To separate proteins by their sizes Non-denaturing PAGE: Native PAGE Principle: To separate proteins by their sizes and charges

11 2D PAGE (two-dimensional PAGE) 2D gel is a combination of 1. SDS PAGE 2. Isoelectric focusing (IEF) (To separate proteins by their charges in the electric field)

12 HPLC (High-Pressure Liquid Chromatography) Separation unit Ion exchange Size exclusion Hydrophobicity Affinity Detection unit Absorbance Fluorescence Refractive index (RI) Evaporative light scattering detectors (ELSD)

13 HPLC (High-Pressure Liquid Chromatography) The column is packed with the stationary phase and the mobile phase is forced through a chromatography column.

14 Variable stationary phases in HPLC Size exclusion Ion-exchange Affinity Reversed phase Packed with porous beads To separate samples based on their sizes Packed with charged functional groups To separate samples based on their charges Packed with substrates, such as metals To separate samples based on their affinity to the substrates Packed with hydrophobic akyl chains To separate samples based on their hydrophobicity

15 Differential and gradient centrifugation Nature Reviews Molecular Cell Biology 6, (September 2005)

16 Visualization Staining e.g. dye and metals Reporter genes e.g. beta-galactosidase (LacZ) and GFP Labeling e.g. fluorophores (biological or chemical)

17 Protein staining in polyacrylamide (or agarose) gels Staining methods Coomassie Brilliant Blue Staining Silver Staining Fluorescence Staining e.g. Sypro dyes

18 Reporter genes Reporter genes The genes that enable the detection or measurement of gene expression. Can be fused to regulatory sequences or genes of interest to report expression location or levels. Reporter genes include genes that code for fluorescent protein and enzymes that convert invisible substrates to luminescent or coloured products. e.g. lacz (encode beta-galactosidase) & GFP

19 Reporter gene: beta-galactosidase A. C. elegans D. In situ hybridization B. Antibody staining C. β-galactosidase activity (lacz) Limitations Samples to be fixed and then permeabilized Time frame has to be done from many different individuals.

20 Green Fluorescent Proteins, GFP Biological fluorophores Can be overexpressed along or in fusion with target proteins, such as Green fluorescent protein (GFP) was cloned from the jellyfish Aequorea victoria and used as a gene expression reporter

21 Types of Fluorophores for labeling Organic dyes Synthetic and small compounds, such as fluorescein isothiocyanate (FITC), rhodamine (tetramethyl rhodamine isothiocyanate, TRITC) Biological fluorophores Can be overexpressed along or in fusion with target proteins, such as Green fluorescent protein (GFP) was cloned from the jellyfish Aequorea victoria and used as a gene expression reporter

22 Detection and Identification Via antibody (protein peptides or epitome tags) Sequence or Mass (label free) Techniques Western Blot Enzyme-Linked Immunosorbent Assay (ELISA) Mass spectrometry Protein array

23 Western Blot Western Blot A analytical technique used to detect specific proteins by 1.Separation: electrophoresis 2.Transfer: from PAGE to PVDF (or nitrocellulose) 3.Detection/Analysis: immunostaining ELISA) (e.g.

24 Western Blot Transferred proteins s PVDF is tougher!! Layer of blocking protein (milk or BSA) Primary Ab for HSF1 is monoclonal anti-hsf1/mouse Secondary Ab is goat anti-mouse Ab conjugated with HRP (Horseradish peroxidase) Substrate HRP catalyses the oxidation of luminol, converting luminol into an excited state which emits light as it decays to the groud state.

25 Protein tagging systems- tools for isolation and purification and identification Small tags Histidine tag (6xHis, 10xHis) His tag-nickel column (elution with imidazole) FLAG tag (AspTyrLysAspAspAspLys) Longer tags Glutathinone-S-transferase (GST) GST tag-glutathione (Elution with glutathione) Maltose-binding protein (MBP) MBP tag- maltose (elution with maltose) FLAG tag-antibody (elution with FLAG peptides)

26 Multiplex ELISA Enzyme-linked immunosorbent assay, ELISA

27 The basic principles in Mass spec analysis 1. Ionization: (high vacuum, electric field) To convert analyte molecules or atoms into gas-phase ionic species, even breaking the molecules into fragments. This step requires the removal or addition of an electrons or protons. 2. Separation: (high vacuum) The separation and mass analysis of the molecular ions and their charged fragments on the basis of their m/z (mass-to-charge) ratios 3. Measurement: The ion current due to these mass-separated ions is measured, amplified, and displayed in the form of a mass spectrum.

28 Mass spectrometers comprise.. Ionization Separate ions Detect ions Transfer a sample

29 MALDI-TOF Mass spectrometer Tanaka K, Nobel Prize winner In Chemistry, 2002 A matrix can 1. Absorb photon energy 2. Be the solvent for the analyte Sample preparation Mixing with matrix Evaporation of solvent Ionization

30 Electrospray ionization (ESI) mass spectrometry Droplet formation Droplet shrinkage

31 Peptide mass fingerprinting Figure 9.12 Mass Spectrometry of Peptides Because mass spectrometry is so sensitive, the use of large whole proteins is limited. Instead, peptide fragments are generated by protease digestion. The peptides are easily separated with HPLC, and then specific peptides are subjected to mass spectrometry.

32 Peptide mass fingerprinting

33 Interaction Co-immunoprecipitation (Pull-down) Yeast two hybrid (Y2H) Protein array

34 Co-immunoprecipitation to determine whether two molecules are interacting Figure 9.24 Co-immunoprecipitation To determine whether protein A and B interact within the cytoplasm, each protein is fused to a different tag for easy isolation. Each fusion protein is expressed in mammalian cells, which are then lysed to release the cell proteins. The cells must be lysed gently to avoid disrupting the protein interactions. The fusion proteins are isolated using the tag sequence. Each tagged protein and all its associated proteins are isolated independently. For example, on the left, Flag-tagged protein A is isolated with an antibody to the Flag sequence, and on the right, His6-tagged protein B is isolated with an antibody to the His6 sequence. The protein complexes are separated by SDS-PAGE. This example shows the two tagged proteins A and B interacting.

35 Yeast two-hybrid system (Y2H system), Dr Stanley Field Yeast transcriptional activator GAL4 has got two domains: DBD and A When prey/bait bind, DBD/AD work when in close proximity Binding of two proteins activates a reporter gene

36 No interaction No reporter gene

37 Y2H analysis using lacz and HIS3 as reporter gene Beta-galactosidase X-Gal Blue color

38 Two-Hybrid Analysis: Mass Screening by Mating Figure 9.23 Two-Hybrid Analysis: Mass Screening by Mating To identify all possible protein interactions using the two-hybrid system, haploid α yeast are transformed with the Bait library, and haploid a yeast are transformed with the Prey library. When the two yeast types are mated with each other, the diploid cells will each contain a single bait fusion protein and a single prey fusion protein. If the two proteins interact, they activate the reporter gene, which allows the yeast to grow on media lacking histidine (yeast His3 gene) or turn the cells blue when growing on X-gal medium (lacz from E. coli). This process can be done for all 6000 predicted yeast proteins using automated techniques.

39 Forward and reverse phase protein microarray

40 Representative capture agents of protein-detecting microarrays Protein protein interactions and selection: array-based techniques for screening disease-associated biomarkers in predictive/early diagnosis. The FEBS journal, 2010.

41 Quantification Densitometry To quantify protein itself e.g. Protein assay To quantify visualization or identification e.g. Density or intensity detector

42 Five classes of quantitative proteomics Gel-based Involves separation gels and stains Metabolic labeling Involves the incorporation of stable isotope constituents (e.g. 15 N, 2 H, or 13 C) into the growth media Chemical labeling Involves the incorporation of stable isotope tags onto cellular proteins. Label-free No label is incorporated.

43 Gel-based quantitative proteomics Gel types Gel electrophoresis 2D gel electrophoresis (ph gradient + MW separation) Following experiments Stains Coomassie blue silver stains fluorescent stains Cons Multiple proteins in the same spot makes densiometric analysis difficult Low-abundant or membranes proteins are difficult for detection

44 Quantification: Measure the intensity of the dots on 2D gels Proteomics May;9(9): D-PAGE stained by Coomassie Brilliant Blue G-250

45 Metabolic labeling-based quantitative proteomics Nat Rev Mol Cell Biol Dec;7(12): Proteomics 2005, 5, 4 15 e.g. Stable labeling by amino acid in cell culture (SILAC) 15 N ammonium salts 13 C glucose

46 Chemical labeling-based quantitative proteomics Chemical labeling e.g. Isotope coded affinity tag, ICAT The ICAT reagent consists of a reactive group that is cysteine-directed, a polyether linker region with 2 H 8, and a biotin group that allows purification of labeled peptides.

47 Label free-based quantitative proteomics A method without incorporation of labels onto detected proteins in quantitative proteomics. Achieved by superimposing overlapping intensities of the peptides signals from separate LC/MS runs to reference control and changes In principle applicable to any kind of sample, including materials that cannot be directly metabolically labeled (for instance, many clinical samples).

48 Some skills we adapt to determine protein behaviors Separation PAGE.Centrifugation LC.Affinity Quantification To quantify protein itself To quantify visualization or identification Localization To localize visualization and identification Visualization In vivo: Reporter genes In vitro: Staining or labeling Detection and Identification Western Blot Mass spectrometry Protein array Interaction.Array.Pull-down.Y2H

49 Proteomics Proteinprotein interaction Complex proteins Metabolic cascade Organelle composition Localization of organelle proteins Function of transport intermediates Protein profiles Diseases Environmental factors Protein activity patterns Proteomic signatures in diseases From genomics to proteomics, Nature, 2003

50 SILAC-based analysis of protein interaction partners in pull-down experiments The Journal of Cell Biology, Oct , 183(2): 223

51 Proteomics: There are still many blanks for you to fill in Proteinprotein interaction Complex proteins Metabolic cascade Organelle composition Localization of organelle proteins Function of transport intermediates Protein profiles Diseases Environmental factors Protein activity patterns Proteomic signatures in diseases From genomics to proteomics, Nature, 2003

52 Proteomics of organelles and large cellular structures Nat Rev Mol Cell Biol Sep;6(9): Proteomics of organelles and large cellular structures.

53 Quantification: Measure the fluorescence of Green fluorescent protein Plant Cell Rep (2003) 22:

54 Proteomics: There are still many blanks for you to fill in Proteinprotein interaction Complex proteins Metabolic cascade Organelle composition Localization of organelle proteins Function of transport intermediates Protein profiles Diseases Environmental factors Protein activity patterns Proteomic signatures in diseases From genomics to proteomics, Nature, 2003

55 Differential proteomics by using 2D PAGE Figure 9.3 Two-Color 2D-Gel. Proteins from two different conditions (e.g., normal and cancerous) can be compared directly on the same gel by labeling each with a different fluorescent dye. When the gel is visualized to see the dyes, the proteins found only in normal tissue form red spots, the proteins found only in cancerous tissue form green spots, and proteins found in both normal and cancerous tissue look yellow.

56 Water-deficit stress led to tissue-specific changes in protein expression Vitis vinifera Proteomics May;9(9):

57 Quantification: Measure to intensity of bands of western blot Effect of starvation on PPAR-alpha in vivo on PPAR-alpha protein expression in isolated islets

58 Proteomic Pattern Diagostics

59 Proteomics: There are still many blanks for you to fill in Proteinprotein interaction Complex proteins Metabolic cascade Organelle composition Localization of organelle proteins Function of transport intermediates Protein profiles Diseases Environmental factors Protein activity patterns Proteomic signatures in diseases From genomics to proteomics, Nature, 2003

60 考題範例