Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
蛋白質體學 Proteomics 2010 Novel Proteomics Techniques 陳威戎 2010. 11. 23 1 Mass spectrometer schematic Matrix Assisted Laser Desorption Ionization (MALDI) Animation Electrospray ionization (ESI) • Generates ions directly from acidic solution Novel Proteomics Techniques 1. SELDI protein chips (Ciphergen) 2. Multiple Dimensional Liquid Chromatography, MDLC 3. Detect Dynamic Changes in Tissue/Cells (1) Stable-Isotope Labeling (2) Isotope Coded Affinity Tags Technology, ICAT (3) Isobaric Tag for Relative and Absolute Quantitation, iTRAQ 4. Difference Gel Electrophoresis, DIGE Novel Proteomics Techniques 1. SELDI protein chips (Ciphergen) 2. Multiple Dimensional Liquid Chromatography, MDLC 3. Detect Dynamic Changes in Tissue/Cells (1) Stable-Isotope Labeling (2) Isotope Coded Affinity Tags Technology, ICAT (3) Isobaric Tag for Relative and Absolute Quantitation, iTRAQ 4. Difference Gel Electrophoresis, DIGE SELDI protein chip Surface Enchanced Laser Desorption / Ionization Protein chips + MALDI-TOF based instrument Different chromatographic surfaces (e. g. anion exchanger, cation exchanger, reverse phase) 7 SELDI ProteinChip • Chemical Surfaces Hydrophobic Ionic IMAC-3 Mixed • Biochemical Surfaces Antibody DNA Enzyme Receptor Drug8 9 SELDI ProteinChip (Ciphergen) SELDI – surface enhanced laser desorption/ ionization Protein chips 10 SELDI 11 SELDI ProteinChip- suitable for biomarker discovery 12 Novel Proteomics Techniques 1. Protein Arrays ; SELDI protein chips (Ciphergen) 2. Multiple Dimensional Liquid Chromatography, MDLC 3. Detect Dynamic Changes in Tissue/Cells (1) Stable-Isotope Labeling (2) Isotope Coded Affinity Tags Technology, ICAT (3) Isobaric Tag for Relative and Absolute Quantitation, iTRAQ 4. Difference Gel Electrophoresis, DIGE 2. Multiple Dimensional Liquid Chromatography, MDLC 14 2. Multiple Dimensional Liquid Chromatography, MDLC 15 Novel Proteomics Techniques 1. Protein Arrays ; SELDI protein chips (Ciphergen) 2. Multiple Dimensional Liquid Chromatography, MDLC 3. Detect Dynamic Changes in Tissue/Cells (1) Stable-Isotope Labeling (2) Isotope Coded Affinity Tags Technology, ICAT (3) Isobaric Tag for Relative and Absolute Quantitation, iTRAQ 4. Difference Gel Electrophoresis, DIGE 5. Analysis of protein-protein interactions (1) Complex isolation - Coimmunoprecipitation ; Affinity purification (2) Finding partners - Yeast two-hybrid ; Phage display system 6. Post-translational modifications, PTM 3. Detection of dynamic changes in tissue/cells Stable-isotope labeling with amino acids in cell culture (SILAC) 17 Drawbacks: The method does not allow for the analysis of proteins directly from tissue. The stable-isotope enriched media are costly and may themselves affect cellular growth and protein production. The increase in nominal mass because of stableisotope incorporation is not known until the sequence is determined. 18 Isotope Coded Affinity Tags Technology, ICAT 19 Isotope Coded Affinity Tags Technology, ICAT 20 Isotope Coded Affinity Tags Technology, ICAT 21 Advantages: The method is compatible with any amount of protein harvested from bodily fluids, cells or tissues under any growth conditions. The alkylation reaction is highly specific and occurs in the presence of salts, detergents, and stabilizers (e.g. SDS, urea, guanidine-HCl). The complexity of the peptide mixture is reduced by isolating only cysteine-containing peptides. The ICAT strategy permits almost any type of biochemical, immunological, or physical fractionation, which makes it compatible with the analysis of low-abundance proteins. Disadvantages: The size of the ICAT label (~500 Da) is a relatively large modification. The method fails for proteins that contain no cysteines. 22 isobaric Tag for Relative and Absolute Quantitation , iTRAQ 23 isobaric Tag for Relative and Absolute Quantitation , iTRAQ 24 isobaric Tag for Relative and Absolute Quantitation , iTRAQ 25 isobaric Tag for Relative and Absolute Quantitation , iTRAQ 26 isobaric Tag for Relative and Absolute Quantitation , iTRAQ 27 isobaric Tag for Relative and Absolute Quantitation , iTRAQ Non-gel based technique Uses isotope coded covalent tags. Covalent labeling of the N-terminus and sidechain amines of pepitdes from protein digestions with tags of varying mass. Simultaneously identify and quantify proteins from different sources (multiple sample) in one single experiment. Increases confidence in identification and quantitation from MS/MS spectra by tagging multiple peptides per protein. Increases throughput and confidence in results for protein biomarker discovery studies. 28 isobaric Tag for Relative and Absolute Quantitation , iTRAQ Two mainly used reagents: 4-plex and 8-plex. Pooled and fractionated by nano liquid chromatography and analyzed by tandem mass spectrometry (MS/MS) Expands proteome coverage by labeling all peptides, including those with post-translational modifications (PTMs). Offers a simple workflow without sample fractionation for reduced-complexity samples, such as affinity pull-downs. 29 Novel Proteomics Techniques 1. Protein Arrays ; SELDI protein chips (Ciphergen) 2. Multiple Dimensional Liquid Chromatography, MDLC 3. Detect Dynamic Changes in Tissue/Cells (1) Stable-Isotope Labeling (2) Isotope Coded Affinity Tags Technology, ICAT (3) Isobaric Tag for Relative and Absolute Quantitation, iTRAQ 4. Difference Gel Electrophoresis, DIGE 4. Difference Gel Electrophoresis (DIGE) Allows the separation of treated (or diseased) and untreated (or control) samples in a single physical gel. Quick comparison in the differences of the protein profiles of each sample by overlaying the unwrapped maps of treated and untreated samples. It is possible to see which proteins are shared by both, which are present in one sample but not in the other. In a DIGE system, proteins are pre-labelled with fluorescent CyDyes™ such as Cy3, and Cy5 prior to electrophoretic separations. Labelled samples are then mixed before isoelectric focusing, and resolved on the same 2D gel. 31 4. Difference Gel Electrophoresis (DIGE) Key benefits: More confidence- reflects true biological outcomes and is not due to the technical variation Less gels- saves time by reducing the large number of replicates that are used in the conventional, single stain 2D gel method High accuracy- no false negative and no false positive Quantitative data 32 4. Difference Gel Electrophoresis (DIGE) Sample A pre-labelled with dye A Cy3 Cy5 Sample B pre-labelled with dye B Cy5 Cy3 Differential View Cy3/cy5 staining Cy5/Cy3 staining ... 1 Gel, 2 Samples, 2 Dyes: More Data Reliability with Higher Speed!! 33 4. Difference Gel Electrophoresis (DIGE) In a new DIGE system, proteins are pre-labelled with fluorescent CyDyes™ such as Cy2, Cy3, and Cy5 prior to electrophoretic separations. Labelled samples are then mixed before isoelectric focusing, and resolved on the same 2D gel. Cy2 dye is used to label an internal standard, which consists of a pooled sample comprising of equal amounts of each of the samples to be compared. This allows both inter and intra gel matching, and is used in the standardization of spot volumes in different gels. Spot volumes are expressed as a ratio to the internal standard. Images of each dye are acquired with various lasers using a variable mode imager and images are analyzed with 34 differential image analysis software. 4. Difference Gel Electrophoresis (DIGE) Cyanine Dyes (Cy2, Cy3, and Cy5) Fluorophore Absorption Peak (nm) Emission Peak (nm) Cyanine, Cy2 492 510 Fluorescein, FITC 492 520 Indocarbocyanine, Cy3 550 570 Tetramethyl Rhodamine, TRITC 550 570 Indodicarbocyanine, Cy5 650 670 35 4. Difference Gel Electrophoresis (DIGE) Cyanine Dyes (Cy2, Cy3, and Cy5) Excitation Emission 36 4. Difference Gel Electrophoresis (DIGE) IEF+SDS PAGE Control Cy3 (untreated) (Cy5) Mix Treated Sample Internal standard DeCyder DIA Cy5 (Cy3) Cy2 37 4. Difference Gel Electrophoresis (DIGE) 38 4. Difference Gel Electrophoresis (DIGE) 39 4. Difference Gel Electrophoresis (DIGE) 40