Novel Proteomics Techniques 2018/4/28 蛋白質體學 Proteomics 2012 Novel Proteomics Techniques 陳威戎 2012. 12. 03

Novel Proteomics Techniques 2018/4/28 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 5. Analysis of protein-protein interactions (1) Complex isolation - Coimmunoprecipitation ; Affinity purification (2) Finding partners - Yeast two-hybrid ; Phage display system

Novel Proteomics Techniques 2018/4/28 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 5. Analysis of protein-protein interactions (1) Complex isolation - Coimmunoprecipitation ; Affinity purification (2) Finding partners - Yeast two-hybrid ; Phage display system

SELDI protein chip Surface Enchanced Laser Desorption / Ionization 2018/4/28 Surface Enchanced Laser Desorption / Ionization Protein chips + MALDI-TOF based instrument 表面 修飾 雷射 脫附 離子化 Different chromatographic surfaces (e. g. anion exchanger, cation exchanger, reverse phase)

SELDI Protein Chip Chemical Surfaces Biochemical Surfaces Hydrophobic Ionic IMAC-3 Mixed Biochemical Surfaces          Antibody DNA Enzyme Receptor Drug

SELDI Protein Chip (Ciphergen) 2018/4/28 SELDI Protein Chip (Ciphergen) SELDI – surface enhanced laser desorption/ ionization Five surface types of 8 array ProteinChips® The Protein Chip Arrays distinguish this technology from other mass spectrometry-based systems. The Arrays provide a variety of surface chemistries for researchers to optimize protein capture and analysis. The chemistries include classical chromatographic surfaces such as hydrophobic for reversed-phase capture, cation-and anion exchange surfaced, immobilized metal affinity capture (IMAC) for capturing metal-binding proteins, and pre-activated surfaces to investigate antibody-antigen, DNA-protein, receptor-ligand, etc. Protein chips

SELDI 2018/4/28

SELDI Protein Chip- suitable for biomarker discovery

SELDI-TOF, Biomarker Animation

Comparative MS profiles among patients and normal control

Comparative MS profiles among patients and normal control

Novel Proteomics Techniques 2018/4/28 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 5. Analysis of protein-protein interactions (1) Complex isolation - Coimmunoprecipitation ; Affinity purification (2) Finding partners - Yeast two-hybrid ; Phage display system

2. Multiple Dimensional Liquid Chromatography, MDLC

2. Multiple Dimensional Liquid Chromatography, MDLC

Novel Proteomics Techniques 2018/4/28 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 5. Analysis of protein-protein interactions (1) Complex isolation - Coimmunoprecipitation ; Affinity purification (2) Finding partners - Yeast two-hybrid ; Phage display system

3. Detection of dynamic changes in tissue/cells Stable-isotope labeling with amino acids in cell culture (SILAC)

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 stable-isotope incorporation is not known until the sequence is determined.

Isotope Coded Affinity Tags Technology, ICAT

Isotope Coded Affinity Tags Technology, ICAT

Isotope Coded Affinity Tags Technology, ICAT

Advantages: Disadvantages: 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.

isobaric Tag for Relative and Absolute Quantitation , iTRAQ

isobaric Tag for Relative and Absolute Quantitation , iTRAQ

isobaric Tag for Relative and Absolute Quantitation , iTRAQ

isobaric Tag for Relative and Absolute Quantitation , iTRAQ

isobaric Tag for Relative and Absolute Quantitation , iTRAQ

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.

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.

Novel Proteomics Techniques 2018/4/28 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 5. Analysis of protein-protein interactions (1) Complex isolation - Coimmunoprecipitation ; Affinity purification (2) Finding partners - Yeast two-hybrid ; Phage display system

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.

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

Fluorescence Difference Gel Electrophoresis Here is an outline of the 2D DIGE technique. Two or more protein samples are individually labelled with different fluors and mixed together prior to separation. The fluors currently used are Cy2, Cy3 and Cy5NHS ester. The samples are then separated by conventional IPG isoelectric focusing followed by SDS-PAGE. The individual samples can be viewed separately by using suitably optimised excitation and emission filters. The images can be analysed with normal 2D software.

Sample A pre-labelled with dye A Sample B pre-labelled with dye B 4. Difference Gel Electrophoresis (DIGE) Sample A pre-labelled with dye A Sample B pre-labelled with dye B Differential View Cy5 Cy3 Cy3/cy5 staining Cy5 Cy3 Cy5/Cy3 staining ... 1 Gel, 2 Samples, 2 Dyes: More Data Reliability with Higher Speed!!

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 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 520 Indocarbocyanine, Cy3 550 570 Tetramethyl Rhodamine, TRITC Indodicarbocyanine, Cy5 650 670

4. Difference Gel Electrophoresis (DIGE) Cyanine Dyes (Cy2, Cy3, and Cy5) Excitation Emission

4. Difference Gel Electrophoresis (DIGE) IEF+SDS PAGE Control （untreated） Cy3 （Cy5） Mix DeCyder DIA Treated Sample Cy5 （Cy3） Internal standard Cy2

4. Difference Gel Electrophoresis (DIGE)

4. Difference Gel Electrophoresis (DIGE)

4. Difference Gel Electrophoresis (DIGE)

Samples are labelled with different fluorescent tags, allowing 2 samples to be separated on a single gel, removing gel-to-gel variation. An internal standard, composed of all samples in the experiment, is run on each gel, allowing comparison of samples on different gels. DIGE comparison of depleted plasma Sample 1 – Cy3 Sample 2 – Cy5 Overlay Image

Novel Proteomics Techniques 2018/4/28 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 5. Analysis of protein-protein interactions (1) Complex isolation - Coimmunoprecipitation ; Affinity purification (2) Finding partners - Yeast two-hybrid ; Phage display system

Analysis of protein-protein interactions Complex Isolation • Affinity purification – requires soluble, active protein • Co-immunoprecipitation – requires antibodies Finding Partners • Phage Display – interactions occur in solution • Yeast Two-Hybrid – Interactions occur in vivo

Complex isolation

High-throughput Mass Spectrometric Protein Complex Identification (HMS-PCI) 2018/4/28 Mike Tyers, SLRI Ste12 Ho et al. Nature. 2002 Jan 10;415(6868):180-3

Yeast Two-Hybrid System (in vivo) Interaction of bait and prey proteins localizes the activation domain to the reporter gene, thus activating transcription. Since the reporter gene typically codes for a survival factor, yeast colonies will grow only when an interaction occurs. Activation Domain Prey Protein Reporter mRNA Bait Protein Reporter mRNA Reporter mRNA Binding Domain Reporter mRNA Reporter mRNA Reporter Gene

Yeast Two Hybrid Assay The two-hybrid system is a molecular genetic tool which facilitates the study of protein-protein interactions. If two proteins interact, then a reporter gene is transcriptionally activated. e.g. gal1-lacZ - the beta-galactosidase gene A colour reaction can be seen on specific media. You can use this to Study the interaction between two proteins which you expect to interact Find proteins (prey) which interact with a protein you have already (bait).

Yeast Two Hybrid Assay

Yeast Two-Hybrid assay 2018/4/28 SNF4 1. B SNF1 A 3. 2. GAL4-DBD Transcription activation domain UASG 4. Fields S. Song O. Nature. 1989 Jul 20;340(6230):245-6. PMID: 2547163 GAL1 Allows growth on galactose

Phage display system (in vitro) 2018/4/28 Phage display system (in vitro) Biopanning SCIENCE VOL 298 18 OCTOBER 2002 James F. Smothers, Steven Henikoff, Paul Carter Smith first demonstrated this technique by genetically inserting a fragment of the bacterial enzyme Eco RI into the pIII coat protein of M13. He then used an antibody to the enzyme to enrich (or preferentially select) for the enzymedisplaying phage so that it was 1000 times more abundant than wild-type phage (1). Moreover, these mutated phage were still infectious and capable of passing along their genetic information to their progeny. Soon thereafter, Smith and colleagues constructed the first library of phage in which pIII was fused to random peptide sequences (2). An antibody was again used as the interacting target protein, but this time it selected from ~107 competing peptides, each of which could potentially form a specific complex with the antibody. After successive rounds of enrichment, several peptides representing the antibody’s interaction site (epitope) were recovered. Jim Wells and colleagues advanced the technology by displaying two different human growth hormone variants on phage and selecting for the variant with higher binding affinity for the hormone receptor (3). of fusion peptides and proteins, which also improves their chances of finding selective interactions. Proteins can also be fused to for next cycle ex unbound phage un Elute bound phage and infect host Sequence target binders Phage minor coat protein SCIENCE VOL 298 18 OCTOBER 2002

Applications for Phage display system

2018/4/28

Interaction/Pathway Databases 2018/4/28 Arguably the most accessible data source, but... Varied formats, representation, coverage Pathway data extremely difficult to combine and use Pathway Resource List (http://cbio.mskcc.org/prl/)

http://bind.ca A free, open-source database for archiving and exchanging molecular assembly information. BIND is managed by the Blueprint Initiative at Mount Sinai Hospital in Toronto. The database contains Interactions/Reactions Molecular complexes Pathways BIND has an extensive data model, GNU software tools and is based on the NCBI toolkit; extended recently to XML/Java The ~175000 BIND records are curated and validated. Bader GD, Betel D, Hogue CW. (2003) BIND: the Biomolecular Interaction Network Database. Nucleic Acids Res. 31(1):248-50 PMID: 12519993

BIND Interaction Types