2. Electrophoretic separation of proteins by charge (isoelectric

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PROTEOMICS: LARGE-SCALE PROTEIN IDENTIFICATION & ANALYSIS. 2D-gel electrophoresis & mass spectrometry. 1. Isolate proteins from tissue (organism, condition…) of interest. 2. Electrophoretic separation of proteins by charge (isoelectric focusing) and by size (SDS-PAGE). - PowerPoint PPT Presentation

Text of 2. Electrophoretic separation of proteins by charge (isoelectric

  • 2. Electrophoretic separation of proteins by charge (isoelectricfocusing) and by size (SDS-PAGE)2D-gel electrophoresis & mass spectrometry 3. Peptide fragmentation of individual protein (with proteases eg trypsin)1. Isolate proteins from tissue (organism, condition) of interestFig. 6.10see Fig. 6.11PROTEOMICS: LARGE-SCALE PROTEIN IDENTIFICATION & ANALYSIS

  • 4. Determine precise peptide mass by MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometry5. Compare aa sequences to genomic data to correlateprotein with its gene Fig. 6.12

  • STRUCTURAL PROTEOMICS- large-scale determination of protein structuresStart with gene of interest cloning, expression, purification of protein- crystallize protein & X-ray diffraction analysisNMR spectroscopy - for small proteins or domains (in solution)Protein data bank: www.rcsb.org/pdb/X-ray crystallographyNov 2000 = 13,750 structuresNov 2001 = 16,550Nov 2006 = 40,132Nov 2008 = 54,559Nov 2009 = 61,418Nov 2011 = 76,814

  • How similar are hemoglobin a-chain and b-chain?hemoglobin tetramerAnalysis of protein sequences and structures

  • Identification of - protein motifs, catalytic centres- binding to ligands, drugs- interaction with other macromolecules- relatedness to other proteins (homology modelling)clues from protein sequence/structure about biological functionExPASy (Swiss Institute of Bioinformatics) www.expasy.org/EMBL-EBI (European Bioinformatics Institute) www.ebi.ac.uk/Tools/ppsearch/For example: www.ncbi.nlm.nih.gov/structure

  • How to find proteins that interact with protein of interest? 1. Phage display- generate phage library producing collection of fusion proteins between phage coat protein & test protein from genome of interest- hybrid protein will be displayed on outer surface of phageFig. 6.14- then screen library to find ones having expressed protein which interacts with test protein of interest

  • mRNAgeneTranscription factors have 2 domains DNA binding domainActivator domainRegulatory cis-element2. Yeast 2-hybrid systemIf TF domains physically separated, no transcriptionBackground info about transcription in eukaryotesBut if bait & prey interact to bring TF domains close together, then transcription occursmRNA

  • Determining protein-protein interactions using yeast 2-hybrid system- use separate vectors to prepare [1] bait fused to DNA binding domain of a yeast transcription factor- fuse (1) to gene for protein X = bait Fig. 6.15 [2] shotgun library of possible prey fused to activation domain of yeast TFprey generate library where (2) is fused to random coding sequences from organism of interest (eg. human)

  • - co-transform yeast cells (which lack this transcription factor TF)if protein X and prey (from library) interact, the 2 domains of yeast TF will be close together (& functional), so activate reporter geneFig. 6.15eg if use lacZ reporter gene blue colour of yeast colony

  • If bait protein does not interact directly with protein(s) in a complex, they may not be isolatedFig.6.18Fig.6.173. Affinity column chromatographyor use co-immunoprecipitation- protein B (bait) attached on column to fish out the protein (or proteins) which specifically bind to it(p.182)

  • If 2 genes are functionally-related, expect them to be co-inherited and may be physically close in genome (as well as co-inherited) Gene fusion/fission (2 short genes in some organisms vs. one long gene in others) 10 different organismsHarrington FEBS Lett. 582:1251, 20084. Computational approaches to predict protein-protein interactionsDo genes 1 and 2 (orange & green) pass this test? - premise that composite (naturally-fused) proteins have direct physical interaction (or functional association)

  • 3. Bioinformatics approach to predict protein-protein interactions- search for one large gene in organism X vs. two separate smaller genes in YFig.6.19in yeastin E.colihis2his10 HIS2 Search of complete genomes of E.coli, Haemophilus, Methanococcus & yeast- found 215 cases of fused vs. split stateEnright et al. Nature 402: 86, 1999

  • Fig. 6.20Yeast protein-protein interaction map (from experimental data)red dots = essential proteins (so knockout is lethal)green = non-lethal; orange = slow growth; yellow = unknown effect- lines connecting dots represent known protein- protein interactions 20012002colour-coded forbiological function

  • - triplet repeat (CAG) expansion disease (p.510)Protein interaction network in Huntingtons diseaseFigure 2. Protein Interaction Network for Huntingtons Disease Comprehensive PPI network for htt [huntingtin protein] Y2H interactors [35 bait and 51 prey proteins & verified in pull down assays], red diamonds; previously published interactors, blue squares; interactors identified from databases HRPD, MINT, and BIND, bridging any two proteins in the extended network, green triangles Htt interactors previously reported and recapitulated in our screens. Goehler et al. Mol Cell 15:853, 2004

  • Marcotte Nature 402:83, 1999www.yeastgenome.org

  • RNA-seqmicroarray profiling They double-checked some by RT-PCRPapers related to questions on 2d mid-term test

  • Hawrylycz et al. Nature Sept. 2012 RNA sequencing methods, which were cost-prohibitive and technologically immature when the project was initiated, hold great promise for elucidating [brain] transcriptional regulation in the future. a small number of high-quality, clinically unremarkable brains profiled with DNA microarrays for quantitative gene-level transcriptome coverageDopamine pathway genes Different parts of brain

  • Actin genes Transcriptional profiles of multi-gene family members (in different parts of human brain)Hawrylycz et al. Nature Sept. 2012different parts of braindifferent genes

  • Saha Haemophilia 17: e928, 2011 Factor VIII (F8) blood clotting geneExample of highly polymorphic human geneNumber of tandem repeats (GTGTGT) in intron 1 varies among populations in India3 of its introns have microsatellites which differ in copy number among individualsAllele frequency for microsatellite in intron 1TB: Tibeto-BurmanAA: Austro-Asiatic

  • In this study, we sequenced 99% of all three [such] unfinished gaps on human chr 20..The finished human genome-assemblies comprise several hundred un-sequenced euchromatic gaps, which may be rich in long polypurine/polypyrimidine stretches.

  • Douglas et al. G3:Genes Genomes Genet 2:1279-89, Oct. 2012Barcode tags used in gene overexpression experiments - similar in design to gene deletion experiments discussed in Topic 7- to track whether having too much protein X is lethal under certain growth conditions

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