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UMD CMSC 838T - CMSC 838T Lecture 12

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1CMSC 838T – Lecture 12CMSC 838T – Lecture 12X Proteomics0 Study of the proteomeX Methods0 2D electrophoresis0 Mass spectrometry0 Protein arrays &microarraysCiphergen ProteinChipAutoloaderCiphergen Protein ArrayCMSC 838T – Lecture 12ProteomicsX The study of the proteome (all proteins) of an organism0 What proteins are present?0 What is the 3D structure of each protein?0 How do proteins interact (networks)?0 How do proteins correlate with DNA?X The proteome is dynamic0 Varies with time, in response to environment0 Proteins are very cell / tissue-specificX The proteome is large0 100,000+ human proteins (vs. 30,000+ genes)0 Due to alternative splicing, post-translation modifications2CMSC 838T – Lecture 12Proteomics – Experimental MethodsX 2D electrophoresis0 Separating expressed proteinsX Mass spectroscopy0 Identifying expressed proteinsX Protein arrays / microarrays0 Identifying expressed proteins & interactionsX Other techniques0 X-ray crystallography0 NMR spectroscopy0 Edman degradation0 Etc…CMSC 838T – Lecture 12ProteomicsX Overview0 2D electrophoresis0 Mass spectrometry0 Protein arrays / microarrays3CMSC 838T – Lecture 12Proteomics – 2D ElectrophoresisX Property of proteins0 Some amino acids are acidic / basic (donate / accept H+)0 Collection of amino acids in protein determines its pI valueO pI = pH at which molecular charge = zeroX 2D electrophoresis0 Separate proteins according to both pI & molecular weightCMSC 838T – Lecture 12Proteomics – 2D ElectrophoresisX Method1. Extract & prepare protein sample in solution2. Separate proteins (in each dimension)I. Based on pHX Using isoelectric focusing (IEF)X Using immobilized pH gradient (IPG) stripsII. Based on molecular weight (size)X Using gel electrophoresis3. Stain proteins to enable visualizationsize →pH →proteinspH →proteins4CMSC 838T – Lecture 12Proteomics – 2D ElectrophoresisCMSC 838T – Lecture 12Proteomics – 2D Electrophoresis2D PAGE (polyacrylamide gel electrophoresis) exampleshuman liverhuman kidneypH →size →5CMSC 838T – Lecture 12Proteomics – 2D ElectrophoresisX Observations0 Able to separate proteins with pI around 0.0025 pH units0 Relative amounts of protein quantified by intensity0 Provides clear pattern of protein expression0 Comparisons can identify protein expression differences0 Good for identifying novel proteinsX Limitations0 Unable to identify all proteins based on pI / size alone0 Cannot handle extremely acidic / basic proteins0 Misses some large proteins & membrane proteins0 Limited reproducibility of 2D gels0 Relatively slow, expensive processCMSC 838T – Lecture 12ProteomicsX Overview0 2D electrophoresis0 Mass spectrometry0 Protein arrays / microarrays6CMSC 838T – Lecture 12Proteomics – Mass Spectrometry (MS)X Mass spectrometer0 Particles are 1. Ionized (charge added) 2. Accelerated through magnetic fieldX Path taken is function of mass / charge ratio3. Vary magnetic field4. Record quantity of particles for different m/z ratiosparticles magnetic fieldionized particleno chargechargeddetectorCMSC 838T – Lecture 12Mass Spectrometer7CMSC 838T – Lecture 12Proteomics – Mass SpectrometryX Goal0 Identify proteins separated by 2D electrophoresisX Method1. Excise individual dots from 2D electrophoresis2. Digest protein into fragments with enzyme (e.g., trypsin)3. Ionize protein fragments (without breaking)• Matrix Assisted Laser Desorption Ionization (MALDI)• Electrospray Ionization (ESI)4. Accelerate through mass spectrometer5. Produces peptide mass fingerprintmass / charge →intensity →fragmentsCMSC 838T – Lecture 12Proteomics – Mass SpectrometryX Peptide mass fingerprint8CMSC 838T – Lecture 12Proteomics – Mass SpectrometryX Identifying peptide mass fingerprint0 Compare withO Fingerprint for actual protein in databaseO Predicted fingerprint for predicted / hypothetical proteinX Precompute predicted fingerprints for efficiency0 May fail to distinguishO Post-translation modifications to proteinX Protein databases / web servers (e.g., SWISS-2D PAGE)0 For each protein, record itsO Protein pI, molecular weight, peptide mass fingerprint…O Experimentally determined location in 2D gel0 Tools to computationally predict same informationCMSC 838T – Lecture 12ProteinPeptidesEnzymatic CleavageMS (ESI-MS/MALDI)Mass SelectedMS/MStranslateA Non-redundantProtein SequenceDatabase Computer DerivedPeptide Fingerprintin silico digest computer-derived cleavage patternn peptidesn computer-derived MS/MS spectraCompareCompare eachProtein IDPeptide fragment fingerprint - size aloneMS/MS - actualpeptide sequenceGENOME-LANDIdentifying Peptide Mass Fingerprints9CMSC 838T – Lecture 12H2NNNNOHR1OHR2OHR3OHR4OxyzabcResidue MassH+H2NR2O+R1Ob2 ion +H3NR3ONHR4OOHy2 ionHN+H3CHHImmonium IonCharge retained on N terminusb ion seriesCharge retained on C terminusy ion seriesPeptide FragmentationCMSC 838T – Lecture 12Proteomics – Mass SpectrometryX Observations0 Ionization methods (MALDI, ESI)O Developed in 1980’s, significant improvementO Combine with capillary electrophoresis for high throughput0 Very precise measure of mass / charge ratios (5 ppm)0 Extremely sensitive (can detect < 10-18moles of protein)X Tandem mass spectrometry (MS-MS)0 Feed output of mass spectrometer to 2ndmass spectrometer0 Increases precision of measurements10CMSC 838T – Lecture 12ProteomicsX Overview0 2D electrophoresis0 Mass spectrometry0 Protein arrays / microarrays CMSC 838T – Lecture 12Proteomics – Protein Arrays / MicroarraysX Goal0 High-throughput analysis of protein expression / interaction0 Adapt approach similar to DNA microarrays0 Improves on speed vs. 2D electrophoresisX Approach0 No equivalent of hybridization for proteins0 Exploit other biochemical binding reactionsO Antibody–antigenO Receptor–ligandO DNA–protein…11CMSC 838T – Lecture 12Proteomics – Antibody / Antigen Bindingbinding sitesCMSC 838T – Lecture 12Proteomics – Protein Arrays / MicroarraysX Method1. Place on glass slide many probes at known locationsO Chemical probesX Ionic, hydrophobic, hydrophilic…O Biochemical probesX Antibody, receptor, DNA…2. Mix protein samples with probes, bind3. Wash off remaining proteins4. Collect & identify bound proteins with mass spectrometerO Surface Enhanced Laser Desorption / Ionization (SELDI) X Produces0 Protein expression profile0 Protein interaction with probes12CMSC 838T – Lecture 12Protein Microarrays – Antibody ProbesX Using antibody probes0 Antibodies


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