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by: Kavon Huel


Kavon Huel
GPA 3.59


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Class Notes
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This 44 page Class Notes was uploaded by Kavon Huel on Friday September 18, 2015. The Class Notes belongs to BCH 6107 at University of Florida taught by Staff in Fall. Since its upload, it has received 8 views. For similar materials see /class/206963/bch-6107-university-of-florida in Biochemistry and Molecular Biology at University of Florida.

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Date Created: 09/18/15
A Proteomics I 2DGels V Optimizing Your 2D Gel Better sample preparation 0 Solublization 0 Sample cleanup quick cleanup kits 0 Simpli cation l Subcellular Fractionation l Affinity Column Enrichment Better gel reproducibility o Immobilized pH gradients o Precast gels Better Detection Methods 0 High sensitivityspeci city stains 0 Differential lnGel Electrophoresis 39Extractjom Urea vs UreaThiourea Rat Liver 8 M urea 7 M urea 2 M thiourea Salt Effects E Coli Extract No San 30 mM NaCI Keys to Protein Detection Compatibility with SDSPAGE Detection threshold I Dynamic range ofdetection Linear range of detection I Reproducibility 0 From geltogel o Interprotein variability Compatibility with downstream analysis 0 Microsequencing 0 Mass Spectrometry Organic Protein Dyes I Coomassie Brilliant Blue Most Common 0 Triphenylmethane family 0 Anionic dye used in acidic media I Fix Protein gt Stain gt Destain I Detection limits 1pg protein 0 Not really sensitive enough for proteomic analysis 0 Less sensitive to glycoproteins amp extreme pls o Destain introduces variability I Colloidal Coomassie Brilliant BlueG 0 Forms microprecipitates in ammonium sulfate 0 Significant increase in sensitivity 20 ng protein Metal Reduction Silver Stain I Incompatible with SDS chloride 0 Fix proteins a Remove Salt a Stain a No Elution Sensitize the gel 0 Sensitizers bind to proteins 0 Sensitizers also bind to or react with Ag2 Impregnate gel with AgNO3 or AmmoniaAg2 Develop o Formaldehyde in alkaline carbonate for AgNO3 o Formaldehyde in weak acid for AmmoniaAg2 Metal Reduction Silver Stain Sensitivity 15 ng protein Linear Range 10fold I Not compatible with downstream analysis Less abundant proteins can be overdetected Lots of silver is not always fun to handle Homemade stains will typically be more sensitive due to short reagent halflife Fluorescent Dyes I Environment sensitive fluorescence o Weakly uorescent in water 0 Highly uorescent in apolar media detergents I Higher sensitivity 0 SYPRO Orange Red Molecular Probes 48ng o SYPRO Ruby Molecular Probes 1ng o DeepPurple GE Healthcare lt1ng o Krypton Pierce Biochem 025ng I Linear range over 3 orders of magnitude I and Limited shelflife Selective Fluorescent Stains I Made by lnvitrogen I ProQ Diamond Stains Phosphoproteins I ProQ Emerald Stains Glycoproteins I ProQ Amber Stains Transmembrane Prot I ProQ Sapphire Stains Polyhistidine Prot I Tend to be very touchy stains 2D Gel Databases I Now that you have your stained gel Many online databases with annotated images of stained 2D gels Tend to be silver stained gels Not all spots are annotated Usually sorted by sample source liver heart Increased reproducibility has greatly aided the utility of these databases 2D Differential InGel Electrophoresis System devised by GE Healthcare Covalent labeling ofdifferent samples with different uorescent dyes Mix samples after labeling Run normal 2D lsoDalt gel I Use imager Typhoon to read uorescence Use computer software to analyze data Minimal Labeling Dyes H3N iprotein 0 pH 85 y 7 o ON quot 5 0 protein 0 NHS reactive group I Amino group of Lysine is labeled 12protein I Dye is charge matched to preserve pl I All dyes are weight matched Cy2Cy3Cy5 Courtesy of G E Healthcare Saturation Dyes s TCEP 1 h 37 C r 7 7 pH 80 r 37 C 30 min 0 o uk pH 80 O umN o S Maleimide reactive group 0 Courtesy of G E Healthcare CyDyes vs Silver Stain E coli lysate 50ug loading pH 47 u in 4 sum Cy5 Labeled Sample Silver Stained Gel Courtesy of GE Health care 2D DIGE Procedure Normal Sample lnternalStindard Diseased Sample Label amp c 2 mix cya 39039 y 39039 cy5 extracts 2 l 2DE separation 39quotSigfy i zl i quot I I I Li M w Courtesy of GE Health care The Internal Standard Sample 2 Cy5 Standard CyT39V392 Sample 1 Cy3 Gel A H 6 Ci Standard Cy2 Sample 3 Cy3 Sample 4 Cy5 Gel B lt9 t Without internal standard With internal standard Conclusion expression decrease in sample 3 Counesy of GE Healthcare InterGel Spot Normalization Eravh View Master Na 27 Eravh View Master Na 27 Abundan e Slan dard lsed Lng Comm rrealeqa Not normalized to standard Normalized to standard Counesy of GE Healthcare What Does 2D DIGE Give You I High sensitivity o 0251 ng protein Minimal Dyes 0 As low as 001 ng for BSA Saturation Dyes Linear range to 35 orders of magnitude Ability to easily overlaycompare gels Standardized intergel quantitative analysis Compatibility with downstream analysis 0 Microsequencing 0 Mass Spectrometry MultiDimentional Gel Electrophoresis What s Wrong With Just 1D Comigration of Protein How many proteins in a cell weigh 50 kDa How many proteins in a cell have a pl64 Picking Your Dimensions 2D Separation based on similar properties will generate a diagonal line Most common choice is EFSDSPAGE Other choices include o IEFNative PAGE 0 2D SDSPAGE Caspase cleavage 0 3D gels lsoDalt The 2D Gel 1St Dimension SCAlEFlPGIEF 0 Tube gels lt1 mm 5 cm diameter 0 Slab gels slice strips after gel run 2nd Dimension SDS PAGE 0 Always slab gel Mol weight amp pl are intrinsic independent physical properties of proteins Cannot invert order of separation as SDS is not compatible with IEF The lsoDalt Protocol Perform isoelectric separation Equilibrate IEF striptube 0 SDS Denaturecoat proteins 0 Buffer pH68 Replaces SCA in SCAIEF o Bromophenol Blue Loading Dye Apply equilibrated strip to top of SDS gel 0 Uniform or gradient acrylamide gel 0 No stacking gel needed for small diam IEF Perform SDSPAGE Sample Preparation Small imperfections in sample can lead to small imperfections in protein separations Small imperfections are amplified with every additional separation dimension Samples must be compatible with the conditions of each separation step 0 Equilibration steps 0 Enzymatic treatments Bustin Disulfides BMercaptoethanol 15 0 Can ionize at basic pH 0 Ionized BME will ruin pH gradient for IEF DTT 50mM 0 Less prone to ionization than BME 0 Not as effective at breaking disulfide bridges Phosphines Most effective 0 TBP very toxic insoluble o Triscarboxyethylphosphine I Soluble in water I Compatible with acrylamide polymerization Sample Denaturation Chaotropes o Urea soluble to 8M 9M in H20 0 Thiourea soluble to 1M in H20 0 Urea Thiourea5M 8M 2 M Detergents o Tween weak gt SDS strong 0 No net charges allowed SDS is out o No long chain hydrophobics complex w urea o Tritons NP40 CHAPS nOctylglucoside Carbamylation Urea H NH4OCN39 0 Free Cyanate reacts with 1 amines Lys 0 Neutralizes charges shifts pl 0 Increase in Temp pH shifts Eq to more ON Fixes 0 Use very pure urea 0 Never heat solutions over 37 C 0 Freeze urea stock solutions at 20 C 0 Include cyanate scavengers 1 aminesSCA Protease Contamination Proteases not always denatured easily 0 Recorded protease activity in 9M urea 0 Recorded protease activity in SDS Fixes o Protease lnh Can modify other proteins bad 0 Use absolutely strongest denaturant possible I UreaThiourea I Boiling SDS 0 Solubilize at high pH 7 Mcleic AcidPolysaccharide Polyanions o Bind to proteins and ampholytes 0 Can clog pores of acrylamide gels 0 Causes major streaking on 2D gels Fixes o Nuclease columns Chew it up 0 Hydroxyapatite columns Take it out o CsClCsZSO4 gradients Spin it down V Nucleic Acid Effects T RabilluudKM h u q vulemsin nnn I ts amp Lipids Salts Lipids I Inten eres with IEF I Can cause precipitation I Deadens pH gradient 0f hYdFOPhObiC PrOteinS at the Edges I Causes protein loss I Causes unfocusing andor streaking I Fixes I Fixes o Dialysis o Increasedetergent 0 Protein precipitation PFOtein PreCiPitation V 7 Salt Effects r RabiiiuudKM h u q ruiemsm nnn A PostTranslational Modifications V PTM Comes in Many Flavors Phosphorylation Glycosylation AcetylationMethylation Lipoylation Myristoylationlsoprenylation Proteolytic cleavage Ubiquitinylation PhosphoProblems I Low abundance relative to nonPO4 I PhosphatasesKinases in protein extracts I Misbehave in 2D gels I Misbehave in RPHPLC Can inhibit local trypsin cleavage I Can alter ionization efficiency in MS I Phosphates are very labile in ClDPSD Phospho Enrichment I AntiPhosphoSerPhosphoThrPhosphoTyr o Immobilized antibody af nity columns 0 Enhances signal simpli es sample composition I lMACFet3Ga3 o Phosphopeptides have high af nity for Fequot3Ga3 o Enhances signal simpli es sample composition I BEliminationBiotinylation o Removes PO4 better behavior 0 Replaces with Biotintag af nity enrichment I Tandem LC with SCXRP I Commercial Enrichment Kits Phosphate Removal Phosphatase No analytical benefit BEHn na on o Generates dehydroalanine amp dehydrothronine 0 These products are reactive will Xlinkadsorb BEliminationMass Tagging 0 Done after or without enrichment o RXN with 24pyridylethanethiol 0 Characteristic mass loss can also isotope label 3EliminationBiotinylation Allows enrichment of phosphopeptides 0 Mass spectra can be scanned for loss of biotin group O 3Elimination I Tagging lat40H2 CH gt lt3 gt CH CH2 cHz CHZ I I o S s t o r 0 T cHz I o CHI 1342 I06 31TH fl 6 N N M Quadrum Pvuleume Research Masssmcuumelm mm Phosphoprotein Detection I AntiPO4 protein western blots2D blots o MALDlTOF analysis of phosphospot Protein ID 0 Nonspecific antibodies 0 Signal intensity problems abundanceionization ProQ Diamond Stain PO4 sensitive stain o MALDlTOF analysis of phosphospot Protein ID 0 Stains are not perfect nonspecific not omniscient 0 Signal intensity problems abundanceionization I Mass Spectrometry MS 0 PrecursorIon Scanning o NeutralLoss Scanning PrecursorIon Scanning TripleQuadrupole is best suited for analysis Q1 is scanned Q3 is set to transmit one mz CID is still carried out in Q2 Q3 mz is characteristic loss for a PTM For phosphopeptides o mz 79 PO339 o mz 63 PO239 0 Q3 must be run in Negative ion mode I When target mz is detected peptide can be resampled for further MSn analysis NeutralLoss Scanning I Similar in concept to precursorion scanning Some losses in CID generate neutral ions Neutral ions will not appear on spectrum Again TripleQuad is best suited Q1 amp Q3 are scanned offset by defined mz Only those peptides with neutral loss of defined mz will be transmitted to detector For phosphopeptides mz 98 for H3PO4 Glycoprotein Analysis Glycosylated proteins don t behave ideally in most types of separations Gel LC Low abundance of modi ed protein Many proteins are multiply glycosylated Heterogeneity complicates analysis 0 Variations in extent glycosylationsialylation o Resultant heterogeneity in chargeionization Significant suppression is observed in MALDI Glyco Enrichment Affinity chromatography 0 maminophenylboronic acid likes glycans o Lectin columns Conconavalin A Wheat Germ Periodate OxidationBiotinylation SolidPhase Extraction of Glycoproteins SPEG Subcellular fractionation o Membrane bound associated o Integral membrane proteins 0 Secreted proteins Biotin Labeling of Glycans Biotin Labeled Glycan Glyoan Periodate Oxidized Glycan 0 o Periodate 0 am Hy mzide D H C H lt gt 8 g k l l 7 NH NH HNKNH l l OH OH 0 o o c c o H y H l l k l o Hc Hzc squotmmzomzmzmAalsz l l CH1 CH2 l l Hao l l 9H 0er H 9 i H N H H N lt 5 s gt N N H H H H W NWSlgmaaldrlCh com SPEG Proceedure W W Siam oxidation Step3 proteolysis NW Step2 coupling removal of non M glycosylated peptides M MM 0 a I r m 39 E Zang andAebersuld Methul arm Gunman 177485 Glycan Removal I Chemical Removal 0 Alkaline Belimination OIinkage preferred 0 Periodate Oxidation No linkage preference 0 Trifluoromethanesulfonic acid Destroys glycan o Hydrazinolysis Can damage protein I Enzymatic Removal 0 Protein Nglycosidase F PNGF Nlinkage only 0 Lots of other more specific glycosidases Glycoprotein Detection I Chemicalenzymatic Glycan Removal 0 2D gel LC analysis before amp after 0 MALDlTOF of eXglycoprotein Protein ID I ProQ Emerald Glycoprotein Stain 0 Some nonspecific staining not omniscient o MALDlTOF of eXglycoprotein Protein ID I Periodate OxidationBiotinylation 0 Run 2D gel oftreated sample 0 Use an avidin conjugate detection system I Mass Spectrometry MSn lon Scanning Analysis of Released Glycan I Normal phase HPLC I NPRP Tandem HPLC I Chemicalenzymatic glycan sequencing I Mass spectrometry o Requires more sample 0 Must operate in both and ion modes MS Ion Scanning Typically using precursorion scanning NAcetylglucosamine mz 204 Gluc Gluc NAcetyl mz 366 Sialylated glycopeptides mz 292 Still have all the dif culties in isolating and ionizing a glycopeptide Other PTM Proteolysis 2D SDSPAGE Stable modi cations are detected in 2nd D MS 0 Some Alkylations o Ubiquitinylation residual ubiquitin peptide Labile modi cations can be detected in MSn by precursorion or neutralloss scanning o Sulfonation o Nitrosylation Specific PTM elimination followed by labeling is also common for dif cult cases A Mass Spectrometry V What is Mass Spectrometry Mass Spectrometry MS The generation of gaseous ions from a sample separation of these ions by masstocharge ratio and measurement of relative abundance MasstoCharge mz 0 Mass in Uni ed Atomic Mass Units u Dalton Da 0 The amu is now officially frowned upon Molecular Ion M1 0 Results from loss of single electron mz m Resolving Power mAm Resolution Am at a given m de ned in ppm The Output Called Mass Spectrum Peak height is proportional to ion abundance Peak clusters result from isotope effects 0 Cluster distribution should follow isotope abundance 255 an 1314 quot13 629 mm 39 I l ii if i i Rziawe Abundance mi 5434 Hm I 9253 my I am mu man man The Anatomy of a MassSpec Sample Inlet Introduce analyte Ionization Source Generate charged moI Ion Accelerator Mass Analyzer Masscharge separation Detector Sample Ionization Source Mass Analyzer Detector Inlet A Ion ccelerator 1 i j v Ionization Sources I Electron Ionization Chemical Ionization Atmospheric Pressure Chemical Ionization Electrospray Ionization Desorption Ionization 0 Laser Desorption o MatrixAssisted Laser Desorption 0 Fast Atom Bombardment Ion Acceleration Newly formed ions are accelerated into vacuum by high voltage application 0 Most protein applications voltage is negative 0 For specialized work voltage is positive Kinetic Energy KE imparted by voltage 0 KE is constant for all 1 charges in same voltage 0 Translational KE 12mv2 o SoSmall m Big v amp Big m Small v v22Vm Mass Analyzers I Magnetic Sector Timeof Flight Quadrupole Octapole lon Trap 0 Quadrupole Ion Trap 0 Fourier Transform IonCyclotron Resonance Orbit Trap Sector Mass Analyzer SingleFocus MS One mz at a time Ions are accelerated into a curved path through a homogenous magnetic eld B Magnet attractive force Bzv Centrifugal force on ion mv2r To avoid annihilation on tube wall Bzvmv2r mz Bzrz 2V By scanning B the entire spectrum of mz is obtained sequentially Sector MS Schematic mgmmh l i Hi lit ii iii VI in bi v u it lill rhldiill dmmm uh f mm H q 1 pump 7 illwir39mliir r Lllc 1m mmpk i mum TimeOfFlight Mass Analyzer l Uses a pulse of ion mixtures not steady stream Ions accelerated into drift tube by a pulsed electric field called the ionextraction fied Drift Tube is usually 12 m long under vacuum Ions traverse the drift tube at different speeds Ltv22Vm TOF Reflectronllon Mirror Heterogeneity in starting positionvelocity of ions reduces resolution Set of charged plates that reverses travel direction Faster ions penetrate farther c Slower ions penetrate less All ions of same mz are accelerated the same Net effect focuses signal and increases resolution a a cl Quadrupole Mass Analyzer Electric not magnetic field separates ions Two pairs of rods connected to both a DC and RF voltage Oscillating RF voltages are 180 out of phase Ions are subjected to complex forces 0 Forward motion isnot affected by fields 0 Sum of forces generates corkscrevxl forward path Only specific mz ratio is allowed to pass Scanning RF frequency scan mz ratios Quadrupole Schematic Ion Trap Mass Analyzer Electricmagnetic fields trap store eject ions Requires an inIine quadrupole to act as mass prefilter Contains a single ring electrode and a top and bottom cap electrode Varying RF frequency will vary the mz ratios that are trapped Additional fragmentation can be performed on ions stored in the ion trap Orbit Trap Mass Analyzer API Ion Source LTEl Mass Analyzer 39 39H I 7 A r 1fl39l T r r r T a I39 11 I quot l39ll fj I I 1 l Dvllilmp x 91 Z x isy I Detectors Electron Multipliers EM 0 DiscreteDynode EM 0 ContinuousDynode EM Faraday Cup Microchannel Plate Array Detector Electron Multiplier Most common detector Similar to Photomultiplier Tube or PMT Reactive surface that emits electrons when struck by high velocity particle Multiple surfaces are used in sequence amplifying the initial response Degree of amplification is called the gain Signal is proportional to impact energy incidence angle and particle type Electron Multiplier Diagrams High Voltage Typical Hnnlulp High quotwage Slgnal 0m Faraday Cup Least expensive detector Metal or Carbon cup Captured ions transfer charge to cup Current generated is directly proportional to number of ions and number of chargesion o No mass discrimination 0 Low sensitivity Faraday cups can be used to calibrate other MS detectors Microchannel Plate Detectors Spatially resolved array of EM channels I Not used as frequently yet Allows 3 D analysis of data 0 Intensity 0 Time 0 Space CHANNEL CHANNEL OUTPUT WALL EECTRODE STRIP CURRENT m VD INPUT ELECTRON a INPUT SIDE ELECTRODE Copyright Del Mar Ventures Sector MS Using MCP undergraauazemazmmemaAnaysysmen Rubmsun SkeHv39FvamE KFvame m5 A 2D1 Ggel Mass Spectrometry V Pick a Spot Any Spot l 2D gel analysis generates spots of pure protein Visualize by stain l Pick by hand Robotic spot picking 0 True Omics technology Highthroughput en masse 0 Can be user directed or automated o Who Cares About MW Mass spectrometric analysis on pure whole protein will generate o Accurate molecular mass 0 Isotope distribution SDSPAGE gives MW to 1030 accuracy Does a MWto 1Da and isotope distribution assist in protein identi cation Peptide Mass Fingerprinting Specific limited fragmentation of pure protein 0 Enzymatic Trypsin Endopeptidase LysC AspN 0 Chemical CyanogenBr Met NBS Trp Cleanup of resultant peptide mixture Mass Spec MALDlTOF of peptides Spectrum gives unique peptide fingerprint Bioinformatics takes over 0 Fingerprint databases 0 Theoretical in silico protein digestion More Omics InGel Proteolytic Digestion Simplest procedure is to digest ingel Trypsin digestion is most common Small 23 kDa Easily diffuses into gel High specificity Cterm of R K except XPro Resistant to buffer salts chaotropes Pure protein is readily available Generates 5002500 kDa fragments Peptide Mass Fingerprint 2256 Dana Cytolysin III Tryptic Digest 779 4555 1777 Emu 7924363 Post Source Decay MALDI ain t so soft after all FivePoint Palm Exploding Heart Technique After acceleration ions collide with matrixgas Collisions generate subpeptide fragments Fragments have same vas parent different KE Since TOF uses velocity as indicator of mass Use ion gatequot as mass filter Use gridded reflector to spread fragments PSD Reflection amp Detection MALDITOF PSD Instrument 100 smail PSD mgmems iarge area MOP detector us cm2 s1A sample a es gt U s 32 1 beam blanking I Lance ngu ii i r i r via mm 111511 mm 50 11 i i W 03 large stable precursor ions PSD fragments 39 LEN I 39 n quot AH m 1 39 i r U u2 NJ HZIi i l mp mf Peptide Fragments Fragmentation most often at peptide bond Generates series of b and y ions Other minor fragments can occur PostTransl Mod are usually lost Not ID d 2 1 y y R1 0 R2 0 R3 0 I II H2N c C N c C 1 N c 0 COOH H H H H H 2 b1 b2 Alternative Fragmentation mum mum R mum Fraumcnlmiun N larmma R NH HNcum nanccozn n R Iquot Hmrcnnrcam Handle cog M 3 R n HQNicHR CNH1 ocu g cozn M PSD Peptide Sequencing F IKTNP EPILVDT TGS a i 3 i i 5 i i 3 a g ero bz b7er Ym 2 b 5J1 7 l biHO 5 yx y in S 2 I 1 x mu EDD Bun mun nun Mun mun mun mun m Molecular Scanning I New approach not quite perfected yet Whole 2D gel analysis Tryptic digest of entire 2D gel 0 Parallel lnGel Digest PIGD o OneStep DigestionTransfer OSDT 0 Double Parallel Digestion DPD Transferto PVDF membrane Direct analysis of membrane by MALDITOF o Membrane adhered to plate with vaccuum grease 0 Matrix deposited on membrane 0 Largest membrane analyzed 4 cm2 Molecular Scanning Proteomics equivalent to microarray ln orderto catch up More complete digestion More complete transfer to PVDF Better visualization of peptides on PVDF Better resolution In gel amp In MS Larger MALDI platforms More automation of each step I When optimized has potential to increase highthroughput protein identi cation 10X 0 00000


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