PROTEOMICSPROTN SCI BCH 6107
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This 9 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 4 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
Tandem Analysis LCquotMSn V Bells amp Whistles For What Simplicity Sensitivity Selectivity Simplicity I LCMS has a practical peptide maximum 0 Too many proteinspeptides swamps the analyzer 0 At best mixtures of 400500 peptides are workable Fractionating samples increases Simplicity o Leads to higher overall peptide analysis limit 0 Generates better certainty in protein ID I PreTrypsin subcellular SEC af nity PostTrypsin CXAX RPLC af nity ICAT o LCLCMSn Sensitivity Tandem LC allows better peptide separation o Reveals lower abundance peptides 0 Better protein ID sensitivity Affinity puri cation enriches sample 0 Low abundance proteins become concentrated o More sensitive and selective protein ID Tandem MS or MS provides more data 0 Peptide fragmentation data generates sequence info 0 Peptide sequence gives better protein ID with fewer usable peptide peaks Collision Induced Decay I How do we get more info out of MS 1D Mass Spectrometry 0 Protein Mass 0 Tryptic Peptide Masses Peptide Mass Fingerprint I Collision Induced Decay CID 0 Introduction of large inert neutral atoms Ar Xe etc PeptideAtom collisions induce peptide fragmentation Peptides typically fragment along peptide bond Forms series of b amp y ions 0 O O 2ndD Mass Spectrometry b amp y ion mass b amp y lon Review Same as those seen in MALDI PSD b ions retain amino terminus y ions retain carboxy terminus 2 1 y y R1 0 R2 0 R3 0 I II I II HzN c cN c cN c C COOH I I H H H H H b amp y Peptide Sequence IKTNP FEPILVDTTGS x y ier 0 2 b77112 Ym hr 20 7 W ling y y I l I mu sun am mun 12m mu 15m mm mm MSquot Triple Quadrupole Q1 Peptide mass filter Q2 RF only Everything transmitted 2 Acts as collision cell for CID o Introduce inert collision gas He Ar Xe Q3 Scan CID fragment masses MSquot QuadrupoleTOF Same concept aumumz MS YOF MS as Triple Quad H Remove Q3 add TOF Higher Sensitivity o Simultaneous analysis of fragments o Requires less sample not steady stream o TOF reflectron inherently more sensitive No upper mz limit Greater mass accuracy MSquot QuadrupoleIon Trap Ion traps can store and accumulate ions CID is carried out inside ion trap Fragments are selectively ejected 0 Scan all resonance ejection frequencies 0 Generate full fragment mass spectrum Advantages o Attomole sensitivity can be obtained 0 Multiple rounds of fragmentation can be applied Disadvantages o PTM scanning precursorneutral loss are not practical with QTraps Accurate Quantitation in MS I Relative abundance not perfect 0 0 Variations in ionization sample preparation Low signal high complexity decrease accuracy Isotope Enrichment O 000 Metabolic labeling with stable isotopes Isolate protein Mix trypsinize Mass spectra will have mass doublets Relative intensity of doublets relative abundance I ICAT iTRAQ Tagging Isotope Coded Affinity Tag Uses protein reactive group to tag samples Include an affinity tag for sample enrichment 0 Usually biotin group 0 Some biotin tags are chemically removable Contain different isotopes 1H2H or 12C13C Labeled peptides from different samples differ by de ned mass amounts Results in peak splitting 0 Relative abundance in sample Relative signal in MS 0 Normalizes for variations in ionization sample prep etc ICAT DIH NonCleavable Heavy reagent d8 ICAT X deuterium ICAT Reagents Light reagent dOICAT X hydrogen 0 JL NWOAOwOWNJKl S u XX XX Biotin Linker heavy or light Thioi speci c tag reactive group N ICAT 120130 Cleavable 227236amu Patti I Isotope Coded Tag Huavy in C 23 amu Light 9x quotC 227 am iTRAQ Similar in theory to ICAT tags Uses a set of different isobaric tags 0 Reporter mass tag 0 Mass balance Tag is liberated upon CID Reporter masses detected in a noisesignal free region of mass spectra 120 mz Peptide by ion peaks are not split as in ICAT iTRAQ Tag Anatomy IsobaricTag Total mass 145 Reporter lMass ll tliiu ll7l A E PBG Peptide Reactive I Y Balance Mass 3i 1an 28 l MSMS Fragmentation Site iTRAQ Mass Spectrum Full scan mass spectrum obtained via HPLC tandem mass spectrometric analysis on QSTAR XL A ma a H1quot jig T MSMS of mZ 4839 h
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