Mass spectrometrybased methods for protein identification and :5基于质谱的蛋白质鉴定和方法.ppt
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1、MS-based methods for protein identification&phosphorylation site analysis,Mass Spectrometer(MS),MS(fragments)./(m/z)signal./(Abundance)mass spectrum,mass spectrum.,MS Component,MS component,Sample inlet MS Ion source.MS analyzer Mass analyzer m/z ratio Ion detector,signal Vacuum system MS 10-4 10-9
2、Torr Data System MS,Ion source,Gaseous sample introduction-EI(electron ionization)-CI(chemical Ionization)Liquid sample introduction-FAB(fast atom bombardment)-ESI(electrospray ionization)(soft ionization)Solid sample introduction-MALDI(soft ionization)(matrix-assisted laser desorption/ionization),E
3、I(electron ionization),filament(+)sample M+spectrum M+e-M+2e-,CI(chemical Ionization),filament,106 reagent gas reagent gas ion sample gas sample gas fragmentation reagent gas ion complex.EI.R(CH4)+e-R+2 e-R+M M1+N1 M1+M2+N2,M A L D I,MALDI process,matrix sample 100010,000:1(acidic organic solvent-TF
4、A+MeCN)probe matrix(UV at 337nm,IR at 2.94um)sample-matrix matrix sample.protonation/deprotonation,cation attachment/cation detachment,oxidation/reduction.,Matrix in MALDI,Sinapinic acid:peptide,protein2kDa2,5-Dihydroxybenzoic acid:glycoprotein,glycolipids,carbohydrateA-Cyano-4-hydroxycinnamic acid:
5、low MW peptides,peptide2-Benzoic acid:sulfonated dyesNicotinic acid/Anthranilic acid(1;1):oligonucleotide,sialylated glycopeptides3-Hydroxypicolinic acid:oligonucleotide adducts,oligonucleotide2,4(6)Trihydroxyacetophenone:oligonucleotide,proteins 1-30kDa,E S I,ESI(electrospray ionization),capillary
6、droplet drpolet capillary orifice inert gas(or heat)desolvation Desolvation ion charge“Coulombic expolsion”droplet ion gas phase.smaple,multiple charge peptide.,E S I,Mass analyzer and Detector,MALDI MS(Linear),TOF(time of flight),Ion source tube(field-free drift tube).ion source m/z.TOF m/z,T O F,M
7、ass resolution,Resolution=m/m,MALDI-TOF MS resolution,Reflectron(ion mirror)-m/z reflectron reflectron detector Time-lag focusing(delayed extraction)-,MALDI MS(Reflectron),Quadruple analyzer(mass filter),4 molybdenum,(1,2)dc voltage(3,4)radio frequency voltage.Dc voltage 0 RF voltage m/z ion ion sou
8、rce detector quadropole mass scanning mass spectrum.,PSD(postsource decay),The metastable fragmentation of ions after full acceleration that occurs in the field free region of TOF-MSIf peptides are subjected to PSD they will fragment predominantly along th polypeptide backbone,thus generating series
9、 of fragment ions which,in principle,contain the amino acid sequence information of the peptideTo obtain primary structural information,MALDI MS(Reflectron),MS/MS,ESI TQ-MS,ESI TQ-MS,CID(collision-induced dissociation)The term used to describe fragmentation in MS/MS experiment.The precursor ion is i
10、solated and allowed to collide with neutral gas molecules in a collision cell.The translational energy of the precursor ion is converted to internal energy after the collisions resulting in fragmentation of the precursor ion.quadruple mode-MS mode-MS/MS mode-Neutral loss scan mode-Precusor(or parent
11、)ion scanning-In-source CID:fragmentation occurs in the high-pressure region of an ESI source as a result of collision with atmospheric gases.,Ring electrode end-cap electrode,quadruple dc voltage RF voltage.RF only trap,ESI IT-MS,ESI IT-MS,Tandem mass spectrometer in which ions can be accumulated a
12、nd stored prior to analysisThe ion trap is both a mass analyzer and collision cell“Resonance ejection”refers to ions becoming unstable in the trap and being ejected axially through th end-cap electrodes where they are detected Through this process of trapping and selective ejection of ions,ions of s
13、pecific m/z can be isolated in the trap,Mass spectrum of CO2,Total ion chromatogram,EI-MS spectrum of propionic acid,PA,Data base,Experimental data,Propionic acid,M+,MALDI-MS spectrum of protein,MS/MS spectrum of peptide,Peptide mass searching,Peptides are generated by digestion of the protein of in
14、terest using specific cleavage reagents(usually enzymes)The masses of these peptides are accurately determined experimentally using MALDI-MS(or ESI-MS)Theoretical peptide masses are calculated for each sequence entry in the database using the same cleavage specificity as the reagent employed experim
15、entally.A score(or ranking)is then calculated to provide a measure of fit between the experimentally derived and calculated peptide masses.,Measured peptide mass sequence,The additional masses are due to posttranslational or artifactual modifications or post-translational processingUnspecific proteo
16、lysis had occurred or contaminating protease was presentProtein was part of a mixture of contaminating proteins,Critical experimental parameter in peptide mass searching,The accuracy of the peptide-mass measurement-time-lag focusing/delayed extraction-internal standard with isotope The specificity o
17、f the enzyme(or chemical reagent)employed-missed cleavage,ragged termini control program,Orthogonal method in peptide mass searching,Site-specific chemical modificationDetermination of partial amino acid composition of the peptideIdentification of the N-terminal amino acid residueIdentification of d
18、ifferent cleavage sites within the peptidesIdentification of the C-terminal residue(s),Fragment ion,“b ion”-the fragment with c-terminal deletions and intact N-terminal“y ion”-the fragment with N-terminal deletions and intact C-terminal internal fragment-internal acyl ion-immonium ion(represent indi
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