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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
19、vidual amino acid),Uninterpreted fragment ion searching,Fragmentation can be induced by PSD-MALDI-MS as well as by CID in triple quadruple or ion-trap mass spectrometerFragment ion spectra contain reduntant pieces of information,How to interpret fragment ion,Manual interpretationInterpret with datab
20、ase-A partial manual interpretation of the spectrum to identify consecutive elements of a particular(b or y)ion series-Uninterpreted fragment ion search program(SEQUEST),De novo sequencing,Data base independent“peptide ladder sequencing”-different peptide in length by one amino acid-by chemical(Edma
21、n)N-terminal blocking:Gln(128.13),Lys(128.17),Ile,Leu-enzymatic degradation:Ile and Leu,Gln and Lys-are analyzed by MALDI-TOF MSCID spectra(fragment ion spectra)-are manually interpretated-missing frgament(incomplete ion series)trypsin H218O(50%H218O+50%H216O,intact C-terminal peptide ion series hav
22、e doublet by 2u)-methyl esterfication of the carboxyl groups in the peptide(14u increase,derivatized and underivatized),Peptide ladder sequence,Phosphorylation site analysis strategies,Complication of phosphoprotein analysis-the frequently low stoichiometry of phosphorylation-the presence of multipl
23、e,differentially phosphorylated formsIn vitro analysis-scale up of protein by kinase reaction-comparison with 2D-PP maps of in vivo(confirmation of identity indirectly)-MS analysis,Detection and isolation of phosphoproteins,For the analysis of the site(s)of protein phosphorylation-purification of ph
24、osphoprotein-enzymatic or chemical fragmentation of the phosphoprotein-Isolation,separation,analysis of peptideIsolation-separation of proteins by gel electrophoresis-fragmentation of the phosphoprotein band or spot-extraction of the generated phosphopeptideMore positive identification-32 P radiolab
25、elling:in vivo(32 PO4),in vitro(-32PATP)-western blotting:particularly tyrosine phosphorylated protein,Separation of phosphopeptides,-S/N-radiolabel activity phosphopeptide-separation phosphorylation-nonpeptide contaminants phosphopeptide,Phosphopeptide separation techniques,By 2-dimensional phospho
26、peptide mapReversed-phase HPLCHigh-resolution gel electrophoresisImmobilized metal affinity chromatogrphy(IMAC)Phosphopeptide,separation,Separation by 2D-PP,1st dimension by electrophoresis on thin-layer cellulose plate+2nd dimension by TLC on the same plate information-radiolabelled spot phosphoryl
27、ated sites-radiolabelled spot intensity peptide phosphorylation-relative state of hydropathy between phosphopeptieMS analysis after extraction from plate-protease sensitive and reproducibile by radiolabelling,Separation by RP-HPLC,Reproducible and simplecolumn radioactivity count fraction count radi
28、oactive fraction-very hydrophilic phosphopeptide,very hydrophobic phosphopeptide-2D-PP resolution-phosphopeptide will stick to metal surface-ESI MS on line(LC-MS/MS)-isotope,Separation by high-resolution electrophorsis and IMAC,High-resolution gel electrophoresis-2-DE-phosphopeptide IMAC-sequence no
29、nphosphorylated peptide phosphorylated peptide-separation and enrichment 1)phosphopeptide metal(Fe3+,Ga3+)chelating 2)elution by phosphate or increased pH 3)acidic amino acid enrichment,Detremination of the type of phosphorylated amino acid,phosphorylated site polypeptide phosphorylated residues ass
30、ignment Technique 1)phosphoamino acid analysis-32P-amino acid(hydrolysate of 32P-labeled phosphoprotein or phosphopeptide)autoradiography-phosphoamino acid standard ninhydrin staining-sample standard(1site/phosphopeptide)2)phosphoamino acid-specific immunodetction-antibodies specific for particular
31、phosphoamino acid-antibody,Determination of the site of phosphorylation,Chemical phosphopeptide sequencing-phosphopeptide sequencing by step-wise chemical degradation(nonradioactive,radioactive methods)-analyzed as phenylthiohydantoyl derivatives-not available in very limited amountMass spectrometri
32、c analysis of phosphopeptides-phosphopeptide 1pmole 2D-PP map extraction,MS-two basic theme 1)chemical lability of the phosphate ester bonds 2)the detection of the mass added to a peptide(80u)-product ion scan in a tandem MS phosphorylation site phosphorylated amino acid type,Mass scan for phosphope
33、ptides analysis,In-source CID-identify phosphopeptides by observation of H2PO4-(97U),PO3-(79U)and PO2-(63U)-detect phosphopeptides in negative ion mode and then switch to positive ion mode Neutral loss scan-positive ion mode with ESI in a TQ MS-Q1,Q3 are scanned over different m/z ranges-neutral los
34、s of phosphoserine and phosphothreonine:98,Mass scan for phosphopeptides analysis,Presursor ion scan-negative ion ESI(positive ion mode)-Q1:continous scan,Q2:ion fragmentation Q3:79m/z(PO3-)ion Product ion scanning-in-source CID,neutral loss and precursor ion scanning phosphorylated residue identify
35、-3 peptide fragment ion,Mass scan for phosphopeptides analysis,Post-source decay MALDIEnzymatic and chemical dephosphorylation-MALDI-TOF phosphopeptide mass+phosphate MALDI-TOF mass-nonphosphorylated peptide phosphopeptide-identification of phosphorylation sites using MS/MS,Emerging methods and futu
36、re directions in phosphoprotien analysis,phosphoprotein in vivo 32P-labeled phosphoprotein in vivo in vitro in vivo in vivo 32P-labeled protein-FT-ICR-MS,microcapillary HPLC-at level of tens of attomoles Single automated LC-MS/MS-presence of phosphoprotein,mass of peptide,CID spectrum of phosphopeptide,Present and future challeges and opportunities,Protein identification and characterization has to be performed in a high-throughput manner,efficiently and with high accuracy and sensitivity Robotic system2D-chromatography MS/MS,
