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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
2、 system MS 10-4 10-9 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-assi
3、sted laser desorption/ionization),EI(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
4、 I,MALDI process,matrix sample 100010,000 : 1 (acidic organic solvent- TFA+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, protein2kDa 2,5-Dihy
5、droxybenzoic acid : glycoprotein, glycolipids, carbohydrate A-Cyano-4-hydroxycinnamic acid : low MW peptides, peptide 2-Benzoic acid : sulfonated dyes Nicotinic acid/Anthranilic acid(1;1) : oligonucleotide, sialylated glycopeptides 3-Hydroxypicolinic acid : oligonucleotide adducts, oligonucleotide 2
6、,4(6) Trihydroxyacetophenone : oligonucleotide, proteins 1-30kDa,E S I,ESI(electrospray ionization), capillary 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
7、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,Mass resolution,Resolution = m/m,MALDI-TOF MS resolution,Reflectron(ion mirror) - - m/z reflectron reflectron detector Time-lag focusing(delayed extraction) - ,MALDI MS(Reflectr
8、on),Quadruple analyzer(mass filter),4 molybdenum , (1,2) dc voltage (3,4) radio frequency voltage . Dc voltage 0 RF voltage m/z ion ion source detector quadropole mass scanning mass spectrum .,PSD(postsource decay),The metastable fragmentation of ions after full acceleration that occurs in the field
9、 free region of TOF-MS If peptides are subjected to PSD they will fragment predominantly along th polypeptide backbone, thus generating series of fragment ions which, in principle, contain the amino acid sequence information of the peptide To obtain primary structural information,MALDI MS(Reflectron
10、),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 isolated and allowed to collide with neutral gas molecules in a collision cell. The translational energy of the precursor ion is converted to internal e
11、nergy after the collisions resulting in fragmentation of the precursor ion. quadruple mode - MS mode - MS/MS mode - Neutral loss scan mode - Precusor(or parent) ion scanning - In-source CID : fragmentation occurs in the high-pressure region of an ESI source as a result of collision with atmospheric
12、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 and stored prior to analysis The ion trap is both a mass analyzer and collision cell “Resonance ejection” refers to ions becoming u
13、nstable 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 specific m/z can be isolated in the trap,Mass spectrum of CO2,Total ion chromatogram,EI-MS spectrum of propionic acid,PA,Data b
14、ase,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 interest using specific cleavage reagents(usually enzymes) The masses of these peptides are accurately determined experimentally
15、 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 experimentally. A score(or ranking) is then calculated to provide a measure of fit between the experimentally derived and calculate
16、d peptide masses.,Measured peptide mass sequence ,The additional masses are due to posttranslational or artifactual modifications or post-translational processing Unspecific proteolysis had occurred or contaminating protease was present Protein was part of a mixture of contaminating proteins,Critica
17、l experimental parameter in peptide mass searching,The accuracy of the peptide-mass measurement - time-lag focusing/delayed extraction - internal standard with isotope The specificity of the enzyme(or chemical reagent) employed - missed cleavage, ragged termini control program,Orthogonal method in p
18、eptide mass searching,Site-specific chemical modification Determination of partial amino acid composition of the peptide Identification of the N-terminal amino acid residue Identification of different cleavage sites within the peptides Identification of the C-terminal residue(s),Fragment ion,“b ion”
19、 - 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 individual amino acid),Uninterpreted fragment ion searching,Fragmentation can be induced by PSD-MALDI-
20、MS as well as by CID in triple quadruple or ion-trap mass spectrometer Fragment ion spectra contain reduntant pieces of information,How to interpret fragment ion,Manual interpretation Interpret with database - A partial manual interpretation of the spectrum to identify consecutive elements of a part
21、icular (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(Edman) N-terminal blocking : Gln(128.13),Lys(128.17), Ile, Leu - enzymatic degradation :
22、Ile and Leu, Gln and Lys - are analyzed by MALDI-TOF MS CID spectra(fragment ion spectra) - are manually interpretated - missing frgament(incomplete ion series) trypsin H218O (50% H218O+50%H216O, intact C-terminal peptide ion series have doublet by 2u) - methyl esterfication of the carboxyl groups i
23、n 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 multiple, differentially phosphorylated forms In vitro analysi
24、s - 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 phosphoprotein - enzymatic or chemical fragme
25、ntation of the phosphoprotein - Isolation, separation, analysis of peptide Isolation - separation of proteins by gel electrophoresis - fragmentation of the phosphoprotein band or spot - extraction of the generated phosphopeptide More positive identification - 32 P radiolabelling : in vivo(32 PO4), i
26、n 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 phosphopeptide map
27、 Reversed-phase HPLC High-resolution gel electrophoresis Immobilized 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
28、ated sites - radiolabelled spot intensity peptide phosphorylation - relative state of hydropathy between phosphopeptie MS analysis after extraction from plate - protease sensitive and reproducibile by radiolabelling,Separation by RP-HPLC,Reproducible and simple column radioactivity count fraction co
29、unt radioactive 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 - phosph
30、opeptide IMAC - sequence nonphosphorylated 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 pol
31、ypeptide phosphorylated residues assignment 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 im
32、munodetction - antibodies specific for particular 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 derivati
33、ves - not available in very limited amount Mass spectrometric 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 phos
34、phorylation site phosphorylated amino acid type ,Mass scan for phosphopeptides 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
35、mode with ESI in a TQ MS - Q1, Q3 are scanned over different m/z ranges - neutral loss 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 Produ
36、ct ion scanning - in-source CID, neutral loss and precursor ion scanning phosphorylated residue identify - 3 peptide fragment ion ,Mass scan for phosphopeptides analysis,Post-source decay MALDI Enzymatic and chemical dephosphorylation - MALDI-TOF phosphopeptide mass + phosphate MALDI-TOF mass - nonp
37、hosphorylated peptide phosphopeptide - identification of phosphorylation sites using MS/MS,Emerging methods and future 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 le
38、vel 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 system 2D-chromatography MS/MS,