MS-based methods for protein identification & phosphorylation site analysis 생명과학부...
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- Slide 1
- MS-based methods for protein identification &
phosphorylation site analysis
- Slide 2
- M ass S pectrometer(MS) MS (fragments) . / (m/z) signal . /
(Abundance) mass spectrum , mass spectrum .
- Slide 3
- MS Component
- Slide 4
- MS component Sample inlet MS Ion source . MS analyzer Mass
analyzer m/z ratio Ion detector , signal Vacuum system MS 10 -4 ~
10 -9 Torr Data System MS
- Slide 5
- 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)
- Slide 6
- EI(electron ionization) filament (+) sample M + spectrum M + e
- M + + 2e -
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- CI(chemical Ionization) filament , 10 6 reagent gas reagent gas
ion sample gas sample gas fragmentation reagent gas ion complex .
EI . R(CH 4 ) + e - R + + 2 e - R + + M M1 + + N1 M1 + M2 + +
N2
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- M A L D I
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- MALDI process 1.matrix sample 1000~10,000 : 1 (acidic organic
solvent- TFA+MeCN) 2. probe matrix 3. (UV at 337nm, IR at 2.94um)
sample-matrix matrix sample . 4. protonation/deprotonation, cation
attachment/cation detachment, oxidation/reduction .
- Slide 10
- Matrix in MALDI Sinapinic acid : peptide, protein>2kDa
2,5-Dihydroxybenzoic 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 2,4(6)
Trihydroxyacetophenone : oligonucleotide, proteins 1-30kDa
- Slide 11
- E S I
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- 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 .
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- E S I
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- Mass analyzer and Detector
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- MALDI MS(Linear)
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- TOF(time of flight) Ion source tube(field-free drift tube) .
ion source m/z . TOF m/z
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- T O F
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- Mass resolution Resolution = m/ m
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- MALDI- TOF MS resolution Reflectron(ion mirror) - - m/z
reflectron reflectron detector Time-lag focusing(delayed
extraction) -
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- MALDI MS(Reflectron)
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- Slide 22
- 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 .
- Slide 23
- Slide 24
- PSD(postsource decay) The metastable fragmentation of ions
after full acceleration that occurs in the field 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
- Slide 25
- MALDI MS(Reflectron)
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- MS/MS
- Slide 27
- ESI TQ-MS
- Slide 28
- 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 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) ion scanning - In-source CID :
fragmentation occurs in the high-pressure region of an ESI source
as a result of collision with atmospheric gases.
- Slide 29
- Ring electrode end-cap electrode , quadruple dc voltage RF
voltage . RF only trap
- Slide 30
- ESI IT-MS
- Slide 31
- 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 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 specific m/z can be isolated in
the trap
- Slide 32
- Mass spectrum of CO 2
- Slide 33
- Total ion chromatogram EI-MS spectrum of propionic acid PA
- Slide 34
- Data base Experimental data Propionic acid M+
- Slide 35
- Slide 36
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- MALDI-MS spectrum of protein
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- MS/MS spectrum of peptide
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- Slide 40
- Peptide mass searching 1.Peptides are generated by digestion of
the protein of interest using specific cleavage reagents(usually
enzymes) 2.The masses of these peptides are accurately determined
experimentally using MALDI-MS(or ESI-MS) 3.Theoretical peptide
masses are calculated for each sequence entry in the database using
the same cleavage specificity as the reagent employed
experimentally. 4.A score(or ranking) is then calculated to provide
a measure of fit between the experimentally derived and calculated
peptide masses.
- Slide 41
- 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
- Slide 42
- 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 of the enzyme(or chemical reagent) employed - missed
cleavage , ragged termini control program
- Slide 43
- Orthogonal method in peptide 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)
- Slide 44
- Slide 45
- 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 individual amino acid)
- Slide 46
- Slide 47
- Slide 48
- Uninterpreted fragment ion searching Fragmentation can be
induced by PSD- MALDI-MS as well as by CID in triple quadruple or
ion-trap mass spectrometer Fragment ion spectra contain reduntant
pieces of information
- Slide 49
- How to interpret fragment ion 1.Manual interpretation
2.Interpret with database - 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)
- Slide 50
- 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 : 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 H 2 18 O (50% H 2 18 O+50%H 2 16 O, intact C-terminal
peptide ion series have doublet by 2u) - methyl esterfication of
the carboxyl groups in the peptide (14u increase, derivatized and
underivatized )
- Slide 51
- Peptide ladder sequence
- Slide 52
- Slide 53
- Phosphorylation site analysis strategies Complication of
phosphoprotein analysis - the frequently low stoichiometry of
phosphorylation - the presence of multiple, differentially
phosphorylated forms In vitro analysis - scale up of protein by
kinase reaction - comparison with 2D-PP maps of in vivo
(confirmation of identity indirectly) - MS analysis
- Slide 54
- Detection and isolation of phosphoproteins For the analysis of
the site(s) of protein phosphorylation - purification of
phosphoprotein - enzymatic or chemical fragmentation 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 PO 4 ), in vitro([- 32 P]ATP) -
western blotting : particularly tyrosine phosphorylated
protein
- Slide 55
- Separation of phosphopeptides - S/N - radiolabel activity
phosphopeptide - separation phosphorylation - nonpeptide
contaminants phosphopeptide
- Slide 56
- Phosphopeptide separation techniques By 2-dimensional
phosphopeptide map Reversed-phase HPLC High-resolution gel
electrophoresis Immobilized metal affinity chromatogrphy(IMAC)
Phosphopeptide , separation
- Slide 57
- Separation by 2D-PP 1 st dimension by electrophoresis on
thin-layer cellulose plate + 2 nd dimension by TLC on the same
plate information - radiolabelled spot phosphorylated sites -
radiolabelled spot intensity peptide phosphorylation - relative
state of hydropathy between phosphopeptie MS analysis after
extraction from plate - protease sensitive and reproducibile by
radiolabelling
- Slide 58
- Separation by RP-HPLC Reproducible and simple column
radioactivity count fraction count 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
- Slide 59
- Separation by high-resolution electrophorsis and IMAC
High-resolution gel electrophoresis - 2-DE - phosphopeptide IMAC -
sequence nonphosphorylated peptide phosphorylated peptide -
separation and enrichment 1) phosphopeptide metal(Fe 3+,Ga 3+ )
chelating 2) elution by phosphate or increased pH 3) acidic amino
acid enrichment
- Slide 60
- Detremination of the type of phosphorylated amino acid
phosphorylated site polypeptide phosphorylated residues assignment
Technique 1) phosphoamino acid analysis - 32 P-amino
acid(hydrolysate of 32 P-labeled phosphoprotein or phosphopeptide)
autoradiography - phosphoamino acid standard ninhydrin staining -
sample standard ( 1site/phosphopeptide) 2) phosphoamino
acid-specific immunodetction - antibodies specific for particular
phosphoamino acid - antibody
- Slide 61
- 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 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 phosphorylation site phosphorylated amino acid type
- Slide 62
- Mass scan for phosphopeptides analysis In-source CID - identify
phosphopeptides by observation of H 2 PO 4 - (97U), PO 3 - (79U)
and PO 2 - (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 loss of phosphoserine and phosphothreonine :
98
- Slide 63
- Slide 64
- Slide 65
- Mass scan for phosphopeptides analysis Presursor ion scan -
negative ion ESI( positive ion mode ) - Q1 : continous scan, Q2 :
ion fragmentation Q3 : 79m/z(PO 3 - ) ion Product ion scanning -
in-source CID, neutral loss and precursor ion scanning
phosphorylated residue identify - 3 peptide fragment ion
- Slide 66
- Mass scan for phosphopeptides analysis Post-source decay MALDI
Enzymatic and chemical dephosphorylation - MALDI-TOF phosphopeptide
mass + phosphate MALDI-TOF mass - nonphosphorylated peptide
phosphopeptide - identification of phosphorylation sites using
MS/MS
- Slide 67
- Emerging methods and future directions in phosphoprotien
analysis phosphoprotein in vivo 32 P-labeled phosphoprotein in vivo
in vitro in vivo in vivo 32 P-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
- Slide 68
- 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