醫學新知導論 mass spectrometry in biotechnology/proteomics yi-ting chen ( 陳怡婷 ), ph.d....
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醫學新知導論醫學新知導論
Mass Spectrometry in BiotechnMass Spectrometry in Biotechn
ology/Proteomicsology/Proteomics
Yi-Ting Chen ( 陳怡婷 ), Ph.D.
Molecular Medicine Research Center
Chang Gung University
Dec. 4, 2008
Outline• Introduction of mass spectrometry
– principle– Instrumentation
• Proteomics– Introduction– Mass spectrometry in proteomics– Protein identification– Quantitative proteomics– Post-translational modification
• Application of mass spectrometry in biotechnology– Food science– Environment chemistry– Drug analysis
The Five Mass Spectrometry Nobel Prize Pioneers
WHAT IS A “MASS SPECTROMETER ”...?
MS
The black box problem…...
ESI MALDI LC/MS LC-MSMSESI
ESI Ion trap TOF QTOFquad.
ESI CRIMS EI APCIFAB
FT-ICR
qQTOF
SELDI
…many black boxes !
“A mass spectrometer measures the molecular weight….”
“...A MS analysis gives
the mass-to-charge ratio (m/z) of ions…in gas phase”.
Data Processing
Ion Source
Analyzer
ion separation
vacuum
Detector
Pumping system
Sample introduction
Introduction(solid, liquid, gas)
Separation technique (HPLC, CE, GC)
ESI, nano ESI, MALDI, FAB, EI, CI, APCI, SIMS…
TOF, quadrupole, Ion trap, FT, magnetic sectorQQQ, Q/TOF, Q/IT…
h
Nd:YAG Laser355 nm
Sample ions, MH+
Ground Grid
Sampleplate
High voltage
Sample & matrix
To Mass Analyzer
Matrix Assisted Laser Desorption Matrix Assisted Laser Desorption Ionization (MALDI)Ionization (MALDI)
HPLC - MS
• Ion Source ( 離子源 )– 大氣壓下游離法 (Atmosp
heric Pressure Ionization, API)
• 電灑法 – Electro-spray, ESI
• 大氣壓化學游離法– Atmospheric Pressure
Chemical Ionization, APCI
Analyte Polarity
ESI
APcI
Molecular Weight
1000
1000000
EI600
Electro-spray, ESI ( 電灑法 )
Desolvation
泰勒錐Taylor cone Cone
voltage
pumping
HPLC - MS
• Electro-spray, ESI ( 電灑法 )
Capillary ~3 kV
As droplets evaporate, the electric field increases and ions move towards the surface.
Ions evaporate from the surface
Solvent evaporationCoulombic explosion
0
20
40
60
80
100
%Int.
5000 10000 15000 20000Mass/Charge
Data: 1015pl30001.O7 15 Oct 2002 16:22 Cal: 16 Oct 2002 8:12
8480.6
5653.4
16953.04239.4
MALDI-TOF spectra of apomyoglobin
INSTRUMENT: Kratos Axima-CFR
Sample: 1 pmole apomyoglobin (horse skeletal muscle)
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
+101695.9
+111541.8
+121413.5
+131304.8
+141211.7
+15
1131.0
+16
1060.4
+17
998.1
+18
942.8
+19893.2
+20848.6
+21808.2
+22771.5
+23738.0+24
707.4+25
679.1
+91884.5
ACTUAL SPECTRUM
+91884.3
16000 16400 16800 17200 17600 18000 18400
mass
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
16951.5
AFTER DECONVOLUTION
ESI-ion trap spectra of apomyoglobin
INSTRUMENT: Thermoquest LCQ-classic
Sample: 1 pmole apomyoglobin (horse skeletal muscle)
HPLC -MS• Atmospheric Pressure Chemical Ionization, APCI ( 大
氣壓化學游離法 )
• http://www.chm.bris.ac.uk/ms/theory/apci-ionisation.html
HPLC- MS
TechniqueFlow Rate
(ml/min)M.W Range
Species
Produced
ESI 0.001-0.3 < 2000000(M+H)+
(M-H)-
(M+nH)n+
APCI 0.2-2.0 < 1000(M+H)+
(M-H)-
ESI and APCI differ in…• How ions are generated
• ESI - solution phase ionization• APCI - gas phase ionization
• Analyte compatibility• ESI - polar compounds and large biomolecules• APCI - less polar, smaller compounds (relative to th
ose ionized by ESI) that have some volatility
• Flow rate compatibility• ESI - 0.001 to 1 mL/min• APCI - 0.2 to 2 mL/min
HPLC -MS• Effect of Flow Rate
Flow rate (ml/min) (50/50 ACN/H20)
0 0.5 1.0 1.5 2.0
50
100
Rel
ativ
e in
tens
ity
APCI
ESI
HPLC -MS• Quadrupole
To Detector
-+
-+
From Source
X
Y
Volta
ge o
n Ro
ds-v
e
0
+ve
RF Cycle
X
Y
MALDI Time-of-flight (TOF) MSMALDI Time-of-flight (TOF) MS
source
TOF
Mass (m/z)
inte
ns
ity
Laser
The ions enter the flight tube with the lighter ionstravelling faster than the heavier ions
m/z is mass-to-charge ratio of the ionE is the extraction pulse potentials is the length of flight tube over which E is appliedd is the length of field free drift zonet is the measured time-of-flight of the ion
Ion Trap
Fourier-transform Ion Cyclotron Resonance (FT-ICR)
High resolution and accuracy
Single MS Analyzer
mass scanning mode
m1m3m4 m2
m3
m1
m4
m2
single mass transmission mode
m2 m2 m2 m2m3
m1
m4
m2
m/z
HPLC-MS/MS Triple Quadrupole
1. Samples from the liquid 1. Samples from the liquid introduction system enter the introduction system enter the ionisation source at atmospheric ionisation source at atmospheric pressurepressure
1. Samples from the liquid 1. Samples from the liquid introduction system enter the introduction system enter the ionisation source at atmospheric ionisation source at atmospheric pressurepressure
2. Ions are transferred 2. Ions are transferred to the analysers to the analysers through a hexapole through a hexapole lenslens
2. Ions are transferred 2. Ions are transferred to the analysers to the analysers through a hexapole through a hexapole lenslens
3. The ions are filtered in 3. The ions are filtered in MS1 according to their mass MS1 according to their mass to charge ratio (m/z)to charge ratio (m/z)
3. The ions are filtered in 3. The ions are filtered in MS1 according to their mass MS1 according to their mass to charge ratio (m/z)to charge ratio (m/z)
4. The mass separated 4. The mass separated ions undergo CID in the ions undergo CID in the hexapole collision cellhexapole collision cell
4. The mass separated 4. The mass separated ions undergo CID in the ions undergo CID in the hexapole collision cellhexapole collision cell
5. The fragment ions are 5. The fragment ions are filtered in MS2 filtered in MS2 according to their mass according to their mass to charge ratioto charge ratio
5. The fragment ions are 5. The fragment ions are filtered in MS2 filtered in MS2 according to their mass according to their mass to charge ratioto charge ratio
6. The ions are detected by 6. The ions are detected by an off-axis photomultiplier an off-axis photomultiplier detector.detector.
6. The ions are detected by 6. The ions are detected by an off-axis photomultiplier an off-axis photomultiplier detector.detector.
MS/MS Scan Functions
mass scan modesingle mass transmission
m2 m2 m2 m2m3
m1
m4
m2
Collision Chamber (gas)Collision Chamber (gas)
++
+
+
+
+
N2
+ + + ++
Q1 Q3Product Ion Scan (PI) Fix ScanMultiple Reaction Mode (MRM) Fix FixPrecursor Ion Scan (PS) Scan FixNeutral Loss Scan (NL) Scan Scan
Product Ion Scanning
mass scan modesingle mass transmission
m2m2 m2m2 m2m2 m2m2m3m3
m1m1
m4m4m2m2
Collision Chamber (gas)
++++
++++
++
++
NN22
++++ ++ ++
Parked on Precursor ion
Parked on a product Ion CAD
Q1 Q3Q2
Most sensitive scan type for detection of known components
- Q1 is set on the parent ion m/z (usually multiply charged for peptides)- ions are fragmented in Q2 collision cell - Q3 is set on the diagnostic fragment m/z
Fundamental to Absolute Quantitation is the Triple Quad Scan termed Multiple Reaction Monitoring (MRM)
Precursor Ion Scanning
Scan Precursors Select ProductCAD
Q1 Q3Q2
Only ions passed through Q1 that produce the PTM-specific fragment mass (e.g. 79 for phosphorylation or 204 for glycosylation) will produce signal at the detector
http://www.sciencemag.org/cgi/content/full/291/5507/1221/F1
• Proteomics– Introduction– Mass spectrometry in proteomics– Protein identification– Quantitative proteomics– Post-translational modification
• Proteome : In 1993, the term “Proteome”, by Marc Wilkins and Keith Williams, was referred to the systematic identification of the entire protein population expressed by a genome or by a cell or tissue type.
• Proteomics : The subject of proteomic analysis of the proteome (PROTEin complement expressed by a genOME or by a cell or tissue type).
(Wilkins et al., 1995 Biotechnology and Genetic Engineering Reviews 13, 19-50.)
Definition of ProteomicsDefinition of Proteomics• Yates defined proteomics as the scientific discipline of characterizin
g and analyzing the proteins, protein interactions, and protein modifications of an organism.
• Gygi and Aebersold defined proteomics as the ability to systematically identify every protein expressed in a cell or tissue tissue as well as to determine the salient properties of each protein, i.e., abundance, state of modification, involvement in multiprotein complexes, etc.
• Wagner defined the proteome is the entire profile of all the proteins expressed by a cell or a tissue under strictly defined conditions at a given time.
-proteomics aims to: -separate identity and characterize proteins on a large scale -define levels of proteins in cells / tissues and how these change -investigate protein complexes -elucidate protein functions, pathways, and interrelationships
蛋白體的發展里程碑蛋白體的發展里程碑
Why analyze gene expression at the protein level?
Y
YRNA Proteins Modified
ProteinsDNA Biological
Function
Transcription Translation Post-TranslationModification
Genome Transcriptome Proteome
<30,000 Genes > 1,000,000 Proteinsx 5 to 50
functionallinks per protein
Why the study of proteins is so challenging?
A polypeptide can fold to generate a particular three-dimensional structure specified by its amino acid sequence.
The structural description of proteins is described in terms of four levels of organization.
MS-based ProteomicsMS-based Proteomics
Qualitative
Protein identificationPeptide Mass Fingerprint (PMF)
MS/MS ion searching
De novo Sequencing
Post Translation Modification (PTM)
Quantitative
Protein quantitation
ICAT
iTRAQ
SILAC
O16/O18
Non-labelling tech.
MRM
The general Property Differences The general Property Differences between DNA and Proteinbetween DNA and Protein
mRNA level expressed protein level
Kind of Material DNA PROTEINResidues 4 20
Size define large
Concentration even wide range
Solubility in H2O highly highly-poorly
Isoelectric point 4.5 2-13
Cleaving enzyme ~100 a few
Amplification >106 no
Modification Methylation Several
Goal of Genomics and Proteomics
• What do biologists want?• Identify proteinssequence of primary structure
and look up genomic and protein database• Characterize proteinsanalyze biologically relev
ant modifications• Look for differetially-expressed proteins as biom
arkers• Look for protein complexes and networksbiolo
gical function(new drug!)
Protein Extract
Analyze spots by AAA SequencingMass spectrometry
Separation based on pI
Separation based on
size
Separate proteins on 2-D gels
Conventional ApproachConventional Approach
1510.010.0 7.57.5 6.36.3 6.06.0 5.85.8 5.55.5 5.25.2 5.05.0 4.04.0 3.03.09.09.0 8.08.0
100100
6060
5050
4040
3535
3030
2525
2020
Mol
ecul
ar W
eigh
tM
olec
ular
Wei
ght
ppII
11
88
77
66
55 1111
44
22 33
3030
99
2020
18181717
1616
15151010
14141313
1919
1212
128128
127127
126126
125125
121121
124124 122122
8787
123123
2828
2727
2626
25252121
2424
2323
2929
22223838
3737
36363434
313135353333
3939
3232
4848 4747
46464545
4141
4444
4343
4949
4242
7272
7878
7777
76767575
7171
7474
7373
6969
4040
7070
6868
6767
666665656161
64646363
7979
6262
6060
58585757
5656
5555
5151
5454
5353
5959
52525050
80808888
129129
8686
8585
8181
8484
8383
8989
8282
9898
97979696
9595
9191
9494
9393
9999
92929090
108108
106106
105105
101101
104104103103
109109102102
100100
118118
117117
116116
107107
111111
114114
113113
119119
112112
110110
115115
151151153153
152152
120120138138
137137
136136 135135
131131
134134
133133
139139
132132
130130
148148 147147
146146
145145
141141
144144
143143
149149
142142
140140
150150
154154
155155
156156
Excise spot;wash; digest
Run 2D gel; Stain/Image
Extract peptides;MALDI-TOF analyzeor qQ-TOF analyze
Database search
Edman DegradationAAA CompositionImmunoblotMS
1045.5584
1183.64981199.6755
1430.7831
1457.6913
1485.72441488.7815
1552.7802
1675.73701876.9218
1894.9346
1909.9414
2167.19222299.1675
2599.2658
1000 1500 2000 2500 Mass (m/z)
Protein ID : Experimental Approach
Peptide-Mass Fingerprinting (PMF)Peptide-Mass Fingerprinting (PMF)
Protein
ProteinSequence
M/Z
M/Z
ProteolyticPeptides
FragmentMass Spectrum
Theoretical FragmentMass Spectrum
TheoreticalProteolytic
PeptidesGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLKKHGATVLTALGGILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKHPGDFGADAQGAMNKALELFRKDMASNYKELGFQG
GLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLK...
Advantages & Disadvantages of 2D gel Advantages & Disadvantages of 2D gel ApproachApproach
• Excellent resolving power• Visual display of protein patterns• Well established technique and Relatively inexpensive to ge
t started with manual techniques• Poor ability to handle certain classes of proteins
• membrane, basic, acidic, high and low molecular weight proteins
• Multiple spots correspond to the same protein or multiple protein products co-migrate to the same spot
• Cannot visualize low abundant proteins; Only abundance proteins are identified
• Time consuming and difficult to automate• Limited recovery of analyte for further analysis• Poor reproducibility; limited dynamic range; certain protei
n stain poorly or not at all.• 2-D gel does not possess the sensitivity and the dynamic ran
ge needed for isolating different proteins
Limitation of 2-D Gel Based Proteomics Platform
• Mouse liver protein database by F. Hoffmann-La Roche Pharmaceutical Research published in Electrophoresis, 2001 showed that analysis of approximately 5800 spots excised from 14 2-D gels resulted in the id of 2,500 proteins which are the products of 328 genes.
• Likewise, in a study published in 2002, only 278 genes whose protein products were identified from rat liver.
Protein Detection MethodsProtein Detection Methods
• Coomassie Blue 0.1 g/band - 1 g/band
• Fluorescent Stain 1 - 10 ng/band
• Silver Stain 1 - 10 ng/band
• 1 ng of a 10kDa 100 femtomoles• 1 ng of a 100kDa 10 femtomoles
High-Performance Liquid Chromatography
LoadingSample
Packing Material
ElutionSample
Impurity 1
Impurity 2, 3
A compound
HPLCLC/MS
Multi Dimensional Liquid Multi Dimensional Liquid ChromatographyChromatography
• Size – Gel filtration columns
• Affinity
• Charge – Ion exchange columns• Hydrophobicity – HIC columns, Reverse phase
Size Exclusion
• Separation of proteins based on their size: The beads are composed of dextran polymers (sephadex), agarose (Sepharose), polyacrylamide (Sephacryl or BioGel P). Each bead contains pores of approximate macromolecule sizes. Larger molecules will travel through less pores, thus migrate faster. Smaller molecules will travel through more pores, thus migrate slower. The molecules passively distribute between the volume outside the porous beads (V
0) and the volume inside the beads (Vi) dependent on their ability to enter the pores. If the total volume of the column is Vt,
Vt = Vo + Vi
Reference: 清華大學 分子與細胞生物研究所暨生命科學系張大慈教授V0
Vi Vt
UV
ab
sorb
ance
Larger Protein
Smaller Protein
Affinity Chromatography
Affinity chromatography makes use of the affinity between a ligand and the protein of interests. Ligands are usually immobilized through covalent bonds on insoluble matrix, such as cellulose or polyacrylamide. The protein of interests become bound to the matrix while other proteins flow through the column. After washing the protein bound to the matrix can be eluted by adding completing groups such as the free ligand, or reagents that disrupt the interactions. Examples of ligand-protein interactions include those between antibodies and antigens, those between Ni+ and poly-histidine tag.
UV
ab
sorb
ance Free ligand concentration
protein
ligand
Ion Exchange Chromatography
• Electrostatic properties of a protein determine the type of ion exchange resins it interacts with. In principle:
– Protein is positively charged if solution pH < pI; It should bind to negatively charged resins, or cation exchanger;
– Protein is negatively charged if solution pH > pI; It should bind to positively charged resins, or anion exchanger;
– Note that in practice, protein surface has local charges that may different from total charge of the protein.
• Examples of cation exchangers include : carboxymethyl (CM) and sulfopropyl (SP)
• Examples of anion exchangers include: diethylaminoethyl (DEAE), quaternary amine (QAE)
proteinsurface-
--++
+-
nonpolar
nonpolar
Ion Exchange Chromatography
• Ion exchange chromatography makes use of electrostatic properties of the protein of interests. Charged polymers are usually immobilized through covalent bonds on insoluble matrix, such as cellulose or polyacrylamide. The protein of opposite charge become bound to the matrix while other proteins flow through the column. After washing, the protein bound to the matrix can be eluted by adding salts.
UV
ab
sorb
ance Increasing salt concentration
protein B
Protein B
protein B
Protein B
Protein A
ProteinANa+ SO3+--CM
Hydrophobic Interaction
• Hydrophobic interaction chromatography makes use of the hydrophobic patches on protein and their interactions with hydrophobic resins.
• For instance, phenyl sepharose is a strong hydrophobic resin that is made by covalently attach phenyl group to agarose supporting matrix. Proteins bind to phenyl group by virtue of hydrophobic interactions. Such interactions are most favored in the presence of high salts. Thus a typical procedure for running hydrophobic chromatography is to first bind proteins under high salt condition and then elute the bound proteins by running a salt gradient from high to low concentrations.
decreasing salt concentration
UV
ab
sorb
ance
proteinsurface-
--++
+-
nonpolar
nonpolar
General approachesGeneral approaches
Protein Extract
Chromatographic Fractionation
2-D GelElectrophoresis
1-D GelElectrophoresis
Biological Pre-fractionation of sample(organs, tissues, cell types, subcellular components)
Enzymatic or chemical Digest
Digest
Chromatography
Label with amino acidtargeted affinity reagentssuch as iTRAQTM
Mass spectrometryElectrospray &/or MALDI
Digest
Multidimensional LCDigest
Protein Protein Identification by Peptide Fragment Fingerprinting
Protein
ProteinSequence
M/Z
M/Z
ProteolyticPeptides
FragmentMass Spectrum
Theoretical FragmentMass Spectrum
TheoreticalProteolytic
PeptidesGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLKKHGATVLTALGGILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKHPGDFGADAQGAMNKALELFRKDMASNYKELGFQG
GLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLK...
PeptideFragments
TheoreticalPeptide
Fragments
LFKLFKGHPELFKGHPETLEGHPETLEKPETLEKETLEK...
SampleIons
LINAC
IRPDetector
Accelerator
Accelerator
Ion Mirror
Curtain Gas
Ground
Pusher Plate
Puller Grid
ESI-Q/TOFESI-Q/TOF
MALDI-TOF/TOFMALDI-TOF/TOF
Source #2
x,y Deflectors #2
Reflector
LinearDetector
ReflectorDetector
Source 2FocusingCID cell
TIS
x,y Deflectors #1
Source 1Focusing
Sample plate & stage
V1
V2
Source #1
Laser
nanoRPLC-ESI-Tandem MSnanoRPLC-ESI-Tandem MS
Total Ion Chromatogram
TOF MS Spectrum
MS/MS Spectrum
N- and C-terminal Peptides
N-term
inal
pep
tides
C-te
rmin
al p
eptid
es
Terminal peptides and ion types
Peptide
Mass (D) 57 + 97 + 147 + 114 = 415
Peptide
Mass (D) 57 + 97 + 147 + 114 – 18 = 397
without
N- and C-terminal Peptides
N-term
inal
pep
tides
C-te
rmin
al p
eptid
es
415
486
301
154
57
71
185
332
429
N- and C-terminal Peptides
N-term
inal
pep
tides
C-te
rmin
al p
eptid
es
415
486
301
154
57
71
185
332
429
N- and C-terminal Peptides
415
486
301
154
57
71
185
332
429
N- and C-terminal Peptides
415
486
301
154
57
71
185
332
429
Problem:
Reconstruct peptide from the set of masses of fragment
Mass Spectra
G V D L K
mass0
57 Da = ‘G’ 99 Da = ‘V’LK D V G
• The peaks in the mass spectrum:– Prefix
– Fragments with neutral losses (-H2O, -NH3)
– Noise and missing peaks.
and Suffix Fragments.
D
H2O
Protein Identification with MS/MS
G V D L K
mass0
Inte
nsity
mass0
MS/MSPeptide Identification:
Sequence from ID
Fragments window shows theoretical ions that match to
spectrum
Label spectrum
easily from fragments window
Mascot Search Mascot Search ParametersParameters
Mascot Search ResultMascot Search Result
Quantitative proteomics
isobaric Tag for Relative and Absolute Quantitation • O16/O18
• ICAT• cICAT• iTRAQ• ICPL• 2D-DIGE• 2MEGA-labeling
1. Reporter Group: Fragmentation of reagent produces a charger reporter ion of mass 114, 115, 116, or 117.
2. Balance Group: Balance mass changes in concert with reporter mass to maintain a total mass of 145.
3. Isobaric Tag: Labeling/mixing of up to four samples with each tag produces peptides with identical m/z. No increase in MS complexity.
4. Protein Reactive Group: The reactive group covalently links the iTRAQTM Reagent to the peptides at the free amines (N-terminus and lysine side chain).
Isobaric Tagging - General Method (4-Plex)P E P T I D E
P E P T I D E
P E P T I D E
+
+
+
+
P E P T I D E
-PRG114 31
-PRG115 30
-PRG116 29
-PRG117 28
1347.0 1349.6 1352.2 1354.8 1357.4 1360.0Mass (m/z)
1352.84
-Reporter-Balance-Peptide INTACT- 4 samples identical m/z
MS
114
115
116
117
111.0 112.8 114.6 116.4 118.2 120.0
Mass (m/z)
114
116
115
117
- Reporter ions DIFFERENT
Mix -N H
P E P T I D E114 31-N H
P E P T I D E115 30-N H
P E P T I D E116 29-N H
P E P T I D E117 28 MS/MS
9.0 292.8 576.6 860.4 1144.2 1428.0
Mass (m/z)
8396.7
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sity y8
P
A b2
y10
q,H
72.1 b4
b1
45.1
b7
LT 112.
1
y3 b10
y5 b9
y2 b6
74.1 13
52.8
y6
142.
1
39.0 y4 b
8
y9 y11
- Peptide fragments EQUAL
P E P T I D EP E P T I D E
Precursor Ion Scan Precursor Ion Scan 用途用途•Small Molecule – 在一堆混合物中尋找代謝物 (Metabolite)標的– 在一堆混合物中尋找合成副產物 (side product)標的– 需知道主結構 (指標性子離子 ) 的分子量– 需要High sensitivity, High speed
•Proteomics– 在一堆混合物中尋找PTM標的– 需知道主結構 (指標性子離子 , 如磷酸根 ) 的分子量– 需要High sensitivity, High speed
Scan Precursors Select ProductCAD
Q1 Q3Q2
Only ions passed through Q1 that produce the PTM-specific fragment mass (e.g. 79 for phosphorylation or 204 for glycosylation) will produce signal at the detector
Precursor ion scan of –79Negative ion mode
Full scan MS (400-1700)Positive ion mode
-casein tryptic digest
Precursor ion scan of –79Negative ion mode
Full scan MS (400-1700)Positive ion mode
-casein tryptic digest
Precursor Ion Scanning (PTM-specific scan)
e.g. Identification of Phosphopeptides in a mixture
phosphopeptides
Matrix Crystals
Embedded Proteins
Chip Surface
Surface Enhanced Laser Desorption/Ionization (SELDI)
• Desorption/ionization method of non-volatile compounds in which the sample presenting surface plays an active role in the extraction, presentation, structural modification, amplification, and/or ionization of a given sample
Invented by T. William Hutchens and Tai-Tung Yip in early 1990s
ProteinChip Technology: Protein Binding
• Crude sample is placed (and processed) on a ProteinChip Array
• Proteins bind to chemical or biological “docking sites” on the ProteinChip surface
ProteinChip Arrays:Available proprietary
surfaces
Hydrophobic Ionic
Chemical Surfaces
Antibody DNA Enzyme Receptor
Biochemical Surfaces
Drug
IMAC
Si
HOOH
Si
OH
SiSi
HO
Hydrophilic
My+ My+
ProteinChip Technology: SELDI TOF-MS Detection
• Retained proteins are “eluted” from the ProteinChip Array by Laser Desorption/Ionization
• Ionized proteins are detected and their mass accurately determined by Time-of-Flight Mass Spectrometry
Inte
nsi
ty
Molecular Mass (Da)
Dete
ctor
Dete
ctor
Laser
TOF-MS
Data presentation:2500 5000 7500 10000 12500
2500 5000 7500 10000 12500
Ret Map 1-4
Ret Map 1-3
Ret Map 1-4(2)
Ret Map 1-3(2)
comp1Difference map
Patient
Control
GelViewTM
Precursor ion fixed
Product ion fixed
Fragmentation(CAD)
MS/MS Multiple Reaction Monitoring (MRM)
127 Da85 Da68 Da
蛋白體研發相關「技術服務產業」蛋白體研發相關「技術服務產業」
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