Peptide Mass Finger-Printing
Part II. MALDI-TOF
Y o n s e i
P r o t e o m e
R e s e a r c h
C e n t e r
2013 생화학 실험 (1) 6주차 자료
임종선 조교
[email protected]
내선 6625
Yonsei Proteome Research Center
Concept of Mass Spectrometry
Mass Spectrometer?
• Instrument measuring molecular weight (MW) of sample
• Only picomolar concentrations is required
• Accuracy of 0.01% of total weight of sample
• Able to detect amino acid substitution/post-translational
modifications
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Concept of Mass Spectrometry
History of Mass spectrometry
• 1953 : Quadrupole and the ion trap(W. Paul at H.S. Steinwedel).
Nobel Prize to Paul in 1989.
• 1956 : First GC-MS
• 1968 : First commercial quadrupole
• 1975 : First commercial GC-MS
• 1990s : Explosive growth in biological MS, due to ESI & MALDI
• 2002 : Nobel Prize to Fenn & Tanaka for ESI & MALDI
• 2005 : Commercialization of Orbitrap MS
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Concept of Mass Spectrometry
How does Mass Spectrometer work?
Ion
Source
Inlet
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Mass
Analyzer
Detector
Concept of Mass Spectrometry
Types of Machines / Techniques
High Vacuum
System
1. Inlet
2. Ion
Source
HPLC
Flow
injection
Sample
plate
MALDI
ESI
FAB
LSIMS
EI
CI
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3. Mass
Analyze
r
Time of flight
(TOF)
Quadrupole
Ion Trap
Magnetic
Sector
FTMS
4.
Detecto
r
5. Data
System
Microchannel
plate
Photomultiplier
Electron
multiplier
Concept of MALDI-TOF
“Sample Preparation”
Peptide Sample Preparation
gel
Excise
Extract
Wash
Dry
Digest
Reconstruction
Inlet
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Concept of MALDI-TOF
“1. Inlet: MALDI-TOF matrix”
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Concept of MALDI-TOF
“2. Ion Source: MALDI”
(Matrix Assisted Laser Desorption Ionization)
Sample plate
Laser
hn
Ionization is triggered by
a laser beam
AH+
+20 kV
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A matrix is used to
protect the biomolecule
from being destroyed by
direct laser beam and to
facilitate vaporization
and ionization
Concept of MALDI-TOF
“2. Ion Source: MALDI”
(Matrix Assisted Laser Desorption Ionization)
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Question
“2. Ion Source: MALDI”
(Matrix Assisted Laser Desorption Ionization)
Which ion will strike the detector faster?
Laser
The ions enter the flight
tube with the lighter ions
traveling faster than the
heavier ions.
SO, GREEN ION WILL
STRIKE THE DETECTOR
FASTER!
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Concept of MALDI-TOF
“3. Mass Analyzer: TOF”
(Time Of Flight)
+22 kV
Reflectron
TOF
0 kV
1) Ions enter source region,
accelerated toward reflectron.
2) Ions separate in space based on
their relative mass-to-charge (m/z).
0 kV
3) Ions reverse path in reflectron.
Signal
4) Ions impact detector.
0 kV
0 kV
+ + +
LM
H
+20 kV
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Flight time
amp
comp
[Molecular weight]
Concept of MALDI-TOF
“3. Mass Analyzer: TOF”
(Time Of Flight)
Linear TOF
Linear TOF is used in larger molecules.
*we are going to use reflectron TOF.
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Concept of MALDI-TOF
“4. Detection”
Mass accuracy : How accurate is the mass measurement?
Resolution : How well separated are the peaks from each other?
Sensitivity : How small an amount can be detected / analyzed?
Yonsei Proteome Research Center
Concept of MALDI-TOF
“4. Detection”
Massmeasurement
measurement accuracy
depends
on Resolution
Mass
accuracy
depends
on
resolution
High resolution means better mass accuracy
Counts
8000
Resolution =18100
15 ppm error
6000
Resolution = 14200
24 ppm error
4000
2000
Resolution = 4500
55 ppm error
0
2840
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2845
2850
Mass (m/z)
2855
Concept of MALDI-TOF
“4. Detection”
Theoretical MALDI TOF
SPECTRUM
Of ONE PEPTIDE
149876
MH+
Relative Abundance
40000
30000
(M+2H)2+
20000
10000
0
(M+3H)3+
150000
50000
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100000
150000
Mass (m/z)
200000
Concept of MALDI-TOF
“Monoisotopic
mass”
No 13C atoms
(all 12C)
149876
“4. Detection”
One 13C atom
Two 13C atoms
Three 13C
atoms
150000
m/z
We calculate resolution and accuracy with these peaks.
Annotations on spectra will be for the monoisotopic peaks only.
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Concept of MALDI-TOF
“4. Detection”
Assume these are
peaks found at
[M+4H]4+
How to calculate mass?
m/z of monoisotopic peak =
431.73
m/z = 431.73
m/4 = 431.73
m=431.73 x 4=1726.92
You must subtract mass of H+ (1)
[ 1726.92] – 4 = 1722.92
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Question
“4. Detection”
Theoretical
Results
(M+3H)3+
(M+4H)4+ 3334
(M+2H)2+
5+
2501
(M+5H)
5001
2001
Relative
MH+
10001
Intensity(%)
0
2500
5000
10000
Assume this is a result of MALDI-TOF for ONE SINGLE PEPTIDE.
Please calculate the mass of this peptide.
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Solution
“4. Detection”
3334 3+
(M+3H)
2501 4+
5001
2001 (M+4H)
(M+2H)2+
(M+5H)5+
Relative
10001
MH+
Intensity(%)
0
H+
H+
H+
2500
H+
H+
H+
H+
[M + 5H]5+
5000
H+
H+
H+
[M + 4H]4+
10000
H+
H+
[M + 3H]3+
H+
H+
[M + 2H]2+
H+
[MH]+
M/Z = 10,005 / 5
M/Z = 10,004 / 4
M/Z = 10,003 / 3
M/Z = 10,002 / 2
M/Z = 10,001 / 1
M/Z = 2001
M/Z = 2501
M/Z = 3334
M/Z = 5001
M/Z = 10,001
The same protein with a molecular weight of 10,000 contains 5, 4, 3, 2, and 1 charges
Yonsei Proteome Research Center
Concepts of MALDI-TOF
“5. Database”
N
R
K
K
Protein
N
K
K
K
K
Trypsin
K
R K
K
K
K
R
C
R
C
K
R
R
R
R
Tryptic peptide mixture.
Masses measured by MS.
Every peptide has a basic C-terminus.
A protein can be identified in a database by matching masses of a subset of
the tryptic peptides against calculated values.
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Concepts of MALDI-TOF
“5. Database”
intact
protein
enzyme
peptide
fragments
MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS
PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW
EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK
SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE
SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL
LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE
PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK
GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED
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HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT
Concepts of MALDI-TOF
“5. Database”
Database
Gel
In Silico Digestion
In Gel Digestion
848.1
1272.5
492.6
883.2
2978.9
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848.3
1272.7
493.2
882.6
2978.3
364.1
948.9
3128.8
812.6
1432.3
3127.1
996.8
702.4
164.9
2748.2
is identical
to
3514.2
2837.1
263.9
147.4
1429.7
199.6
142.3
640.8
Concepts of MALDI-TOF
“5. Database”
Yonsei Proteome Research Center
실험 방법
• 준비물 :
Desalting : Poros buffer(C18 resin in 70% ACN), Zip tip, 100% ACN,
2% Formic Acid, MATRIX buffer(CHCA 8~10mg in 70% ACN)
순서 : 1. zip tip의 끝 0.3~0.5cm 정도를 구부린다.
2. Poros buffer를 3~4ul넣어 실린지로 밀어준다. Zip tip 끝에 3~5mm정도
충진되도록
3. 100% CAN으로(10ul) C18 resin을 wash해준다.
4. 2% F.A로(20ul) C18 resin을 activation 시킨다.
5. ingel-digested sample을 흘려준다.
6. 2% F.A로(10ul) peptide 외의 chemical을 wash해준다.
7. Matrix buffer로 Matrix와 함께 펩타이드를 회수한다. Plate 위에 buffer를
loading (1ul)
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실험 방법
•
MALDI-TOF analysis
1. Plate를 MALDI 기기 안으로 injection한다.
2. 기기의 laser를 켜고 약 10-30분간 laser를 안정화 시킨다.
3. plating한 Standard sample을 큰 오차범위에서 작은 오차범위로 서서히 좁혀
가며 찍어본다.
- Calibration : 기기의 오차범위를 줄여줌 Resolution / peak intensity 를 확인하
며, laser의 강도 / mirror의 위치를 조정하여 분석에 최적화시킨다.
4. Strandard sample을 분석한 후 분석하고자 하는 원래의 시료를 분석한다.
5. 분석 후 결과 spectrum을 추출 프로그램(Data explorer)을 사용하여,
spectrum list를 작성한다.
6. Data search engine (profound ; Mascot; MS-fit)에 입력한다. 이 떄 시료에
사용한 효소, 시약으로 인한 modify를 지정하고, 시료의 종(taxonomy) 등을 설
정하여 준다.
7. 분석된 결과를 토대로 유의성 여부를 검토한다.
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