1. Peptide Mass Finger-Printing Part II. MALDI-TOF
실험조교: 정혜림
Aging Genomics Proteomics Laboratory (Professor Paik)
New Science Building Room #305

2. 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

3. 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

4. Concept of Mass Spectrometry
How does Mass Spectrometer work?
Ion
Source
Mass
Analyzer
Detector
Inlet

5. Concept of Mass Spectrometry
Types of Machines / Techniques
High Vacuum System
2. Ion
Source
3. Mass
Analyzer 4.
Detector
5. Data
System
1. Inlet
HPLC
Flow injection
Sample plate
MALDI
ESI
FAB
LSIMS EI CI
Time of flight (TOF)
Quadrupole
Ion Trap
Magnetic Sector
FTMS
Microchannel plate
Photomultiplier Electron multiplier

6. Concept of MALDI-TOF “Sample Preparation” Inlet
Peptide Sample Preparation
gel
Digest
Excise
Wash
Reconstruction
Extract
Dry
Inlet

7. “1. Inlet: MALDI-TOF matrix”
Concept of MALDI-TOF
“1. Inlet: MALDI-TOF matrix”

8. (Matrix Assisted Laser Desorption Ionization)
Concept of MALDI-TOF
“2. Ion Source: MALDI”
(Matrix Assisted Laser Desorption Ionization)
Sample plate
Laser hn
Ionization is triggered by a laser beam
A matrix is used to protect the biomolecule from being destroyed by direct laser beam and to facilitate vaporization and ionization
AH+
+20 kV

9. (Matrix Assisted Laser Desorption Ionization)
Concept of MALDI-TOF
“2. Ion Source: MALDI”
(Matrix Assisted Laser Desorption Ionization)

10. 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!

11. Concept of MALDI-TOF “3. Mass Analyzer: TOF” (Time Of Flight) H+
+22 kV
1) Ions enter source region, accelerated toward reflectron.
Reflectron TOF
2) Ions separate in space based on their relative mass-to-charge (m/z).
0 kV
0 kV
3) Ions reverse path in reflectron.
4) Ions impact detector.
Flight time
Signal
0 kV
0 kV L+ M+ H+
[Molecular weight]
+20 kV
amp
comp

12. 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.

13. 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?

14. High resolution means better mass accuracy
Concept of MALDI-TOF
“4. Detection”
Mass measurement accuracy depends on resolution
High resolution means better mass accuracy
2000
4000
6000
8000
Counts
2840
2845
2850
2855
Mass (m/z)
Resolution = 14200
Resolution = 4500
Resolution =18100
15 ppm error
24 ppm error
55 ppm error

15. Theoretical MALDI TOF SPECTRUM
Concept of MALDI-TOF
“4. Detection”
Theoretical MALDI TOF SPECTRUM
Of ONE PEPTIDE
MH+
10000
20000
30000
40000
149876
Relative Abundance
(M+2H)2+
(M+3H)3+
150000
50000
100000
150000
200000
Mass (m/z)

16. Concept of MALDI-TOF “4. Detection”
One 13C atom
“Monoisotopic mass”
149876
Two 13C atoms
No 13C atoms (all 12C)
Three 13C atoms
150000
m/z
We calculate resolution and accuracy with these peaks.
Annotations on spectra will be for the monoisotopic peaks only.

17. Assume these are peaks found at [M+4H]4+
Concept of MALDI-TOF
“4. Detection”
Assume these are peaks found at [M+4H]4+
How to calculate mass?
m/z of monoisotopic peak =
m/z =
m/4 =
m= x 4=
You must subtract mass of H+ (1)
[ ] – 4 =

18. Please calculate the mass of this peptide.
Question
“4. Detection”
Theoretical Results
(M+3H)3+
3334
(M+4H)4+
MH+
(M+2H)2+
2501
(M+5H)5+
10001
5001
2001
Relative
Intensity(%)
2500
5000
10000
Assume this is a result of MALDI-TOF for ONE SINGLE PEPTIDE.
Please calculate the mass of this peptide.

19. Solution “4. Detection” 3334 2501 (M+3H)3+ 10001 (M+4H)4+ 5001 2001
Relative
Intensity(%)
(M+5H)5+
2500
5000
10000 H+ H+ H+ H+ H+
[M + 5H]5+
[M + 4H]4+
[M + 3H]3+
[M + 2H]2+
[MH]+
M/Z = 10,005 / 5
M/Z = 2001
M/Z = 10,004 / 4
M/Z = 2501
M/Z = 10,003 / 3
M/Z = 3334
M/Z = 10,002 / 2
M/Z = 5001
M/Z = 10,001 / 1
M/Z = 10,001
The same protein with a molecular weight of 10,000 contains 5, 4, 3, 2, and 1 charges

20. Tryptic peptide mixture. Every peptide has a basic C-terminus.
Concepts of MALDI-TOF
“5. Database” N K K R K K K R K K
Trypsin K K K R R R
Protein R R N C K K R
Tryptic peptide mixture.
Masses measured by MS.
Every peptide has a basic C-terminus. C
A protein can be identified in a database by matching masses of a subset of the tryptic peptides against calculated values.

21. Concepts of MALDI-TOF “5. Database” intact protein peptide fragments
enzyme
MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS
PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW
EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK
SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE
SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL
LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE
PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK
GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED
HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT

22. Concepts of MALDI-TOF “5. Database” Gel is identical to Database
In Silico Digestion
In Gel Digestion
848.3
1272.7
493.2
882.6
2978.3
364.1
948.9
3128.8
3514.2
2837.1
263.9
147.4
1429.7
199.6
142.3
640.8
848.1
1272.5
492.6
883.2
2978.9
812.6
1432.3
3127.1
996.8
702.4
164.9
2748.2
is identical to

23. Concepts of MALDI-TOF
“5. Database”

24. Desalting method Calibration method
Techniques for MALDI-TOF
Experimental Methods – Tips
Desalting method
1) C18 Zip-Tip: Tip containing immobilized C18 2) TFA: Trifluoroacetic acid 3) Matrix solution: matrix mixed with 50% ACN with water
Calibration method
1) Standard solution: Examples include Angiotensin II, ACTH fragment

25. 보고서작성
Tips
Format: 실험제목, 실험일자, 제출자 및 공동실험자, 목적, 원리, 시약 및 기자재, 방법, 결과, 고찰, 참고문헌 순서로 써주세요.
(실험일자에 요일 꼭 써주세요!)
(방법은 실험교본 그대로 말고! 직접 실험한 방법!)
(고찰 section에 꼭 필요한 내용: DTT와 IAA 사용하는 이유, desalting method 설명, calibration 하는 이유와 방법 설명, 결과에 대한 설명)
DUE DATE: 4월 17일 (월요일 저녁 8시까지)
SUBMISSION LOCATION: 과학원 S305 