蛋白質體學
Proteomics 2011
1. Mass Spectrometry- Review
陳威戎
2011. 12. 05
1
Mass Spectrometry- Review
1. Mass spectrometer compositions
2. Mass spectrum at a glance
3. Difficulty of sample ionization
4. Matrix Assisted Laser Desorption/Ionization
(MALDI)
5. Electrospray Ionization (ESI)
6. Mass analyzer types- TOF, Q, Iontrap
7. Type of mass spectrometry
8. MALDI-TOF MS vs. LC-ESI-Q-TOF MS/MS
Mass spectrometer composition
Mass spectrum at a glance
A. The Y axis is relative intensity
B. The X axis is m/z
C. The tallest peak in the
spectrum: "base peak“
D. The counts associated with the
tallest peak in the spectrum
E. Other peak(s)
Difficulty of sample ionization
• Solid phase  Gas phase
– polar, non-volatile
– molecules : kDa
– gas phase
• “Soft” Methods:
– Matrix Assisted Laser Desorption Ionization
(MALDI)
– Electrospray Ionization (ESI)
Matrix Assisted Laser Desorption
Ionization (MALDI)
Animation
Matrix Selection
Electrospray ionization (ESI)
•
Generates ions directly from acidic solution
Electrospray ion generation
Cys+Ser+Arg
Nanospary ionization
• Flow Rates:
– ESI: 100 ml/min
– Nanospary: 100-300 nl/min
• Reduce the sample amount
• Increase the analysis time
Mass analyzer types
• Time of flight (TOF)
• Quadrupole
• Ion-Trap
• Consideration:
– Accuracy
– Mass range
Time-of-Flight (TOF)
• Smaller ions faster
• high sensitivity, unlimited mass
range
• Accuracy: MALDI
• Resolution: reflection instruments
Animation
Reflection TOF
Animation
Quadrupole mass analyser
• +(U+Vcos(wt)), -(U+Vcos(wt))
– U: fixed potential
– Vcos(wt): radio frequency (RF) field of amplitude V and frequency w
Ion trap analyzer
• Components:
– ring electrode
– entrance endcap electrode
– exit endcap electrode
• 3D quadrupolar potential field
• Trajectory: trapping potential and
the m/z of the ions
• advantages:
– multiple CID without multiple
analyzers
– compact size
– trap and accumulate ions to increase
the signal-to-noise ratio
Nobel Prize Winners related to MS
Type of Mass Spectrometry
•
•
•
•
•
•
MALDI-TOF
SELDI-TOF
MALDI-Quardrupole-TOF
ESI-Quardrupole-TOF
ESI-Triple Quardrupole
ESI-Iontrap
MALDI-TOF Mass Spectrometry
Reflectron
Detector
Sample plate
Quardrupole-TOF Mass
Spectrometry
Nanospray ESI-Q-Iontrap Mass
Spectrometry
Important performance factors
Higher resolution: Better mass
accuracy
General comparison of mass analyzers
Thermo Finnigan LCQ-Deca
Interpreting Electrospray Mass spectra Calculating mass
Charge State
Calculation
Unprotonated Mass
+1
379.2 x 1 - 1
378.2
+2
190.1 x 2 - 2
378.2
Isotopic Effect
Multiple charged state
hen egg lysozyme
Platform II, BMB, The University of Leeds
25-Jan-200010:00:37
LYS01A 1 (1.392) Sm (SG, 2x1.00); Sb (10,10.00 )
A9;1590.6
100
A8
1789.2
A:
Scan ES+
2.16e6
14305.67±0.42
A10
1431.6
A11
1301.4
%
A12
1193.1
1601.4
1801.3
A7
2044.6
1441.3
A13
1101.5
2049.7
1310.5
0
m/z
1000
1200
1400
1600
1800
2000
2200
• Lys and Arg
• (M+nH)n+ in positive ionisation mode
• The m/z values can be expressed as follows:
2400

m Mw  nH 

z
n

Mw  nH 
1431.6 

n
Mw  n  1) H  
1301.4 
n  1


n 1431.6   nH   Mw  n  11301.4  n  1H 
n 1431.6  1301.4   1301.4  H 
n
1301.4  1.008
 10
1431.6  1301.4
( Mw  nH  )
1431.6 
n
 1431.6 x10  Mw  10 x (1.008)
 Mw  14316  10.08  14305.9 Da  14.3kDa
• Theoretical Mw of hen egg lysozyme (based on
average atomic masses): 14305.1438 Da.
Mass spectra comparison between
MALDI-QTOF and ESI-QTOF
Protein identification
蛋白質體學研究工作站
MALDI-TOF MS
Picker & Digestion
robotics
Database Search Workstation
LC-Triple Quad MS
質譜儀 (MS)
• MALDI：
• ESI：
Matrix-assisted laser
desorption/ionization
Electrospray ionization
基質輔助雷射脫附/離子化
電噴灑法離子化
固態樣品
液態樣品
優點: 對鹽類耐受度高
優點: 可產生穩定的複數帶
電離子, 降低質荷比(m/z),
有利於巨大分子之分析
缺點: 僅生成單電荷離子,
不易對大分子進行分析
缺點: 對鹽類耐受度低
基質輔助雷射脫附離子化質譜儀 (MALDI-TOF MS)
液相層析電灑法離子化串聯式質譜儀 (LC-ESI MS/MS)
蛋白質身份鑑定- 胜肽質量指紋 (PMF)
Protein identification by peptide
mass fingerprinting (PMF)
蛋白質身份鑑定- 胜肽質量指紋 (PMF)
Gel
?
Database
Protein
tryptic
digestion
MS Analysis
1
783.9147
375.2561
3
stored data or theoretical peptides “
3235.2259
1234.5396
1234.5391
819.4146
340.3865
783.9142
324.7345
305.7118
299.2559
375.2587
783.9358
504.6673
340.3466
305.7113
783.9444
3235.2256
1234.5396
2
647.4367
296.5783
蛋白質身份鑑定- 串聯式質譜定序法 (MS/MS)
Protein identification by MS/MS
蛋白質身份鑑定- 串聯式質譜定序法 (MS/MS)
MS/MS spectrum of GLSDGEWQQVLNVWGK
Peptide Sequence from CID: b, y series