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Electrospray Ionization (ESI)Electrospray Ionization (ESI)
Matrix Assisted Laser Desorption Ionization (MALDI)Matrix Assisted Laser Desorption Ionization (MALDI)
Primary Ionization Techniques for Primary Ionization Techniques for MoleculesMolecules
LaserLaser
matrix + analytematrix + analyte
Sample supportSample support
aa
mm
mm
mm
mm
mm
mm
mmmm aa
aa
aa
aa
++
++
++
++mm
aamm++
Matrix Assisted Laser Desorption Matrix Assisted Laser Desorption Ionization (MALDI)Ionization (MALDI)
Matrix Assisted Laser Desorption Matrix Assisted Laser Desorption Ionization (MALDI)Ionization (MALDI)
h
Laser
+20 kV
Variable Ground Grid Grid
AH+
Sample plate1. Sample (A) is mixed with
excess matrix (M) and dried on a MALDI plate.
2. Laser flash ionizes matrix molecules.
3. Sample molecules are ionized by proton transfer from matrix:
MH+ + A M + AH+.
MALDI/TOF Mass SpectrumMALDI/TOF Mass SpectrumR
ela
tive
Abu
nda
nce
m/z
0
10000
20000
30000
40000
50000 100000 150000 200000
(M+H)+
(M+2H)2+
(M+3H)3+
MatrixMatrix
HO
CH C(CN)COOHOH
HO
COOH
HO
CH3O
CH3O
CH CHCOOH
-cyano-4-hydroxycinnamic acid 2,5-dihydroxybenzoic acid(2,5-DHB)
Sinapinic acid(3,5-Dimethoxy-4-hydroxy cinnamic acid)
HO
CH C(CN)COOHOH
HO
COOH
HO
CH3O
CH3O
CH CHCOOH
-cyano-4-hydroxycinnamic acid 2,5-dihydroxybenzoic acid(2,5-DHB)
Sinapinic acid(3,5-Dimethoxy-4-hydroxy cinnamic acid)
Sample Dilution/ConcentrationSample Dilution/Concentration•Dilute samples to the concentrations shown in the table below.
•If the sample concentration is unknown a dilution series may be needed to produce a good spot on the MALDI plate.
Compound Concentration
Peptides and proteins 0.1 to 10 pmol/µL
Oligonucleotides 10 to 100 pmol/µL
Polymers 100 pmol/µL
Note: highly dilute samples can be concentrated by Speed-Vac or Solid Phase Extraction.
Calibration of the mass scaleCalibration of the mass scale
The mass-to-charge ratio of an ion is proportional to the square of its time of flight in the analyzer (“drift time”).
2
22
L
Kt
z
m
t = Drift timeL = Drift lengthm = MassK = Kinetic energy of ionz = Number of charges on ion
h1. Sample (M) is mixed with
excess matrix (X) and dried on a MALDI plate.
The plate is loaded onto the sample stage in the Ion Source
Ground Grid
Sample plate
to Mass Analyzer
Sample & Matrix
MALDI:MALDI: Matrix Assisted Laser Matrix Assisted Laser Desorption IonizationDesorption Ionization
h
Laser
2. Laser flash produces matrix neutrals (X), matrix ions (XH)+, (X-H)- , and sample neutrals (M).
3. Sample molecules are ionized by proton transfer from matrix ions:
XH+ + M X + MH+.
X-H- + M X + M-H-
MALDI:MALDI: Matrix Assisted Laser Matrix Assisted Laser Desorption IonizationDesorption Ionization
h4. Ion Extraction:
High voltage is applied to the sample plate, accelerating ions out of the Ion Source into the Flight Tube.
MH+
+20 kV
MALDI:MALDI: Matrix Assisted Laser Matrix Assisted Laser Desorption IonizationDesorption Ionization
Flight Tube
Detector
Ion Source
Time-of-Flight Mass AnalyzerTime-of-Flight Mass Analyzer
Principle: If ions are accelerated with the same potential at a fixed point and a fixed initial time and are allowed to drift, the ions will separate according to their mass to charge ratios.
20-25 kV
++
Flight Tube
Detector
Ion Source
The ions enter the flight tube with the lighter ionstravelling faster than the heavier ions to the detector
+
+ +
Time-of-Flight Mass Analyzer
Flight Tube
Detector
Ion Source
The lighter ions strike the detector before the heavier ions.This “time of flight” (TOF) can be converted to mass
+
++
Time-of-Flight Mass Analyzer
Laser
Sampleplate
Analytemolecules in matrix
Accelerationgrids
Drift tube Ion detector
Mass spectrum
Vacuum system
Vacuumlock
Principle of MALDI-TOF-MSPrinciple of MALDI-TOF-MS
HiRes mass spectrum
Iondetector
MALDI ionsource
Ion reflector
The reflector focuses ion of same mass but different velocity on
detector; high resolution is obtained
Laser
High resolution TOF-MS with ion High resolution TOF-MS with ion reflectorreflector
Daughter ion mass spectrum
Iondetector
Ion reflector
MALDI ionsource
CID
MS/MS spectrum of daughter ionsis measured in a single acquisition;
no pasting of segments;low sample consumption,
high speed, high sensitivity
LID
Laser
TOF/TOF-MS/MS withTOF/TOF-MS/MS with
Parent ionselector
+
-1000 V -100 V
Primary Ion from Flight Tube
L
D= 6-25 u
Ions are detected with a Microchannel PlateIons are detected with a Microchannel Plate
+
-1000 V -100 V
L
D= 6-25 u
Ions are detected with a Microchannel PlateIons are detected with a Microchannel Plate
+
-1000 V -100 V
e-
L
D= 6-25 u
Ions are detected with a Microchannel PlateIons are detected with a Microchannel Plate
Multification by secondary emission
Secondary emissive materials: Beryllium oxide, magnesium oxide etc
+
-1000 V -100 V
e-e- e-
e-
L
D= 6-25 u
Ions are detected with a Microchannel PlateIons are detected with a Microchannel Plate
+
-1000 V -100 V
e-e- e-
e-
L
D= 6-25 u
Ions are detected with a Microchannel PlateIons are detected with a Microchannel Plate
+
-1000 V -100 V
e-e- e-
e-
L
D= 6-25 u
e-
e-
e-
e-
e-
e-e-
e-
~103
Amplification
Ions are detected with a Microchannel PlateIons are detected with a Microchannel Plate
Why interested in MALDI-TOF MSWhy interested in MALDI-TOF MS 분자량 측정
큰분자량 물질 분석
혼합물 분석 : 한 종류의 성분이 아닌 몇 종류가 혼재해 있어도 분석이 가능함
미량분석 : 매우 민감하여 미량의 시료도 분석 가능 함 : 펩타이드 경우 fmol 분석 가능
데이터 분석이 쉬움 : 분자 구조가 깨어 지지 않고 , 보통 다 전하를 (multiple charging) 띠지
않으므로 데이터가 다른 질량 분석기에서 보다 단순하여 해석이 용이함
염의 영향이 적음 : 생체단백질 분리에 이용되는 완충용액 , 염 등에 LC/MS 보다 영향을 덜 받음
분석이 신속함 : 시료와 Matrix 섞어 sample plate 에 떨어뜨려 용액을 말리는 시간 ( 약 5~10
분 ), MALDI-TOF 로 분석하는 시간 (1 분 이내 )
기기 사용 및 유지하기 위한 비용이 저렴 : LC/MS, GC/MS 처럼 질소 또는 아르곤 가스를 사용하지 않
고 , 미량의 Matrix 와 ACN, TFA 등을 이용함으로 시약 비용도 저렴함