Document

advertisement
Primary Ionization Techniques for
Molecules
Electrospray Ionization (ESI)
Matrix Assisted Laser Desorption Ionization (MALDI)
Matrix Assisted Laser Desorption
Ionization (MALDI)
Laser
m
m
+
a
a
+
m a
m
+
a m
a
a
+
m
m
m +
m
m
matrix + analyte
Sample support
Matrix Assisted Laser Desorption
Ionization (MALDI)
Laser
Sample plate
hn
1. 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:
AH+
MH+ + A  M + AH+.
Variable Ground
+20 kV
Grid
Grid
MALDI/TOF Mass Spectrum
(M+H)+
Relative Abundance
40000
30000
(M+2H)2+
20000
10000
(M+3H)3+
0
50000
100000
m/z
150000
200000
Matrix
CH O
3
COOH
CH
C (C N )C O O H
CH
CHCOOH
OH
HO
HO
HO
-cyano-4-hydroxycinnamic acid
2,5-dihydroxybenzoic acid
(2,5-DHB)
CH O
3
Sinapinic acid
(3,5-Dimethoxy-4-hydroxy cinnamic acid)
Sample 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.
Time of Flight (TOF)
Calibration 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”).
m
z
2

2t K
L
2
t
L
m
K
z
=
=
=
=
=
Drift time
Drift length
Mass
Kinetic energy of ion
Number of charges on ion
MALDI: Matrix Assisted Laser Desorption
Ionization
Sample plate
hn
1. Sample (M) is mixed with
excess matrix (X) and
dried on a MALDI plate.
to Mass
Analyzer
Sample &
Matrix
Ground
Grid
The plate is loaded onto
the sample stage in the
Ion Source
MALDI: Matrix Assisted Laser Desorption
Ionization
Laser
hn
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: Matrix Assisted Laser Desorption
Ionization
hn
MH+
+20 kV
4. Ion Extraction:
High voltage is applied
to the sample plate,
accelerating ions out of
the Ion Source into the
Flight Tube.
Time-of-Flight Mass Analyzer
Ion Source
Flight Tube
20-25 kV
+ +
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.
Detector
Time-of-Flight Mass Analyzer
Ion Source
Flight Tube
+
Detector
+
+
The ions enter the flight tube with the lighter ions
travelling faster than the heavier ions to the detector
Time-of-Flight Mass Analyzer
Ion Source
Flight Tube
+
+
+
The lighter ions strike the detector before the heavier ions.
This “time of flight” (TOF) can be converted to mass
Detector
Principle of MALDI-TOF-MS
Vacuum
lock
Vacuum system
Sample Analyte Acceleration
plate molecules
grids
in matrix
Drift tube
Mass spectrum
Ion detector
High resolution TOF-MS with ion
reflector
MALDI ion
source
Ion
detector
The reflector focuses ion of same
mass but different velocity on
detector; high resolution is obtained
Ion reflector
HiRes mass spectrum
TOF/TOF-MS/MS with
CID
LID
MALDI ion
source
Parent ion
selector
Ion
detector
MS/MS spectrum of daughter ions
is measured in a single acquisition;
no pasting of segments;
low sample consumption,
high speed, high sensitivity
Ion reflector
Daughter ion mass spectrum
Mass analyzers
Ions are detected with a Microchannel Plate
-1000 V
-100 V
D= 6-25 u
+
Primary Ion from
Flight Tube
L
Ions are detected with a Microchannel Plate
-1000 V
-100 V
+
D= 6-25 u
L
Ions are detected with a Microchannel Plate
-1000 V
-100 V
D= 6-25 u
Multification by secondary emission
+
e-
Secondary emissive materials:
Beryllium oxide, magnesium oxide etc
L
Ions are detected with a Microchannel Plate
-1000 V
-100 V
D= 6-25 u
ee- e-
+
e-
L
Ions are detected with a Microchannel Plate
-1000 V
-100 V
D= 6-25 u
ee- e-
+
e-
L
Ions are detected with a Microchannel Plate
-1000 V
-100 V
D= 6-25 u
eee-
e-
e-
e- e
e-
+
e- e-
ee-
~103
Amplification
L
Why 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 데이터
가 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 plate에 떨어뜨려 용액을 말리는 시간(약 5~10 분), MALDI-TOF
로 분석하는 시간 (1분 이내)
 기기 사용 및 유지하기 위한 비용이 저렴 : LC/MS, GC/MS 처럼 질소 또는 아르곤 가스를 사용하지 않고, 미
량의 Matrix와 ACN, TFA등을 이용함으로 시약 비용도 저렴함
Download
Related flashcards

Symbols

28 cards

Electric power

25 cards

Transducers

13 cards

Electrical components

22 cards

Automation

13 cards

Create Flashcards