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.

Time of Flight (TOF)Time of Flight (TOF)

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

Mass analyzersMass analyzers

+

-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