MASS Spectrometry Physical Basis

Differential deflection of charged species in a magnetic field. Two species, of identical charge and velocity but different mass, will be deflected differentlyby a magnetic field. The species with the smallestmomentum, is deflected to a greater extent thanthat with larger

MASS Spectrometry Physical Basis

Electron Impact ionisation. Sample is bombarded with a high-energy beamof electrons which causes ionisation.These ions are subsequently accelerated and deflected in the analyser

Samplein

Ionisationchamber

Electronsoruce

Chargedparticles

Toanalyseranddetector

Ionisation of lactic acid by electron impact. Latic acid is ionised by electrons forming a molecular ion. This breaks down into a large nmber of fragment ions, each possessing a characteristic mass allowing their identification. The pattern of ions generated is characteristic of the chemical structure of the parent molecule and can therefore be used to calculate this structure

Composition of MASS Spectrometry

Turbo pumpsDiffusion pumpsRough pumps Rotary pumps

•Sample plate•Target•HPLC•GC•Solids probe•DCI

•MALDI•Electro-spray (IonSpray)•APCI•FAB•LSIMS•EI•CI

•TOF•Quadrupole•Ion Trap•Magnetic Sector•FTMS

•Micro-channel Plate•Electron Multiplier•“Hybrid”

•PC’s•Sun SPARKStation•DECStation

InletIon

sourceMass Filter

Detector Data

System

High Vacuum System

• EI (Electron ionization) - Filament 등의 thermionic emission 에 의해 방출되는 열전자를 이용한 이온화 . 이용하기가 매우 간단하고 용이함 . 비선택적 이온화 과정에서 fragmentation 되는 경향이 강함 .

• PI (Photoionization) – Lamp 나 laser 등에서 나오는 빛을 이용한 이온화 . 선택적으로서 들뜬 상태에 대한 정보를 필요로 함 . 많은 장비가 필요하나 fragmentation 이 심하지 않고 다양한 변형이 가능함 .

• ESI (Electrospray ionization) & TSI (Thermospray ionization) – 응축상 시료에 대한 전기장 또는 열을 이용한 분무방식의 vaporization/ionization. Multiply charged ion 이 생성 . Fragmentation 이 심하지 않고 액체등의 시료에 매우 적합함 .

Ionisation Modes

• CI (Chemical ionization) – 이온 / 분자간 화학반응에 의한 이온화 . EI 다음으로 널리 사용되며 양이온뿐만 아니라 음이온도 생성됨 .

• FI (Field ionization) – 양자역학적 electron tunneling 에 의한 이온화 . 거의 fragmentation 이 없슴 . 매우 낮은 감도 .

• FAB (Fast atom bombardment) ionization - 전하교환을 통해 얻어진 고속 원자를 액체 / 고체 시료에 충돌시켜 일어나는 분자의 ejection/ionization. 비휘발성 물질 및 쉽게 분해되는 분자들의 질량 분석에 적합 .

• MALDI (Matrix-assisted laser desorion/ionization) – matrix solution 에 laser 를 조사할 때 일어나는 탈착 및 이온화 . 휘발성이 작거나 잘 녹지 않는 시료에 적합함 . Fragmentation 이 적음 .

FAB

PDI

Continuous flow fast atom bombardment (FAB) Ionisation. Sample, held in a liquid matrix, is bombarded with a beamOf atoms such as xenon. Ions are formed in a dense gas which may be accelerated towards the analyser and detector.In continuous flow (dynamic) FAB, sample may flow continuously in a capillary passing through the probe as shown

Plasma Desorption Ionisation (PDI).252Cf breaks down to form two particles which travel in opposite directions at the same velocity. One of these activates a time counter (at t0) while the other causes vaporisation and ionisation of the sample. Smaller mass ions travel down the drift tube of the TOF analyser more quickly than larger ones. When ions arrive at detector, time of flight (t) is calculated

MALDI

Matrix-assisted laser desorption ionisation (MALDI). A pulse of laser light (UV) is directed at the sample which is Co-crystallised with a suitable matrix component. This matrix component absorbs most of the energy of the laser.Both matrix and sample are vaporised by the laser. In the gas phase, proton exchange occurs between matrix andSample components producing MH+ ions

ESIElectrospary ionisation (ESI). Sample is converted to a fine spray of droplets. Matrix is evaporated from this, leading to Coulombic explosion and the liberation of ions which then are directed to the analyser and detetor. In ion spray ionisation (ISI), the sample is surrounded by a sheath of gas such as N2 which imparts greater energy and hence facilitates faster input of sample to the system

APCI interface 의 원리(atmospheric pressure chemical ionisation)

APPI interface 의 원리(atmospheric pressure photoionization ionisation)

ESI APCI/APPI

휘발성을 요구하지 않는다 휘발성이 있어야 한다 .

열에 민감한 시료 열에 안정한 시료

용액에서 multiple charge 를 형성 작은 분자 , 비극성 분자

이온이 용매에서 형성 이온이 단일전하로 가스상에서 형성

Magnetic sector

Ion trap

MASS Analyzer

Ion trap analyser. Ions are either created within or rapidly infected into the trap. The electrostatic field forces them into specific orbits, depending on the m/z ratio. By varying this electrostatic field, ions may be selectively ejected in order of increasing m/z from the trap and detected

Quadrupole EI/MS

EI/MS source. Sample (shown as circles) is introduced as vapour into evacuated chamber. Electrons diffuse from the heated filament at an energy of 70 eV. The electron beam ionises sample molecules into anions or cations which are accelerated by an electric potential towards the analyser

Quadropole mass analyser. Varying the radiofrequency (RF) component of voltage allows ‘tuning’ for specific m/z ratios. Ions with either larger of smaller m/z collide with rods and are lost

Why interested in MALDI-TOF MS?

• High mass range

• Speed of analysis

• Sensitivity

• Simplicity/ease-of-use

• New research approach

• Robustness/tolerance for sample impurities

• Utility as a complement to other MS methods

• Low cost

• Basic interest in the technique

Useage of MALDI-TOF MS

- 고분자 합성시료 및 생체물질 ( 단백질 , 핵산 , 탄수화물등 ) 분자량 측정

- 단백질 분자량 측정을 통한 post translational modifications 확인 ,

recombinant protein 의 QC 등- 합성 펩타이드의 확인 , 단백질 분해를 통한 서열분석

및 단백질 ID- 핵산의 분자량 측정을 통한 서열분석 , 합성확인 및 S

NP 결정- 핵산 및 단백질 분해에 의한 서열분석 - 미량 생화학 , 석유화학 혼합물의 분자량 측정 - SPR bio chip 을 이용한 분자 상호작용 연구

MALDI-TOF 주요 응용분야Proteins Peptides DNA

Mass Measurement - Post translational modifications 확인 : Phosphorylation - Heterogeneous glycoproteins 분석 - Recombinant protein 의 MW 측정

Protein ID from Proteolytic Fragments (Proteomics) MS 또는 MS-MS 데이터로 database 검색 Mass Measurement 합성 peptide 확인Sequencing - 화학적 또는 효소를 이용하여 생성된 ladder 들 의 분자량 측정으로 sequencing - MS-MS 방법 (PSD, CID) 이용 sequencing

Mass Measurement 합성이 잘 되었는지 확인Sequencing - Exonuclease 에 의해 생성 된 ladder 들의 분자량 측정 후 분자 량 차이에 의해 서 열판독 - Sanger mini-sequencing 반응 후 질량 측정

Sample Prep. of MALDI-TOF MS

- 시료는 phosphate buffers, Ammonium salts or organic amines 의 유도체 ,

detergent 가 포함되었을 경우에는 clean-up 과정이 필요 .

- 시료의 농도는 peptides 와 proteins (0.1 - 10pmol/ ㎕ ) Oligonucleotides (10 - 100 pmol/ ㎕ ) polymers (100 pmol/ ㎕ ) glycoproteins 같은 몇몇 단백질은 10 pmol/ ㎕이상의

농도 필요

MALDI-TOF 질량분석기 MMatrix AAssisted LLaser DDesorption IIonization

TTime OOf FFlight MMass SSpectrometry

Voyager

Voyager DE-STR

Matrix 의 화학구조

COOH

OH

N N

OH O OH

HO

CH3O

CH3O

CH CHCOOH

COOH

OHN

OH

OHHO

COCH3

HO

CH C(CN)COOH

C C

COOH

N

H

H

OH

HO

COOH

Sinapinic acid (3,5-Dimethoxy-4-hydroxy cinnamic acid)

2,5-dihydroxybenzoic acid (2,5-DHB)

2,4,6-trihydroxy acetophenone (THAP)

-cyano-4-hydroxycinnamic acid

trans-3-indoleacrylic acid

2-(4-hydroxyphenylazo)-benzoic acid (HABA)

3-hydroxypicolinic acid (3-HPA)

Dithranol

Matrix-Assisted Laser Desorption Ionization (MALDI)

In Matrix-Assisted Laser Desortion Ionization (MALDI), sample is embeddedin a low molecular weight, UV-adsorbing matrix that enhances intact desorption and ionization of the sample. The matrix is present in vast excess of sample, and therefore isolates individual sample molecules.

TOF 에서 분자 이온들의 분리

KE=1/2mv2

Flight Tube (0.5 – 4m)

DetectorIon Source (4 – 25 KV)

Reflector TOF 내 분자이온의 비행

TOF (Time-of-Flight)

Reflector 또는 ion mirror 는 이온발생 부위에서 동일한 질량의 이온들이 초기 에너지 분포 (initial energy spread) 를 갖는 것을 보정하여 분리능을 증가 시킨다 .

Flight TubeDetector

Ion Source

4-25 kV

Reflector (Ion Mirror)

Interfacing MS with other methods

MS/MS

LC/MS

GC/MS

Electrophoresis/MS

MS / MS 란 ?

MALDI-TOF 에서는 PSD (post source decay), CID (collision induced dissociation) MS/MS 방법이 있음

Drug 또는 peptide 혼합물중 특정 분자의

분자만 선택하여MS/MS 실행Drug 또는 peptide

혼합물

MS MS/MS

+

+

+

++

+

Interfacing MS with other methods

Collision-Induced dissociation(CID) in ESI. Three quadropole analysers are used to select for ions with different m/z ratios.Q1 selects intact ions of specific m/z. A colision gas (argon) in introduced into the second analyser (Q2) called the colision cell. Collision with this gas causes fragmentation of the ion into smaller ions. These are analysed in the third quadropole analyser, Q3. Other formats for this kind of experiment are also possible.

LC/MS system.

The ionspray interface.

LC/MS system suitable for analysis of proteins by electrospray MS.

Experimental setup featuring splitting of solvent flow and use of

small-diameter HPLC.

Analyte-enrichment interfaceFor GC/MS.

Q-TOF mass spectrometer

MALDI-MS for C-terminal sequence Determination of peptides and proteins degraded by carboxypeptidaseY and PBernd Thiede, Brigitte Wittmann-Liebold, Michael Bienert, Eberhard KrauseFEBS Letters 357 (1995) 65-69

Hydrophobic Core around Tyrosine for Human Endothelin-1Investigated by Photochemically Induced Dynamic NuclearPolarization Nuclear Magnetic Resonance and Matrix-AssistedLaser Desorption Ionization Time-of-Flight Mass Spectrometry

Biochemistry 2004, 43, 13932-13936