Mass Spectrometry

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Transcript Mass Spectrometry

Mass Spectrometry

• • • • • Widely used analytical technique Within an accuracy of 0.01% of total weight of sample and within

5 ppm for small organic molecules

Unequaled sensitivity –

Nanomolar range routinely (1 x 10 -9 M)

–

Femtomolar range possible (1 x 10 -15 M)

–

Attomolar range claimed (1 x 10 -18 M)

Diversity of applications –Proteins –Oligonucleotides –Oligosaccharides –Lipids –Others

Proteomics

– Identification of proteins expressed under specific conditions

3 fundamental parts

: Ionization source, the analyzer, the detector

-Ionization source

시료분자를 이온화시키고 더 작은 이온으로 쪼갠다.

-Mass analyzer

이온들을 mass-to-charge(m/z) ratio에 따라 선택적으로 분리

-Ion detector

이온 흐름을 그 양에 비례하게 전기적인 흐름으로 전환, 증폭시켜 signal을 생성

-Vacuum system

Basic components to MS

•Ion source

–

Electrospray(ESI)

–Atmospheric Pressure Ionization (APCI) –Chemical Ionization (CI) –Electronic Ionization (EI) –

Matrix Assisted Laser DesorptionIonization (MALDI) •Mass Selection

–

Quadrupole

–

Time of Flight (TOF)

–Magnetic Sector –Ion Trap –Ion Cyclotron •

Detector

–Phosphor / Photo Diode –

Multi-channel Plate (MCP)

Ion Source: ESI Electrospray ionization(ESI)

-용액 상태의 시료를 이온화(LC-MS) -기존의 방법으로는 얻기 힘들었던 intact 상태의 peptide나 단백질을 이온화 -한 개 이상의 전하를 띤 이온을 생성

Ion Source: ESI

Ion Source: MALDI

M atrix A ssisted L aser D esorption I onization(MALDI)

Laser

m m + m + a m a m m m m m a a + a + m a

matrix + analyte Sample support

Matrix



-cyano-4-hydroxycinnamic acid

CH 3 O CH 3 O

Sample plate

n AH +

Laser

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: MH + + A  M + AH + .

Variable Ground Grid Grid +20 kV

Why MALDI?

-Less sensitive to salts -Lower PRACTICAL detection limits -Easier to interpret spectra(less multiple charges) -Quick and easy -Higher mass detection -Higher Throughput(1000>samples per hour)

MALDI/TOF Mass spectrum

(M+H) + 40000 30000 20000 10000 (M+3H) 3+ 0 50000 (M+2H) 2+ 100000 m/z 150000 200000

The Mass Analyzer: TOF

ime

f

light

(TOF)

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.

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

K L

t L m K z

= = = = = Drift time Drift length Mass Kinetic energy of ion Number of charges on ion

The Mass Analyzer: TOF

Flight Tube Ion Source Detector

The Mass Analyzer: TOF

Flight Tube Ion Source Detector

The Mass Analyzer: Quadrupole Quadrupole(Mass filter)

-4개의 molybdenum 막대로 이루어져 있으 며, 한쌍(1,2)은 DC voltage, 다른 한쌍(3,4) 은 Radio frequency voltage가 가해진다.

-가해지는 전압의 진폭은 선택된 m/z에 해 당되는 ion만 ion source에서 detector까 지 통과하게 한다.

- quadropole의 전압을 바꾸면서 주어진 mass범위의 이온을 scanning 한다.