Mass spectrometry
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Mass Spectrometry. 시스템생물학과 이선아. 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)

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Mass Spectrometry

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Mass spectrometry

Mass Spectrometry

시스템생물학과

이선아


Mass spectrometry1

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 10-9M)

    –Femtomolar range possible (1 x 10-15M)

    –Attomolar range claimed (1 x 10-18M)

  • Diversity of applications

    –Proteins

    –Oligonucleotides

    –Oligosaccharides

    –Lipids

    –Others

  • Proteomics

    –Identification of proteins expressed under specific conditions


Mass spectrometry

-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

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

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 esi1

Ion Source: ESI


Ion source esi2

Ion Source: ESI


Ion source maldi

Ion Source: MALDI

Matrix Assisted Laser Desorption Ionization(MALDI)


Mass spectrometry

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+.

Sample plate

hn

Variable Ground

Grid Grid

+20 kV


Why maldi

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

40000

30000

20000

10000

0

MALDI/TOF Mass spectrum

(M+H)+

Relative Abundance

(M+2H)2+

(M+3H)3+

50000

100000

150000

200000

m/z


The mass analyzer tof

The Mass Analyzer: TOF

Time Of Flight(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.


The mass analyzer tof1

+

+

The Mass Analyzer: TOF

Ion Source

Flight Tube

Detector

+


The mass analyzer tof2

+

+

The Mass Analyzer: TOF

Ion Source

Flight Tube

Detector

+


The mass analyzer quadrupole

The Mass Analyzer: Quadrupole

Quadrupole(Mass filter)

-4개의 molybdenum 막대로이루어져 있으며, 한쌍(1,2)은 DC voltage, 다른 한쌍(3,4)은 Radio frequency voltage가 가해진다.

-가해지는전압의 진폭은 선택된 m/z에 해당되는 ion만 ion source에서 detector까지 통과하게 한다.

- quadropole의 전압을 바꾸면서 주어진 mass범위의 이온을 scanning 한다.


The mass analyzer quadrupole1

The Mass Analyzer: Quadrupole


Detectors

Detectors


Mass spectrometry

+

Ions are detected with a Microchannel Plate

-1000 V

-100 V

D= 6-25 u

Primary Ion from Flight Tube

L


Mass spectrometry

+

Ions are detected with a Microchannel Plate

-1000 V

-100 V

D= 6-25 u

L


Mass spectrometry

+

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


Mass spectrometry

+

Ions are detected with a Microchannel Plate

-1000 V

-100 V

D= 6-25 u

e-

e-

e-

e-

L


Mass spectrometry

+

Ions are detected with a Microchannel Plate

-1000 V

-100 V

D= 6-25 u

e-

e-

e-

e-

L


Mass spectrometry

e-

e-

e-

e-

e-

e-

e-

e-

+

Ions are detected with a Microchannel Plate

-1000 V

-100 V

D= 6-25 u

e-

e-

e-

e-

~103

Amplification

L


Tandem ms ms ms

Tandem MS(MS/MS)


Tandem ms ms ms1

Tandem MS(MS/MS)


Tandem ms ms ms2

Tandem MS(MS/MS)


Mass spectrometry

MALDI TOF MS

Analyte

molecules in matrix

Vacuumlock

Laser

Vacuum system

Ion detector

Sampleplate

Acceleration

grids

Drift tube

Mass spectrum


Mass spectrometry

Laser

High resolution TOF-MS with ion reflector

MALDI ionsource

Ion reflector

Iondetector

HiRes mass spectrum

The reflector focuses ion of same mass but different velocity on detector; high resolution is obtained


Mass spectrometry

LID

Laser

TOF/TOF-MS/MS with

CID

MALDI ionsource

Parent ionselector

Ion reflector

Iondetector

MS/MS spectrum of daughter ionsis measured in a single acquisition;no pasting of segments;low sample consumption, high speed, high sensitivity

Daughter ion mass spectrum


Why interested in maldi tof ms

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 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 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, 미량의 Matrix와 ACN, TFA등을 이용함으로 시약 비용도 저렴함


Mass spectrometry2

Mass Spectrometry 분석

-base peak

-parent peak

-radical cations

-Isotopes


Biomolecule analysis

Biomolecule Analysis

*과거에는?

-Electrophoresis, chromatography, ultracentrifugation

-Not very precise

*MS이용하면?

-Proteins, oligonucleotides, oligosaccharides, lipids

-Detect modifications and sequences


Post translational modifications ptm s

Post Translational Modifications(PTM’s)

  • PTM’s are very important in signaling as well as metabolic pathways (e.g. phosphorylation)

  • Often we want to know not only which modification a protein has undergone, but exactly where in the sequence the modification lies.

  • Many of the search engines allow for “variable” modifications, but very few at one time (combinatorialy explosive)

  • There is great opportunity here for robust searches that find PTM’s reliably!


Protein sequence analysis

Protein sequence Analysis


Peptide sequencing

Peptide Sequencing


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