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)

–

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: ESI

Ion Source: MALDI

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

Sample plate

h

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

T ime O f 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.

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.

The Mass Analyzer: Quadrupole

Detectors

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 e e e e L

Ions are detected with a Microchannel Plate

-1000 V -100 V D= 6-25 u e e e e L

Ions are detected with a Microchannel Plate

-1000 V -100 V D= 6-25 u e e e e e e e e e e e e -

~10 3 Amplification

L

Tandem MS(MS/MS)

Tandem MS(MS/MS)

Tandem MS(MS/MS)

Vacuum lock

MALDI TOF MS

Vacuum system Sample plate Analyte molecules in matrix Acceleration grids 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

LID

TOF /TOF -MS/MS with

CID

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

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 데이 터가 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 않고

,

미 량의

Matrix

와

ACN, TFA

등을 이용함으로 시약 비용도 저렴함

Mass Spectrometry 분석

-base peak -parent peak -radical cations -Isotopes

Biomolecule Analysis

*과거에는?

-Electrophoresis, chromatography, ultracentrifugation -Not very precise *MS이용하면?

-Proteins, oligonucleotides, oligosaccharides, lipids -Detect modifications and sequences

• • • •

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

Peptide Sequencing