Transcript Ion Source: ESI

Mass Spectrometer(MS)
• 분자가 MS 내로 들어가면 분자는 이온화됨과 동
시에 더 작은 이온들(fragments)로 쪼개진다. 쪼
개진 이온들은 그들의 질량/전하(m/z)비에 따라
선택적으로 분리되어 이온수에 비례해 signal을
만든다.
• 이온들의 질량/전하비는 이온들의 생성된 양
(Abundance)의 함수로 표시되어 mass spectrum
이 그려지며, 이 mass spectrum을 이용하여 미
지성분의 정성확인을 할 수 있다.
• 이온은 양이온과 음이온 모두 사용
MS Component
MS component
• Sample inlet
시료도입장치로 시료를 MS내로 효율적으로 보내주는 역할을 한다
• Ion source
시료분자를 이온화시키고 더 작은 이온으로 쪼갠다. 생성된 이온들을
MS analyzer 쪽으로 이동시킨다
• Mass analyzer
이온들을 m/z ratio에 따라 선택적으로 분리시킨다
• Ion detector
이온 흐름을 그 양에 비례하게 전기적인 흐름으로 전환, 증폭시켜
signal을 생성한다
• Vacuum system
MS 내 진공상태를 10-4 ~ 10-9 Torr 로 만들어 주어 최적의 상태로
분석이 진행될 수 있도록 한다
• Data System
MS 내 각 구성성분들의 조절이 가능하며 시료분석과 동시에 데이터
해석을 할 수 있는 곳이다
Sample introduction / ionization method:
Ionization
method
Typical
Analytes
Sample
Introduction
Mass
Range
Electron Impact (EI)
Relatively
small
volatile
GC or
liquid/solid
probe
to
1,000
Daltons
Method
Highlights
Hard method
versatile
provides
structure info
Chemical Ionization (CI)
Relatively
small
volatile
GC or
liquid/solid
probe
to
1,000
Daltons
Soft method
molecular ion
peak [M+H]+
Liquid
Electrospray (ESI)
Peptides
Proteins
nonvolatile
Chromatography
or syringe
to
200,000
Daltons
Soft method
ions often
multiply
charged
Carbohydrates
Organometallics
Peptides
nonvolatile
Sample mixed
in viscous
matrix
to
6,000
Daltons
Soft method
but harder
than ESI or
MALDI
Peptides
Proteins
Nucleotides
Sample mixed
in solid
matrix
to
500,000
Daltons
Soft method
very high
mass
Fast Atom Bombardment
(FAB)
Matrix Assisted Laser
Desorption
(MALDI)
Ion source
• Gaseous sample introduction
- EI(electron ionization)
- CI(chemical Ionization)
• Liquid sample introduction
- FAB(fast atom bombardment)
- ESI(electrospray ionization)(soft ionization)
• Solid sample introduction
- MALDI(soft ionization)
(matrix-assisted laser desorption/ionization)
Ion Source - EI(electron
ionization)
• 전자를 발생시키기 위한 filament가 가열되고
(+) 극판에 전압이 걸리면 가속된 전자들이
흐르고 그 속을 지나던 기체화된 sample은 전
자와 충돌하여 에너지를 얻고 전자 하나를 잃
어 분자이온 M+가 된다
• 이 방법은 큰 에너지를 사용하기 때문에 복잡
한 spectrum을 이루며 분자 이온을 얻기 힘들
다
• M + e- ⇒ M+ + 2e-
Ion Source –
CI(chemical Ionization)
• 가열된 filament에서 발생, 가속된 전자는 106 정도로
많은 reagent gas와 충돌하여 이들을 이온화시키고
이 reagent gas ion은 sample gas와 충돌
• 이 sample gas는 fragmentation 되기도 하고 때로는
reagent gas ion 과 complex를 이루기도 한다.
• 매우 낮은 에너지로 충돌하기 때문에 EI 보다 수백배
이상 많은 수의 분자 이온을 만들어 내기 때문에 분
자량 확인에 많이 쓰인다.
• R(CH4) + e- ⇒ R+ + 2 eR+ + M ⇒ M1+ + N1
M1+ ⇒ M2+ + N2
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
• 시료용액이 고전압이 걸려 있는 capillary를
통과하면서 분무되어 전하를 많이 띤 droplet
이 생성됨
• dropolet이 capillary에서 orifice를 지나면서
inert gas(or heat)에 의해 desolvation
• Desolvation 과정에서 ion의 charge는 더 증
가하고 “Coulombic explosion”에 의해
droplet의 ion이 gas phase로 된다.
• sample에 가해지는 충격이 약하며, multiple
charge를 가진 peptide이온이 생긴다.
Ion Source: ESI
Ion Source: ESI
Ion Source: ESI
Ion Source: ESI
Ion Source: MALDI
Matrix Assisted Laser Desorption
Ionization(MALDI)
Laser
m
m
+
a
a
+
m a
m
+
a m
a
a
+
m
m
m+
m
m
matrix + analyte
Sample support
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)
Matrix
CH
CH O
3
COOH
C(CN)COOH
CH
CHCOOH
OH
HO
HO
HO
-cyano-4-hydroxycinnamic acid
2,5-dihydroxybenzoic acid
(2,5-DHB)
CH O
3
Sinapinic acid
(3,5-Dimethoxy-4-hydroxy cinnamic acid)
Biomolecule Analysis
과거에는?
• Electrophoresis, chromatography,
ultracentrifugation
• Not very precise
MS이용하면?
• Proteins, oligonucleotides, oligosaccharides,
lipids
• Detect modifications and sequences
Biomolecule Analysis
• Mass is one of first measurements to
characterize biopolymers
• Up to 1970s, had to use–
Electrophoresis–Chromatography–
Ultracentrifugation–Not very precise (10
–100% relative error!!)
• May use MS on most biomolecules–
Proteins–Oligonucleotides–
Oligosaccharides–Lipids
• May detect modifications and sequences–
Post translational and other
Peptide Mass Fingerprinting
• Analytical technique for protein
identification (protein sequence)
• Unknown protein of interest cleaved into
peptide by protease
• Collection of peptides resulting from this
cleavage comprise a unique identifier of the
unknown protein
• Mass measured with MALDI-TOF
and ESI-TOF
• in silico compared to the genome
• Computer programs translate the known
genome of the organism into proteins
• Theoretically cut the proteins into peptides with
the same protease (ex.Trypsin: K or R)
• Calculate the absolute masses of the peptides
from each protein
• the masses of the peptides of the unknown
protein vs the theoretical peptide masses of
each protein encoded in the genome
• Results statistically analyzed to find the best
match
In Gel Digestion & Mass Spectrometry
Trypsin Digest
Cut out 2D-Gel Spot
Protein
Peptides
Peptide Mass Fingerprinting
N
K
K
R
K
K
K
R
Trypsin
K
R
Protein
N
K
K
K
R
K
R
R
R
C
K
K
R
C
Tryptic peptide mixture.
Masses measured by MS.
Every peptide has a basic Cterminus.
A protein can be identified in a database by matching masses of a
subset of the tryptic peptides against calculated values.
intact
protein
enzyme
peptide
fragments
MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS
PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW
EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK
SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE
SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL
LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE
PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK
GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED
HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT
Peptide Mass Fingerprinting
2D-Gel
Database
“Spot removal”
In Silico Digestion
In Gel Digestion
MS
848.1
1272.5
492.6
883.2
2978.9
812.6
1432.3
3127.1
996.8
702.4
164.9
2748.2
Is identical to
848.3
1272.7
493.2
882.6
2978.3
364.1
948.9
3128.8
3514.2
2837.1
263.9
147.4
1429.7
199.6
142.3
640.8
Protein sequence Analysis
Protein sequence Analysis
Deduction of Full Amino Acid Sequence of a Protein
by Overlapping the Sequences Obtained from individual Peptides
Edman Degradation Sequentially Removes One Residue at a Time
from the Amino End of a Peptide up to 50 times
Each round
can be complete
within 1 hr and
the Edman degradation
can be repeated
up to 50 cycles
in Practice.
Measured peptide mass 와 sequence가
맞지 않는 경우
• The additional masses are due to
posttranslational or artifactual modifications or
post-translational processing
• Unspecific proteolysis had occurred or
contaminating protease was present
• Protein was part of a mixture of
‘contaminating’ proteins
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!
Phosphorylation site analysis
strategies
• Complication of phosphoprotein analysis
- the frequently low stoichiometry of
phosphorylation
- the presence of multiple, differentially
phosphorylated forms
• In vitro analysis
- scale up of protein by kinase reaction
- comparison with 2D-PP maps of in vivo
(confirmation of identity indirectly)
- MS analysis
Detection and isolation of
phosphoproteins
• For the analysis of the site(s) of protein phosphorylation
- purification of phosphoprotein
- enzymatic or chemical fragmentation of the phosphoprotein
- Isolation, separation, analysis of peptide
• Isolation
- separation of proteins by gel electrophoresis
- fragmentation of the phosphoprotein band or spot
- extraction of the generated phosphopeptide
• More positive identification
- 32 P radiolabelling : in vivo(32 PO4), in vitro([γ-32P]ATP)
- western blotting : particularly tyrosine phosphorylated protein
Separation of phosphopeptides
• 필요한 이유
- 농도를 농축하는 역할을 하여 S/N 비를 높임
- radiolabel의 activity를 이용하여
phosphopeptide의 상대적 또는 절대적인 양
을 구할 수 있음
- separation에 의해 확보된 재현성으로 단백질
의 phosphorylation 상태를 정량적으로 결정
할 수 있음
- nonpeptide contaminants를 제거하여 적은
양의 phosphopeptide의 분석을 용이하게 함
Phosphopeptide separation techniques
By 2-dimensional phosphopeptide map
Reversed-phase HPLC
High-resolution gel electrophoresis
Immobilized metal affinity
chromatogrphy(IMAC)
• Phosphopeptide는 상대적, 절대적으로 적은 양
때문에 분석이 어려우므로 이러한 점을 극복할
수 있는 최적의 separation방법을 선택해야
•
•
•
•
Separation by 2D-PP
• 1st dimension by electrophoresis on thin-layer cellulose
plate
+ 2nd dimension by TLC on the same plate
• information
- radiolabelled spot 수 ⇒ phosphorylated sites 최대수
- radiolabelled spot의 intensity
⇒ peptide 들의 상대적인 phosphorylation 정도
- relative state between phosphopeptide
• MS analysis after extraction from plate
- protease양이 중요
• sensitive and reproducible by radiolabelling
Separation by RP-HPLC
• Reproducible and simple
• column으로 분리하고 radioactivity count로 fraction
⇒ count를 시간의 함수로 하여 radioactive fraction의 수를 알 수 있
음
• 단점
- very hydrophilic phosphopeptide, very hydrophobic
phosphopeptide의 분리가 어렵다
- 2D-PP보다 resolution이 낮다
- phosphopeptide will stick to metal surface
• 장점
- ESI MS와 on line으로 연결하여 사용할 수 있다(LC-MS/MS)
- isotope을 사용할 수 없는 인체 단백질 분석 가능
Separation by high-resolution
electrophorsis and IMAC
• High-resolution gel electrophoresis
- 2-DE
- 특정 phosphopeptide의 손실이 많지만 널리 보급되어
있어 사용하기 좋음
• IMAC
- 같은 sequence를 갖는 nonphosphorylated peptide에
비하여 상대적으로 매우 적은 양의 phosphorylated
peptide의 분석 어려움
- separation and enrichment
1) phosphopeptide와 metal(Fe3+ ,Ga3+)의 chelating
2) elution by phosphate or increased pH
3) acidic amino acid 도 enrichment 되는 단점
Determination of the type of
phosphorylated amino acid
• 이유
가능한 phosphorylated site의 수를 찾아냄으로써 polypeptide
내의 phosphorylated residues의 assignment를 쉽게 할 수 있음
• Technique
1) phosphoamino acid analysis
- 32P-amino acid(hydrolysate of 32P-labeled
phosphoprotein or phosphopeptide) ⇒ autoradiography
- phosphoamino acid standard ⇒ ninhydrin staining
- sample과 standard의 비교 분석(보통 1site/phosphopeptide)
2) phospho-amino acid-specific immunodetection
- antibodies specific for particular phospho-amino acid
- 상업적으로 antibody판매
Determination of
the site of phosphorylation
• Chemical phosphopeptide sequencing
- phosphopeptide sequencing by step-wise chemical
degradation(nonradioactive, radioactive methods)
- analyzed as phenylthiohydantoyl derivatives
- not available in very limited amount
• Mass spectrometric analysis of phosphopeptides
- phosphopeptide의 양이 1pmole이상이면 2D-PP map에서
extraction이 가능하고, MS로 분석이 가능
- two basic theme
1) chemical lability of the phosphate ester bonds
2) the detection of the mass added to a peptide (80u)
- product ion scan in a tandem MS으로 phosphorylation site 확인
⇒ phosphorylated amino acid type을 알고 있으면 더 용이
Mass scan for phosphopeptides analysis
• In-source CID
- identify phosphopeptides by observation of
H2PO4-(97U), PO3-(79U) and PO2-(63U)
- detect phosphopeptides in negative ion mode and
then switch to positive ion mode
• Neutral loss scan
- positive ion mode with ESI in a TQ MS
- Q1, Q3 are scanned over different m/z ranges
- neutral loss of phosphoserine and
phosphothreonine : 98
Mass scan for phosphopeptides analysis
• Presursor ion scan
- negative ion ESI(다시 positive ion mode로 변경)
- Q1 : continous scan, Q2 : ion fragmentation
Q3 : 79m/z(PO3-)를 잃은 ion 만 통과
• Product ion scanning
- in-source CID, neutral loss and precursor ion
scanning는 특수한 경우에만 phosphorylated
residue를 identify
- 상기 3가지 방법을 이용할 수 없는 경우 peptide
fragment ion 전체에 대한 해석이 필요
Mass scan for phosphopeptides analysis
• Post-source decay MALDI
• Enzymatic and chemical dephosphorylation
- MALDI-TOF로 phosphopeptide의 mass 측정
+ phosphate를 제거 후 MALDI-TOF로 mass 측정
- nonphosphorylated peptide에서 phosphopeptide
를 확인하는데 쉽게 이용
- identification of phosphorylation sites using
MS/MS
Emerging methods and future directions
in phosphoprotien analysis
• in vivo와 비교해서 in vitro로 시험하나 동일하다는 보
장이 없음 ⇒ in vivo를 직접
• in vivo 32P-labeled protein을 충분히 얻는다는 것은
어려우므로 분석기기 감도를 높이는 것이 유리
Present and future challeges and opportunities
• Protein identification and
characterization has to be performed
in a high-throughput manner,
efficiently and with high accuracy and
sensitivity
• Robotic system
• 2D-chromatography MS/MS