mass spectrometry widely used analytical technique within an accuracy of 0.01% of total weight of...
TRANSCRIPT
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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
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-3 fundamental parts: Ionization source, the ana-lyzer, 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
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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)
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Ion Source: ESI
Electrospray ionization(ESI)- 용액 상태의 시료를 이온화 (LC-MS)- 기존의 방법으로는 얻기 힘들었던 intact
상태의 peptide 나 단백질을 이온화- 한 개 이상의 전하를 띤 이온을 생성
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Ion Source: ESI
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Ion Source: ESI
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Ion Source: MALDI
Matrix Assisted Laser Desorption Ion-ization(MALDI)
Laser
matrix + analyte
Sample support
a
m
m
m
m
m
m
mm a
a
a
a
+
+
+
+m
am+
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Matrix
HO
CH C(CN)COOHOH
HO
COOH
HO
CH3O
CH3O
CH CHCOOH
-cyano-4-hydroxycinnamic acid 2,5-dihydroxybenzoic acid(2,5-DHB)
Sinapinic acid(3,5-Dimethoxy-4-hydroxy cinnamic acid)
HO
CH C(CN)COOHOH
HO
COOH
HO
CH3O
CH3O
CH CHCOOH
-cyano-4-hydroxycinnamic acid 2,5-dihydroxybenzoic acid(2,5-DHB)
Sinapinic acid(3,5-Dimethoxy-4-hydroxy cinnamic acid)
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hn
Laser
+20 kV
Variable Ground Grid Grid
AH+
Sample plate 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+.
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Why MALDI?
-Less sensitive to salts-Lower PRACTICAL detection limits-Easier to interpret spectra(less multi-
ple charges)-Quick and easy-Higher mass detection-Higher Throughput(1000>samples per
hour)
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MALDI/TOF Mass spec-trum
Re
lativ
e A
bu
nda
nce
m/z
0
10000
20000
30000
40000
50000 100000 150000 200000
(M+H)+
(M+2H)2+
(M+3H)3+
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The Mass Analyzer: TOFTime Of Flight(TOF)
Flight TubeIon Source
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.
20-25 kV
++
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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”).
2
22
L
Kt
z
m
t = Drift timeL = Drift lengthm = MassK = Kinetic energy of ionz = Number of charges on ion
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The Mass Analyzer: TOF
Flight Tube
Detector
Ion Source
+
+ +
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The Mass Analyzer: TOF
Flight Tube
Detector
Ion Source
+
++
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The Mass Analyzer: Quadrupole
Quadrupole(Mass filter)-4 개의 molybdenum 막대로 이루어져 있으며 ,
한쌍 (1,2) 은 DC voltage, 다른 한쌍 (3,4) 은 Radio frequency voltage 가 가해진다 .
- 가해지는 전압의 진폭은 선택된 m/z 에 해당되는 ion 만 ion source 에서 detector 까지 통과하게 한다 .
- quadropole 의 전압을 바꾸면서 주어진 mass범위의 이온을 scanning 한다 .
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The Mass Analyzer: Quadrupole
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Detectors
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+
-1000 V -100 V
Primary Ion from Flight Tube
L
D= 6-25 u
Ions are detected with a Microchannel Plate
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+
-1000 V -100 V
L
D= 6-25 u
Ions are detected with a Microchannel Plate
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+
-1000 V -100 V
e-
L
D= 6-25 u
Ions are detected with a Microchannel Plate
Multification by secondary emission
Secondary emissive materials: Beryllium oxide, magnesium oxide etc
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+
-1000 V -100 V
e-
e- e-
e-
L
D= 6-25 u
Ions are detected with a Microchannel Plate
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+
-1000 V -100 V
e-
e- e-
e-
L
D= 6-25 u
Ions are detected with a Microchannel Plate
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+
-1000 V -100 V
e-
e- e-
e-
L
D= 6-25 u
e-
e-
e-
e-
e-
e-
e-
e-
~103
Amplification
Ions are detected with a Microchannel Plate
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Tandem MS(MS/MS)
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Tandem MS(MS/MS)
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Tandem MS(MS/MS)
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Laser
Sampleplate
Analytemolecules in matrix
Accelerationgrids
Drift tube Ion detector
Mass spectrum
Vacuum system
Vacuumlock
MALDI TOF MS
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HiRes mass spectrum
Iondetector
MALDI ionsource
Ion reflector
The reflector focuses ion of same mass but different velocity on
detector; high resolution is obtained
Laser
High resolution TOF-MS with ion reflector
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Daughter ion mass spectrum
Iondetector
Ion reflector
MALDI ionsource
CID
MS/MS spectrum of daughter ionsis measured in a single acquisition;
no pasting of segments;low sample consumption,
high speed, high sensitivity
LID
Laser
TOF/TOF-MS/MS with
Parent ionselector
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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 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 않
고 , 미량의 Matrix 와 ACN, TFA 등을 이용함으로 시약 비용도 저렴함
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Mass Spectrometry 분석-base peak-parent peak-radical cations-Isotopes
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Peptide Sequencing
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Biomolecule Analysis
* 과거에는 ?-Electrophoresis, chromatography, ul-
tracentrifugation-Not very precise*MS 이용하면 ?-Proteins, oligonucleotides, oligosac-
charides, lipids-Detect modifications and sequences
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Peptide Mass Fingerprint-ing
• Analytical technique for protein identifi-cation (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
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• 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 pep-tides 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
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In Gel Digestion & Mass Spectrometry
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Trypsin Digest
Cut out 2D-Gel Spot
Protein Peptides
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Peptide Mass Fingerprinting
Trypsin
N KK
KK
KK
R
RR
RC
N
C
K
KK
K
K
K R
R
R
RProtein
Tryptic peptide mixture. Masses measured by MS. Every peptide has a basic C-terminus.
A protein can be identified in a database by matching masses of a subset of the tryptic peptides against calculated values.
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MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS
PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW
EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK
SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE
SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL
LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE
PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK
GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED
HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT
intact protein
enzyme
peptide fragments
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Peptide Mass Fingerprinting2D-Gel
In Gel Digestion
MS
848.11272.5492.6
883.22978.9
812.61432.33127.1996.8702.4164.92748.2
848.31272.7493.2882.62978.3364.1948.93128.8
Database
3514.22837.1263.9147.41429.7199.6142.3640.8
Is identical to
In Silico Digestion
“Spot removal”
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Post Translational Modifica-tions(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 modi-fication a protein has undergone, but exactly where in the sequence the modification lies.
• Many of the search engines allow for “vari-able” modifications, but very few at one time (combinatorialy explosive)
• There is great opportunity here for robust searches that find PTM’s reliably!
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Protein sequence Analy-sis
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For sequencing of an entire Protein…??Divide and Conquer !!!
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Deduction of Full Amino Acid Sequence of a Proteinby Overlapping the Sequences Obtained from individual Peptides
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Edman Degradation Sequentially Removes One Residue at a Timefrom 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.
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Lymphomas and Leukemias
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Regulatory Mutations
Chromosome 17
MessengerRNA
Her2 gene Her2 gene amplification
Overexpression
Her2 proteinHer2 protein
Normal expression