mass spectrometry
DESCRIPTION
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) - PowerPoint PPT PresentationTRANSCRIPT
Mass Spectrometry
시스템생물학과이선아
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
-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: ESIElectrospray 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
Ion Source: ESI
Ion Source: ESI
Ion Source: MALDIMatrix Assisted Laser Desorption Ion-
ization(MALDI)
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+.
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)
MALDI/TOF Mass spec-trum
Rel
ativ
e A
bund
ance
m/z
0
10000
20000
30000
40000
50000 100000 150000 200000
(M+H)+
(M+2H)2+
(M+3H)3+
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
++
The Mass Analyzer: TOF
Flight Tube
Detector
Ion Source
+
+ +
The Mass Analyzer: TOF
Flight Tube
Detector
Ion Source
+
++
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 .
The Mass Analyzer: Quadrupole
Detectors
+
-1000 V -100 V
Primary Ion from Flight Tube
L
D= 6-25 u
Ions are detected with a Microchannel Plate
+
-1000 V -100 V
L
D= 6-25 u
Ions are detected with a Microchannel Plate
+
-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
+
-1000 V -100 V
e- e- e-e-
L
D= 6-25 u
Ions are detected with a Microchannel Plate
+
-1000 V -100 V
e- e- e-e-
L
D= 6-25 u
Ions are detected with a Microchannel Plate
+
-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
Tandem MS(MS/MS)
Tandem MS(MS/MS)
Tandem MS(MS/MS)
Laser
Sampleplate
Analytemolecules in matrix
Accelerationgrids
Drift tube Ion detector
Mass spectrum
Vacuum system
Vacuumlock
MALDI TOF MS
HiRes mass spectrum
Iondetector
MALDI ionsource
Ion reflector
The reflector focuses ion of same mass but different velocity on
detector; high resolution is obtained
Lase
r
High resolution TOF-MS with ion reflector
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
Lase
r
TOF/TOF-MS/MS with
Parent ionselector
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
Mass Spectrometry 분석-base peak-parent peak-radical cations-Isotopes
Biomolecule Analysis* 과거에는 ?-Electrophoresis, chromatography, ul-
tracentrifugation-Not very precise*MS 이용하면 ?-Proteins, oligonucleotides, oligosac-
charides, lipids-Detect modifications and sequences
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!
Protein sequence Analy-sis
Peptide Sequencing
