Les détecteurs de masse : une révolution en chromatographie 1ère partie : Introduction à la spectrométrie de masse Pr. Jean-Louis Habib Jiwan UCL – Département.
Post on 15-Dec-2015
225 Views
Preview:
Transcript
Les détecteurs de masse :une révolution en chromatographie
1ère partie : Introduction àla spectrométrie de masse
Pr. Jean-Louis Habib Jiwan
UCL – Département de chimie
habib@chim.ucl.ac.be
Mass spectrometry
Fundamentals
ionization processesinternal energytime scale of events
isotopic distributionisotopic mass
Technical realizations
sample introduction
Ionization methods
vacuum systems
type of mass analyzerscombination of mass analyzersmodes of operation
coupling of separation device
Applications
identificationquantification
Mass spectral interpretation
fragmentation pathwayscharacteristic ionsrules
MS
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry
1897Thomson discovers e-
1912First mass spectrometerThomson
1942First commercialMass spectrometer
1953Quad an Trap descriptionPaul & Steinwedel
1956First GC/MS
1958First commercial TOF
1968First commercial Quad
1974 First LC/MSand FTICR description
1982First complete MS spectrum of Insulinand first commercial triple Quad
1987MALDIKaras & Hillenkamp
1988ESI by FennFirst spectra of protein> 20000 Da
2000Spectrum of noncovalentlyassociated macromolecules> 106 Da
2005OrbitrapMakarov
1918
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
EIMS of 2-methyl-3-pentanone (MW = 100)
O
O
CH2CH3
O
m/z
Rel
ativ
e in
tens
ityMass spectrometry : fundamentals
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
HPLCGC
SampleIntroduction
Ion Source
Analyser(s)
Detector
Vacuum
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry : fundamentals
Electron IonisationChemical ionisationFast Atom BombardmentLaser Desorption IonisationMatrix-Assisted Laser Desorption IonisationAP-MALDIElectrosprayAtmospheric Pressure Chemical IonisationAtmospheric Pressure PhotoionisationDESI , DART
Ion sources
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry : fundamentals
200 000
15 000
1 000
MW
Non Polar Highly Polar
EI/CIAPCI
APPI
ESI
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry : fundamentals
Magnetic and electric sectorsQuadrupolesTime of Flight (Linear, Reflectron)Ion Trap ( 3D, Linear)Fourier Transform instruments :
Ion Cyclotron Resonance, OrbitrapHybrid instruments
Analyzers
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry : fundamentals
Elément isotope % Masse isotopiqu
e
isotope % Masse isotopique
isotope % Masse isotopique
Masse moyenne
C 12C 100 12.0000 13C 1.1 13.0033 12.011
H 1H 100 1.0078 2H 0.015 2.0140 1.0079
N 14N 100 14.0031 15N 0.37 15.0001 14.0067
O 16O 100 15.9949 17O 0.04 16.9991 18O 0.20 17.9992 15.9994
S 32S 100 31.9721 33S 0.789 32.9715 34S 4.44 33.9679 32.066
Cl 35Cl 100 34.9688 37Cl 31.98 36.9659 35.453
Br 79Br 100 78.9183 81Br 97.28 80.9163 79.904
http://physics.nist.gov/PhysRefData/Compositions/index.html
http://www.webelements.com/
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry : fundamentals
For high molecular weights, the difference between thevarious masses becomes important :
Insulin : C257H383N65O77S6
Chemical mass 5807.6559Nominal monoisotopic mass : 5801Exact monoisotopic mass : 5803.6375
The instrument response will depend on its resolution
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry : fundamentals
Resolution
R = m
m
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry : fundamentals
sourcemass
analyzerdetector
ionsresolved
ionssample
Mass spectrum
intensity
m/z
Mass spectrometry : full spectrum
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
CollisionInduced
Dissociation
detector
sourceFull spectrum
MS2 spectrum
Mass spectrometry : tandem mass spec or MS2
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry : scanning mode with a triple quad
MS1
Fragment ion or product ion scan
Precursorselection
FragmentationFragment
detection scan
CID MS2
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry : scanning mode with a triple quad
Parent ion or precursor ion scan
Precursorscan
FragmentationFragment selection
detection
MS1 CID MS2
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry : scanning mode with a triple quad
N COOH
O
CO
R
N COOH
COOH
O
CO
R
- (CH3)3N- R-COOH
m/z = 144
M - 59
COOH
m/z = 85
- N(CH3)3 - R-COOH
CO
R M - 161
Detection of carnitines in urine
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry : scanning mode with a triple quad
FABMS2 spectrumof crude sampledetection of theprecursors of 85
Selective detection at 85 m/z
Scanning
Detection of carnitines in urine
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry : scanning mode with a triple quad
Neutral loss scan
Precursorm scan
FragmentationFragment detection
m-a scan
MS1 CID MS2
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry : neutral loss scan
Acides biliaires dans un échantillon d’urine
m scan
m-a scan
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry : MSn with an ion trap
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Source : documentation Thermo Finnigan
Mass spectrometry : MS2 with an ion trap or a triple quad
1 #1 RT: 0.01 AV: 1 NL: 8.45E5T: + c Full ms [ 50.00-300.00]
60 80 100 120 140 160 180 200 220 240 260 280 300
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
180.08
279.03163.15
242.27
110.9263.86 220.20166.09 267.17162.21
99.3890.40 282.32148.19 208.76 236.33 261.13122.33 283.04181.1478.95 131.24
69.3959.05
1 #42-49 RT: 1.13-1.41 AV: 8 NL: 1.37E5F: + c Full ms2 180.00@20.00 [ 50.00-190.00]
50 60 70 80 90 100 110 120 130 140 150 160 170 180 190
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100R
ela
tive
Ab
un
da
nce
162.02
179.14162.93143.98 148.02131.8685.01 126.06108.75100.9859.99
MS2 of glucosamine in ion trap
- H20
O
CH2OH
H
NH2
HOH
H
HO
H OH
H
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Mass spectrometry : MS2 with an ion trap or a triple quad
NL96-GLA #1-42 RT: 0.01-0.73 AV: 42 NL: 4.36E4T: + c Full cnl 96.00@-15.00[ 126.00-500.00]
130 135 140 145 150 155 160 165 170 175 180 185m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
179.94
180.98
181.65
185.69165.58 174.56151.84 163.13158.81144.78 168.59140.22133.11
MN96-MSMS #3-15 RT: 0.05-0.25 AV: 13 NL: 3.17E4T: + c Full ms2 180.00@-33.00 [ 40.00-187.00]
40 60 80 100 120 140 160 180m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
43.72
68.40
59.8283.77
56.0655.33
79.6095.68
52.87
44.98 67.67130.85117.9096.68
90.35122.29 135.00105.54 144.34 161.38 179.21
MS2 of glucosamine in triple quad
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Elemental composition
N O
O
(CH2)3O
Cl C13H14NO3ClMasse : 267.0662
M+1 : 268.0740M+3 : 270.0640
Peak matching results :
268.0735 12C13H1535ClNO3
270.0705 12C13H1537ClNO3
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Elemental composition
Measured : 150.0701 ± 0.010
C : 0 – 30H : 0 – 60N : 0 – 10O : 0 - 15
14 compositions
Measured : 150.0701 ± 0.050 60 compositions
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Elemental composition
31 compositionsMeasured : 322.0151 ± 0.010
C : 0 – 30H : 0 – 60N : 0 – 10O : 0 - 15
Measured : 322.0151 ± 0.010 152 compositions
C : 0 – 30H : 0 – 60N : 0 – 10O : 0 – 15S : 0 - 10
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Short introduction to proteomics
Protein chemistry Proteomics
Individual protein Complete mixtures
Complete sequence analysis Partial sequence analysis
Emphasis structure and function
Emphasis on identification by database matching
Structural biology Systems biology
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Short introduction to proteomics
Proteinmixture
digestion
Peptidesmixture
separation
separation
Proteins
Peptides MS analysis
MS data
Identification
database search
digestion
Separation: always 2D
2D gel or LC - LC
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
+
2+
3+
2+
3+
3+
+2+
3+
RP - LC
SCXcolumn
Protonatedpeptides
Short introduction to proteomics
1st salt pulse
2nd salt pulse
3d salt pulse
pump
SCXcolumn
RPcolumn
MSsystem
sample
injector
Short introduction to proteomics : mass fingerprinting
Digestion Tryptic peptides
1529.5006
1529.6997
1529.7348
1529.9978
1530.2332
1530.4567
1531.0003
Peptide mass list
…
…
1529.7348measured mass
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Search m/z Mass Tolerance (Da) # Hits
1529 1 478
1529.7 0.1 164
1529.73 0.01 25
1529.734 0.001 4
1529.7348 0.0001 2
IGGHGAEYGAEALER Mouse Hb alpha 1529.7348 (-0.0048)VGAHAGEYGAEALER Human Hb alpha 1529.7348 (-0.0048)
Short introduction to proteomics : mass fingerprinting
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Search m/z Mass Tolerance (Da) # Hits
1529.73 0.1 204
1529.73
1252.70 0.1 7
1529.73
1252.70
1833.88 0.1 1
Short introduction to proteomics : mass fingerprinting
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Short introduction to proteomics : peptide sequencing by MSn
A V A G C A G A R
72 171 242 299 402 473 530 601 769
cumulative mass
N
O
HR1
R2
b
y
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Short introduction to proteomics : peptide sequencing by MSn
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Source : DC Liebler « Introduction to Proteomics », Humana Press 2002
Short introduction to proteomics : peptide sequencing by MSn
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
12
34
time
Full scanMS
m/z
m/z
m/z
m/z
m/z
MS2
fragmentation1
2 3
4
Short introduction to proteomics : peptide sequencing by MSn
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
Source : DC Liebler « Introduction to Proteomics », Humana Press 2002
Further reading
Pr. Jean-Louis Habib Jiwan UCL – Département de chimie IPL mai 2007
- Ed de Hoffmann and V Stroobant « Mass Spectrometry Principles and Applications » 2nd ed, Wiley 2001
- J H Gross « Mass Spectrometry - A textbook » Springer 2004
top related