Les détecteurs de masse : une révolution en chromatographie 1ère partie : Introduction à la spectrométrie de masse Pr. Jean-Louis Habib Jiwan UCL – Département

Les détecteurs de masse :une révolution en chromatographie

1ère partie : Introduction àla spectrométrie de masse

Pr. Jean-Louis Habib Jiwan

UCL – Département de chimie

[email protected]

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


EIMS of 2-methyl-3-pentanone (MW = 100)

O

O

CH2CH3

O

m/z

Rel

ativ

e in

tens

ityMass spectrometry : fundamentals


HPLCGC

SampleIntroduction

Ion Source

Analyser(s)

Detector

Vacuum


Mass spectrometry : fundamentals

Electron IonisationChemical ionisationFast Atom BombardmentLaser Desorption IonisationMatrix-Assisted Laser Desorption IonisationAP-MALDIElectrosprayAtmospheric Pressure Chemical IonisationAtmospheric Pressure PhotoionisationDESI , DART

Ion sources



200 000

15 000

1 000

MW

Non Polar Highly Polar

EI/CIAPCI

APPI

ESI



Magnetic and electric sectorsQuadrupolesTime of Flight (Linear, Reflectron)Ion Trap ( 3D, Linear)Fourier Transform instruments :

Ion Cyclotron Resonance, OrbitrapHybrid instruments

Analyzers



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/



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



Resolution

R = m

m



sourcemass

analyzerdetector

ionsresolved

ionssample

Mass spectrum

intensity

m/z

Mass spectrometry : full spectrum


CollisionInduced

Dissociation

detector

sourceFull spectrum

MS2 spectrum

Mass spectrometry : tandem mass spec or MS2


Mass spectrometry : scanning mode with a triple quad

MS1

Fragment ion or product ion scan

Precursorselection

FragmentationFragment

detection scan

CID MS2



Parent ion or precursor ion scan

Precursorscan

FragmentationFragment selection

detection

MS1 CID MS2



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



FABMS2 spectrumof crude sampledetection of theprecursors of 85

Selective detection at 85 m/z

Scanning

Detection of carnitines in urine



Neutral loss scan

Precursorm scan

FragmentationFragment detection

m-a scan

MS1 CID MS2


Mass spectrometry : neutral loss scan

Acides biliaires dans un échantillon d’urine

m scan

m-a scan


Mass spectrometry : MSn with an ion trap


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 [email protected] [ 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


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 [email protected][ 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 [email protected] [ 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


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



Measured : 150.0701 ± 0.010

C : 0 – 30H : 0 – 60N : 0 – 10O : 0 - 15

14 compositions

Measured : 150.0701 ± 0.050 60 compositions



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


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



Proteinmixture

digestion

Peptidesmixture

separation

separation

Proteins

Peptides MS analysis

MS data

Identification

database search

digestion

Separation: always 2D

2D gel or LC - LC


+

2+

3+

2+

3+

3+

+2+

3+

RP - LC

SCXcolumn

Protonatedpeptides


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


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)



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 : 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




Source : DC Liebler « Introduction to Proteomics », Humana Press 2002



12

34

time

Full scanMS

m/z

m/z

m/z

m/z

m/z

MS2

fragmentation1

2 3

4



Source : DC Liebler « Introduction to Proteomics », Humana Press 2002

Further reading


- Ed de Hoffmann and V Stroobant « Mass Spectrometry Principles and Applications » 2nd ed, Wiley 2001

- J H Gross « Mass Spectrometry - A textbook » Springer 2004

Documents

Les détecteurs de masse : une révolution en chromatographie 1ère partie : Introduction à la spectrométrie de masse Pr. Jean-Louis Habib Jiwan UCL – Département