Spectrométrie de masse: tour d’horizon de la technique et applications Pr. Jean-Louis Habib Jiwan UCL - Chemistry Department [email protected]

Spectrométrie de masse:

tour d’horizon de la technique et applications

Pr. Jean-Louis Habib Jiwan

UCL - Chemistry Department

[email protected]

Layout

Mass spectrometry

Elemental composition

Radiosterilization of solid metoclopramide

Phytosterols

Glucosamine

Carnitines

Proteomics

Pr. Jean-Louis Habib Jiwan UCL - Chemistry Department May 23d 2006

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


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


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

Mass spectrometry


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

Mass spectrometry


HPLCGC

SampleIntroduction

Ion Source

Analyser(s)

Detector

Vacuum

Mass spectrometry


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

Mass spectrometry

Analyzers


Electron IonisationChemical ionisationFast Atom BombardmentLaser Desorption IonisationMatrix-Assisted Laser Desorption IonisationAP-MALDIElectrosprayAtmospheric Pressure Chemical IonisationAtmospheric Pressure Photoionisation

Mass spectrometry

Ion sources


sourcemass

analyzerdetector

ionsresolved

ionssample

Mass spectrum

intensity

m/z

Mass spectrometry


CollisionInduced

Dissociation

detector

sourceFull spectrum

MS2 spectrum

Mass spectrometry : tandem mass spec or MS2




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

Radiosterilization of solid-state metoclopramide


25kGy E Beam irradiated vs unirradiated

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

0.004

0.0045

0.005

3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5

Retention time (min.)

Ab

sorb

ance

at

273n

m

25kGy Gamma irradiated vs unirradiated

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

0.004

0.0045

0.005

3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5

Retention time (min.)A

bso

rban

ce a

t 27

3 n

m



50kGy_001M06 #774-814 RT: 11.82-12.32 AV: 41 NL: 3.72E8T: + c APCI Full ms [ 100.00-900.00]

120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

300.1

227.1

184.1201.0 272.1213.1 254.2 314.0141.0 362.4168.1 400.6286.1154.0 380.1238.2 327.9126.0 343.5

Cl

NH2

OMe

NH

N

O

50kGy_001M05 #308-314 RT: 4.82-4.92 AV: 7 SB: 15 4.63-4.73, 5.06-5.17 NL: 3.13E6T: + c APCI Full ms [ 100.00-900.00]

100 120 140 160 180 200 220 240 260 280 300 320

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

266.0

267.1

193.1205.8150.1

244.0 278.9114.6 167.0 230.2 298.8282.1248.1112.2 194.2 206.3192.1 227.0124.0 171.1 309.8145.3 160.2140.3 319.5



Peak 2

NH2

NH

N

O

O



Peak 350kGy_001M06 #657-671 RT: 10.07-10.29 AV: 15 SB: 42 9.40-9.71, 10.63-10.94 NL: 2.26E6T: + c APCI Full ms [ 100.00-900.00]

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

lativ

e A

bu

nd

an

ce

272.0

274.0

275.0227.1184.0

NH2

NH

NH

O

Cl

O



Peak 4 50kGy_001M06 #740-742 RT: 11,36-11,39 AV: 3 SB: 9 11,19-11,32 NL: 4,78E5T: + c APCI Full ms [ 100,00-900,00]

100 120 140 160 180 200 220 240 260 280 300 320

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

Rel

ativ

e Ab

unda

nce

286,0

288,0

205,4

163,0 201,0 220,3 282,3227,0206,9143,6 293,1104,8 260,7180,9113,2 136,0 183,9 326,8310,0274,0255,2173,0156,2 329,8234,9315,6

NH2

NH

N

O

Cl

O

Phytosterols in Spelt

Spelt is said to have better nutritive value than winter wheat.

Functional properties have traditionally been attributed to spelt.

Lack of scientific evidence for these claims.

What about the phytosterol contents ?

HO

Me H

Me

Me

HH-sitosterol


Fine bran ground to 1 mm sieve

Saponification in KOHaq (500g/l) / EtOH (1/3), overnight, RT, dark

Extraction with diethyl ether

Washing with water

Filtration (MN 616 wa filter)

Evaporation to dryness and redissolution in EtOH

SPE (C18, CHCL3/MeOH; 95/5)

Evaporation to dryness and redissolution in absolute EtOH

Filtration on DynaGardTM filters



29.12

Steryl Glycosides 20.67

31.92

67.63

81.08

Acylated SterylGlycosides

Free Sterols

Steryl Fatty Acid Esters

20.14

39.71

47.76

Rel

ativ

e A

bund

ance

100

00 10 20 30 40 50 60 70 80

LC/APCI-MS TIC trace of 19 standard compounds

SG

FS

ASG

SE



O

OH

CH OH2

O

H H

CH3

CH3

CH3

H CH3CH3

CH3

OH

HO

397

594

m/z

0300 400 500 600200

100

100

Rel

ativ

e A

bund

ance

a

b

m/z200 400300

397

411413

100

0100

OH

H H

CH3

CH3

CH3

H CH3CH3

CH3

Rel

ativ

e A

bund

ance

m/z200 300 400 500 600 700 800

0

100 397

832

c

CO

O

OH

CH2

O

H H

CH3

CH3

CH3

H CH3CH3

CH3

OH

HO

O

(CH )2 14CH3

d

m/z200 300 400 500 600 7000

100

Rel

ativ

e A

bund

ance

369

668651CO

O

H H

CH3

CH3

CH3

H CH3CH3

CH3

(CH )2 7

CH

CH CH 3(CH )2 7

SG

FS

ASG

ES

[M+H-H2O]+



LC/APCI-MS TIC trace

SG

FS



Sat. Sterols Unsat. Sterols

sitostanol 0.25 (0.02) stigmasterol 0.89 (0.02)

campestanol 0.27 (0.01) cholesterol 1.18 (0.03)

cholestanol 0.31 (0.06) -sitosterol 1.41 (0.09)

campesterol 1.42 (0.05)



sterol LOD (ng/µL) LOQ (ng/µL)

cholesterol 0.04 0.08

stigmasterol 0.06 0.11

campesterol 0.04 0.07

-sitosterol 0.04 0.07

cholestanol 0.16 0.32

campestanol 0.19 0.37

sitostanol 0.20 0.40



CombinedSteryl GlycosideandAcylated Steryl Glygoside(µg/g)



Combined FS and ES (µg/g)



Glucosamine

• Stimulates the synthesis of proteoglycans, which, in combination with collagen, form the connective tissue destroyed in osteoarthritis.

• Anti-inflammatory action

~ 21 millions adults in the USA live with osteoarthritis25% of visits to primary care physiciansand half of all non-steroidal anti-inflammatory prescriptions*

* Y. Shao et al J. Pharma. Biomed. Anal. 35 (2004) 625

Dietary supplements as alternative treatment

huge business



Glucosamine


Glucosamine


Glucosamine

D-Glucosamine chlorhydrate (C6H11O5NH2) . HClMW 215.6 83.1% of free base

D-Glucosamine sulfate (C6H11O5NH2)2 . H2SO4 . 2KClMW 605.5 59.2% of free base

Difficulties : ► no chromophore

► highly polar

► positively charged at pH 7

HPLC Methods

O

CH2OH

H

NH2

HOH

H

HO

H OH

H


Glucosamine

Eau-Aceto-2 #8-10 RT: 0.12-0.16 AV: 3 NL: 1.20E8T: + c Q1MS [ 50.00-300.00]

60 80 100 120 140 160 180 200 220 240 260 280 300m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

220.97

179.98

202.98162.00

83.13242.96 278.97

143.96 189.9472.15 233.8084.13 126.01 171.04 267.00212.92 286.9298.15 117.03 258.93 295.9660.20

H2O : CH3CN / 50:50

M+1+CH3CN

M+1

M+Na+CH3CN- H2O

- H2O


Glucosamine

Eau-Metha #1-12 RT: 0.01-0.20 AV: 12 NL: 2.30E8T: + c Q1MS [ 50.00-250.00]

60 80 100 120 140 160 180 200 220 240m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

179.91

161.92

201.88162.92

72.07 143.93 181.91163.9284.04 202.89 217.83 226.9095.99 193.92157.90125.94 242.0460.12 113.97

H2O : MeOH / 50:50

M+1

O

NH3


Glucosamine

NEUTRAL LOSS SCANNING METHOD

TRIPLE QUAD INSTRUMENTS

Precursorm scan

Fragmentation Fragment detectionm-a scan

MS1 MS2CID


Glucosamine

Test02 #133-143 RT: 2.25-2.42 AV: 11 NL: 4.34E7T: + c SIM cnl [email protected][ 179.50-180.50]

179.6 179.8 180.0 180.2 180.4m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

179.89

180.23

180

84


Glucosamine

RT: 0.00 - 14.99 SM: 5B

0 2 4 6 8 10 12 14Time (min)

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

Inte

nsi

ty

2.25

1.91

0.141.66 3.46 3.94 14.804.801.05 5.59 7.05 8.02 14.248.73 9.97 11.45

NL:1.04E5TIC F: MS ArthroMarine03

C18, H2O:MeOH / 50:50200 µl/min10 µl

GLAY = 56026+1570.92*X R^2 = 1.0000 W: Equal

0 10000 20000

ng/m l

0

10000000

20000000

30000000

40000000

Are

a

Y = 56026 + 1571*x R2 = 1.0000

25 – 25000 ng/mlLOQ ~0.5 ng on column


Glucosamine

10mg of powderdissolution in 10 ml MeOH:H2O / 50:50dilution 100 x in PBS

Results and Reproducibility :

1 capsule , 5 injections : 224 ± 5% mg/g

capsule 1 : 233 mg/gcapsule 2 : 231 mg/gcapsule 3 : 233 mg/g

Commercial capsule

Commercial solution 10mg of liquidedilution in 10 ml MeOH:H2O / 50:50dilution 10 x in PBS

5 analysis : 0.52 mg/g ± 10%

Detection of carnitines in urine


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




FABMS spectrumof crude sample



FABMS2 spectrumof crude sampledetection of theprecursors of 85

Selective detection at 85

Scanning


Detection of carnitines in urine : discovery of a new carnitine

FABMS2 spectrumof a crude sampledetection of theprecursors of 85

?


Daughter spectrumof 290 m/z

CHCH2HOOC

CH2(CH3)3NO C

O

CH2 CH2 C

O

O CH2 CH3

ethylsuccinylcarnitine

MSRMNSynthesis



Ethylsuccinylc.

2,6-dimethyl-Heptanoylc.



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

Peptidemixture

separation

separation

Proteins

Peptides MS analysis

MS data

Identification

database search

digestion

Separation: always 2D

2D gel or LC - LC



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

cummulative mass

N

O

HR1

R2

b

y








Future ?


High throughput analysis

Non-covalent association

S. Sanglier et al , JASMAS , 14 (2003) 418

UCL, SC, Mass Spec. Lab.Pr. Em. Ed de Hoffmann, A. Spôte, R. Rozenberg, R. Libert, G. Petitjean

UCL, MD, FARMPMNT Pr. N. DelzenneCHAM Pr. B. Tilquin, A. Maquille, Pr. J. Quetin-Leclercq,

UCL, AGRO, BNUTPr. M. Meurens, Dr. N. Ruibal

Moulin de HollangeDr. D. Delacroix

UCL, Saint-LucPr. Fr. Van Hoof, Pr. Ph. De Nayer

€€€ : Région wallonne, UCL FSR and COSIF


Acknowledgments

Documents

Spectrométrie de masse: tour d’horizon de la technique et applications Pr. Jean-Louis Habib Jiwan UCL - Chemistry Department [email protected]