STANFORD MASS SPECTROMETRY STANFORD MASS SPECTROMETRY STANFORD MASS SPECTROMETRY STANFORD MASS SPECTROMETRY USERS’ MEETINGUSERS’ MEETING

Qualitative Mass Spectrometry for Elucidationfor Elucidation

of Natural & Synthetic Products

For personal use only.Please do not reuse or reproducewithout the author’s permission.

2

O iOverview

• General aspects of mass spectrometry

• Open Access Lab

• Example of Qualitative AnalysisExample of Qualitative Analysis

M S t tMass Spectrometry

• IonizationIonization• Ions are tractable

S iti it• Sensitivity • Destructive analytical method• Selective m/z (MS/MS)

Ionization

• The formation of ions is a critical part of every application of mass spectrometry

• Ion formation has to do with the energy required to produce an ion from a q pneutral molecule or the energy required to transfer pre-existing ions from a liquid p g qor a solid phase into the gas phase

– gas phase ion chemistrygas phase ion chemistryPhysical Review (1925-1930)

dIonization and Energy

• Molecular IonsM + Energy M+. Appearance Energy

M+ Energy M-.

• Pseudo-Molecular IonsM + H+ [M+H]+ Proton Affinity

M+ Na+ [M+Na]+ M Na [M Na]- Odd electron ions

Even electron ions- Even electron ions

Ion Fragmentation

• Ion fragmentation has to do with the energy required to break particular chemical bonds that hold the ion together

• When the amount of energy transfer to the bond is controlled, specific bonds , pcan be broken and molecular structural information can be obtained

I S iIon Suppression

• Competition for either space or charge – Proton affinityy– Surface tension– Viscosity y

• Matrix effect• Media of synthetic reactions• Media of synthetic reactions

– Reactants, products, co-factors

I i I t lIon suppression: Isoproterenol

l dMass Resolution and Accuracy

Magnetic Sector:Resolution is constant with mass

Peak width varies with mass

Quadrupole, Ion Trap, TOF:Resolution varies with Mass

Peak width constant with Mass10% Valley Definition

Peak width constant with MassFull Width Half Mass Definition

lResolution FWHMm/z 300/ 0.7 = R 428

m/z 500/ 0.7 = R 714

m/z 1000/ 0.7 = R 1428

m/z 300/ 0.1 = R 3000

m/z 500/ 0.1 = R 5000

m/z 1000/ 0.1 = R 10,000

FWHM FWHM0.7 0.1

h dMS is a Destructive Method

Open Access LabOpen Access Lab

SUMS• Open Access Lab: Mudd Bldg, room 175Ap g• 200 MS Users• Username user@stanford eduUsername user@stanford.edu

• SUID # Stanford ID card #

maurizio.splendore@stanford.edu

bOpen Access Lab • GC-MS (EI)GC MS (EI)

– Non Polar Compounds– Mass Range 50-550 DaMass Range 50 550 Da

• LC-MS (ESI)– Polar CompoundsPolar Compounds– Mass Range 50-2000 m/z

• DESI-MS/MSDESI MS/MS– Polar Compounds and Solids– Mass Range 50-2000 m/zg

Organometallic CompoundOrganometallic Compound

C34H36N4P2Fe2+ C32H34N3P2Fe+34 36 4 2 32 34 3 2

DESI M S tDESI Mass SpectrumDecrisci100603_23914 #70-104 RT: 1.22-1.96 AV: 29 NL: 7.08E6T: + c ESI Full ms [150.00-2000.00][ ]

80

90

100936.82

C32H34N3P2Fe: C32 H34 N3 P2 Fe1 pa Chrg 1

90

100578.16

60

70

bund

ance

935.99

308.96 50

60

70

80

90

ve A

bund

ance

30

40

50

Rel

ativ

e A

b

613.07

268 25 0

10

20

30

40

Rel

ativ

579.16

580.16579.16577.17 580.16578.17 579.17 580.18578.16

0

10

20268.25

938.77614.13

481.22477.40 753.93 1449.571170.49 1645.931325.78944.64 1893.71

577.0 577.5 578.0 578.5 579.0 579.5 580.0 580.5 581.0m/z

0

200 400 600 800 1000 1200 1400 1600 1800 2000m/z

DESI M S tDecrisci100603_23914 #17-107 RT: 0.51-1.21 AV: 59 NL: 6.71E6T: + c ESI Full ms [150.00-800.00]

DESI Mass SpectrumT: c ESI Full ms [150.00 800.00]

80

85

90

95

100 308.90

C34H36N4P2Fe2+CH3CN

55

60

65

70

75

ndan

ce

288.36

309.09-308.90 = 0.19CH3CN

30

35

40

45

50

Rel

ativ

e A

bu

268.25

20.520 5

CH3CN

5

10

15

20

25309.69

222.88313.88

307.96289.07287.22

217.11 269.01244.98 349.18278.93 329.77267.18233.86 315.86 346.05246.87 291.80211.92 261.04201.07 301.98270.93

20.5

200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350m/z

0211.92 261.04201.07 301.98270.93

X C t ll hX-ray Crystallography

Enteropathogenic Escherichia coliEnteropathogenic Escherichia coli expressing the

Type II secretion systemyp yinduced hemolysis of sheep blood cells

Streptomyces sp. K01-0509

dGuadinomine

bResistant AntibioticsGuad MW Yield ICGuad MW

(g/mol)Yield (mg/L)

IC50(ug/mL)

A 518 0.66 0.02

B 502 1.1 0.007

C 556 0.025 0

D 544 4.4 8.5

Acid 258 1 0

Omura J. Antibiot. 61(4)222, 2008

+/- ESI Mass Spectra Guadinomine A C:\Xcalibur\...\HolmesT_100310_22408_Gua 3/10/2010 3:31:07 PM Guadinomine

519 2HolmesT_100310_22408_Gua #38-52 RT: 1.08-1.41 AV: 7 NL: 4.87E6F: + c ESI Full ms [150.00-1000.00]

60

70

80

90

100

unda

nce

519.20

260.18

+ ESI

519.2

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 10000

10

20

30

40

50

Rel

ativ

e Ab

u + ESI

m/z

HolmesT_100310_22408_Gua #38-53 RT: 1.05-1.44 AV: 8 NL: 1.37E6F: - c ESI Full ms [150.00-1000.00]

70

80

90

100

e

516.91

474.22

516.9474.2

10

20

30

40

50

60

Rel

ativ

e Ab

unda

nce

- ESI

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z

0

Bi th i f G di iBiosynthesis of Guadinomines

• R5 medium, 1 L liquid culture, 30°C shaker, 4 days, aerobic conditions– 0.3M Sucrose, 1.4mM K2SO4, 50mM MgCl2-6H2O, 2 4, g 2 2– 55mM Glucose, 25mM TES Free Acid (Buffer), 2mL Trace Elements

Solution– 4mL 5M CaCl2-2H2O, 7mL 20% L-Proline– 10mL 0 5% KH PO 7mL 1M NaOH– 10mL 0.5% KH2PO4, 7mL 1M NaOH– Sterilize medium by autoclaving

• Extract with 1 L Acetone (acetone lyses the cells)Rotovap acetone leaving 1 mL crude aqueous– Rotovap acetone, leaving 1 mL crude aqueous extract

– Filter & submit for Mass Spec

E t t A l iExtract AnalysisC:\Xcalibur\data\HolmesT_100608_23914_06 6/8/2010 4:11:41 PM R5 Crude 1

HolmesT_100608_23914_06 #91-105 RT: 3.31-3.71 AV: 8 NL: 5.89E5F: + c ESI Full ms [150.00-1000.00]

60

70

80

90

100

danc

e

241.66

264.72

191.66168.65 + ESI

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 10000

10

20

30

40

50

60

Rel

ativ

e Ab

und 168.65

218.73 280.58

+ ESI

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z

HolmesT_100608_23914_06 #89-107 RT: 3.28-3.74 AV: 9 NL: 1.26E5F: - c ESI Full ms [150.00-1000.00]

80

90

100272.76

ESI

10

20

30

40

50

60

70

Rel

ativ

e Ab

unda

nce 386.77288.75

402.69

158.81 673.67759.04

424.71 874.57849.35376.79330.79 665.04 740.55446.65174.77 471.39 677.47 818.33550.36 580.89516.57 902.49658.81 688.67

- ESI

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z

0

10

I d l Improved sample prep

• DOWEX 50W x2 [H+] Ion Exchange Column[ ] g– Strong Cation Exchanger– Elute with 1.5N NH4OH– Neutralize with conc HCl– Neutralize with conc. HCl– Lyophilize and resuspend in minimal amount of H2O– Filter & submit for Mass Spec

C:\Xcalibur\data\HolmesT_100608_23914_01 6/8/2010 2:00:01 PM R5 IEC 1

Guadinomine B: +/- ESI Mass Spectra

HolmesT_100608_23914_01 #99-130 RT: 3.20-4.03 AV: 16 NL: 1.31E6F: + c ESI Full ms [150.00-1000.00]

60

70

80

90

100

unda

nce

337.84216.67

252.16432.21

302.24 503.21

+ ESI

IC50 7 ng/mL

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 10000

10

20

30

40

50

Rel

ativ

e Ab

u

373.39243.43

454.22208.12525.25 674.33185.53 306.59

603.25361.18

+ ESI

m/z

HolmesT_100608_23914_01 #99-138 RT: 3.23-4.30 AV: 20 NL: 6.67E5F: - c ESI Full ms [150.00-1000.00]

70

80

90

100

e

501.27

672.38 - ESI

10

20

30

40

50

60

Rel

ativ

e Ab

unda

nce

430.27

743.38

673.40

431.31 601.42

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z

0

k l dAcknowledgementsAlli Chi Vi t & St ll C t• Allis Chien

• Karolina Krasinska• Ludmila Alexandrova

• Vincent & Stella Coates Foundation

• Pavel Aronov• Chris Adams• Richard Zare• Richard Perry• Robert Waymouth• Antonio De Crisci• Chaitan Khosla• Tracy Holmes II