STANFORD MASS SPECTROMETRY USERS’ MEETING
Transcript of STANFORD MASS SPECTROMETRY USERS’ MEETING
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
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
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
SUMS• Open Access Lab: Mudd Bldg, room 175Ap g• 200 MS Users• Username user@stanford eduUsername [email protected]
• SUID # Stanford ID card #
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
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][ ]
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90
100936.82
C32H34N3P2Fe: C32 H34 N3 P2 Fe1 pa Chrg 1
90
100578.16
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bund
ance
935.99
308.96 50
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ve A
bund
ance
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e A
b
613.07
268 25 0
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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]
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100 308.90
C34H36N4P2Fe2+CH3CN
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ndan
ce
288.36
309.09-308.90 = 0.19CH3CN
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e A
bu
268.25
20.520 5
CH3CN
5
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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
Enteropathogenic Escherichia coliEnteropathogenic Escherichia coli expressing the
Type II secretion systemyp yinduced hemolysis of sheep blood cells
Streptomyces sp. K01-0509
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]
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unda
nce
519.20
260.18
+ ESI
519.2
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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]
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e
516.91
474.22
516.9474.2
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e Ab
unda
nce
- ESI
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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]
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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
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e Ab
und 168.65
218.73 280.58
+ ESI
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HolmesT_100608_23914_06 #89-107 RT: 3.28-3.74 AV: 9 NL: 1.26E5F: - c ESI Full ms [150.00-1000.00]
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100272.76
ESI
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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
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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]
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337.84216.67
252.16432.21
302.24 503.21
+ ESI
IC50 7 ng/mL
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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]
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e
501.27
672.38 - ESI
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430.27
743.38
673.40
431.31 601.42
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0