Ion mobility analyzer – quadrupole mass spectrometer - IOPscience
MASS SPECTROMETRY & STANFORD CHEMISTRY · 4 The Mass Spectrometer: Components 1.Ion...
Transcript of MASS SPECTROMETRY & STANFORD CHEMISTRY · 4 The Mass Spectrometer: Components 1.Ion...
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MASS SPECTROMETRY & MASS SPECTROMETRY & STANFORD CHEMISTRYSTANFORD CHEMISTRY
Allis S. Chien, Ph.D.Friday November 21, 2003
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Brief intro to Mass SpectrometersIonization: ESI & APCISUMS InstrumentationMS – Organic, Organometallic &
BiomoleculeHPLC-MS
MASS SPECTROMETRY & MASS SPECTROMETRY & STANFORD CHEMISTRYSTANFORD CHEMISTRY
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The Mass Spectrometer: Operation
Steps in generating a mass spectrum:
1. Produce ions2. Separate or filter ions3. Detect ions4. Process the data
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The Mass Spectrometer: Components
1.Ion source/interface2.Mass analyzer, including:
a.Mass analyzer (quadrupole, ion trap, TOF, etc.)b.Vacuum systemc.Some electronics
3.Detector4.Data storage, (processing), and output device
(usually a computer)
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Quadrupole Ion Trap
LC Pump ESI Quadrupole Ion Trap
Syringe Pump Detector
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What is API?Atmospheric Pressure Ionization
ESI – Electrospray IonizationSolution-phase process (for the most part)
APCI – Atmospheric Pressure Chemical Ionization
Gas-phase process
An interface between HPLC and Mass Detection
Designed to separate and ionize analytes from HPLC solvents
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ESI Needle+/- 5 kV
Heated Capillary
Taylor Cone
Solvent evaporation and ion release
+ ++++ +
+ +
+ ++++ +
+ +
+
++
+++++ +
++
+ ++++ +
+ +
+ ++++ +
+ +
+ +++
++
++
++
+
++ ++ +
+ +
+++
+ +
++ ++ +
+++
+ +
+
+++
+++
+
+
+
++
+
++
+
++
++
++
+
+
+
+
+
Electrospray – Basic Layout
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APCI: Atmospheric Pressure Chemical Ionization
Mechanism for positive ion formationPrimary ion formation:
Secondary ion formation:
Analyte ion formation:
−•+−
−•+−
+→+
+→+
ee
ee
2OHOH
2NN
22
22
OHOHOHOH 322•+•+ +→+
OH]H[OH 23 ++→+ ++ AnalyteAnalyte
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ESI or APCI ?
Many compounds can be analyzed by both techniques with different sensitivities
ESI is for highly polar compounds
ESI is for molecular weights >1000 amu
ESI is for thermally fragile compounds
APCI generally gives more fragmentation
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Analyte Compatibility
ESIESI
EIEI PBIPBITSPTSP FABFAB
Mol
ecul
ar
Wei
ght
200,000
15,000
1,000
Non Polar
APCI
Polar
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SUMS Instrumentation
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LCQ Classic MS
• Quadrupole Ion Trap LC-MS• ThermoFinnigan Surveyor HPLC &
LCQ “Classic” MS
– MW determination– Analytical LC-MS– MSn
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LCQ Deca XP Plus MS
– Capillary LC-MS– Protein identification & characterization– Complex protein mixture analysis– Assays, quantitation
•LC Packings Capillary HPLC System & ThermoFinnigan LCQ Deca XP Plus MS
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Q-Tof API
MicromassQ-Tof
•High resolution MS•Protein identification & characterization•De novo peptide sequencing•Post-translational modification ID
•Hybrid Tandem Quadrupole – Time of Flight MS
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Mass Spec Examples
Organic CompoundsOrganometallic CompoundsBiomolecules
LC-MSHigh Resolution MS
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Commonly Observed Ions in ESI
NH
O
OH
NH
OHO
O
NH
O
O
O
NH
C27H42N4O8
MW 550.3
100
0
20
40
60
80
Rel
ativ
e Ab
unda
nce
573.1
574.1
551.0[M+H]+
[M+Na]+
Hiroko Tanaka
+ESI
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Commonly Observed Ions in ESI
NH
O
OH
NH
OHO
O
NH
O
O
O
NH
C27H42N4O8
MW 550.3
540 550 560 570 580m/z
0.4
0.0
0.1
0.2
0.3
100
0
20
40
60
80
Rel
ativ
e Ab
unda
nce
573.1
574.1
551.0
549.0
550.0 571.0
[M+H]+
[M+Na]+
[M+Na-2H]-
[M-H]-
Hiroko Tanaka
+ESI
-ESI
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N
O
NH
O
O
O
O
OH
OO
N O
O
O
FF F
FF
N3
98
0
20
40
60
80
Rel
ativ
e Ab
unda
nce
1007.2
951.21023.1677.2 855.3618.3
C48H53N6O11F5
MW 984.4Hiroko Tanaka
Na+ Adduct MS
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N
O
NH
O
O
O
O
OH
OO
N O
O
O
FF F
FF
N3
400 600 800 1000m/z
100
0
20
40
60
80
98
0
20
40
60
80
Rel
ativ
e Ab
unda
nce
1007.2
951.21023.1677.2 855.3618.3
951.1
1007.1
732.1618.1 923.1
534.1C48H53N6O11F5
MW 984.4Hiroko Tanaka
MS/MS Does Not Displace Na+
MS/MS
MS
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APCI of a Non-polar Compound
NN
O
O
C52H74N2O2
MW 758.6
200 400 600 800 1000 1200 1400m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
759.7
Ned Bowden
[M+H]+
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APCI of a Non-polar Compound
NN
O
O
C52H74N2O2
MW 758.6
200 400 600 800 1000 1200 1400m/z
759.7
Ned Bowden
8 760 762m/z
759.7
760.7
761.7
[M+H]+
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APCI of a Non-polar Compound
NN
O
O
C52H74N2O2
MW 758.6Average MW 759.2
200 400 600 800 1000 1200 1400m/z
759.7
Ned Bowden
8 760 762m/z
759.7
760.7
761.7
[M+H]+
759.7
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Cu Isotope PatternSimulat ionCuProf ileResolut ion:
Daltons 1at 5% height
Charges 1Chrg d ist 0Ions 2M in Ion Ab. 1e-020M in Ions 5000M ax Ions. 20000
61 62 63 64 65 66 67m/z
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
Abun
danc
e
62 .9
64.9
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Cu Compound: Theoretical MS
N NCu
O O
H H
C36H56N2O2CuMW 611.37
Russell Pratt
C36 H56 N2 O2 Cu1Simulat ionC36H56N2O2CuProf ileResolut ion:
Daltons 1at 5% height
Charges 1Chrg d ist 0Ions 3328M in Ion Ab. 1e-020M in Ions 5000M ax Ions. 20000
610 612 614 616m/z
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
Abun
danc
e
611.4
613.3
612.4
614.4
615.4616.4 617.4
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Cu Compound: +ESI and -ESI
608 610 612 614 616m/z
6
0
2
4
100
0
20
40
60
80
Rel
ativ
e Ab
unda
nce
612.3
614.3
615.3
610.3
612.3611.2
N NCu
O O
H H
C36H56N2O2CuMW 611.37
Russell Pratt
+ESI
-ESI
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Zr Isotope PatternSimulat ionZrProf ileResolut ion:
Daltons 0.35at 5% height
Charges 1Chrg d ist 0Ions 5M in Ion Ab. 1e-020M in Ions 5000M ax Ions. 20000
88 89 90 91 92 93 94 95 96 97m/z
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
Abun
danc
e
89 .9
93.991.9
90.9
95.9
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Zr Compound: Theoretical MS
ZrO
O
N
N
C28H46N2O2ZrMW 532.26
Kuo-Wei Huang
532 533 534 535 536 537 538 539m /z
533.26
534.26
535.26
537.26
536.27 538.27539.26
Simulated [M+H]+
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Theoretical vs. Experimental
ZrO
O
N
N
C28H46N2O2ZrMW 532.26
Kuo-Wei Huang
532 533 534 535 536 537 538 539m /z
533.26
534.26
535.26
537.26
536.27 538.27539.26
532 533 534 535 536 537 538 539 540
533.1
534.1535.1
537.1
536.1 538.1 539.1
Simulated [M+H]+
Experimental [M+H]+
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Loss of Counterion
500 600 700 800 900 1000 1100 1200m/z
849.3
Richard Decreau
C37H8F15N4FeClMW 884.0
N
N N
NFF
F
F F F F
F F
FFF
F
FF
Fe
Cl
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Loss of Counterion
500 600 700 800 900 1000 1100 1200m/z
849.3
Richard Decreau
[C37H8F15N4Fe]+
MW 849.0
C37H8F15N4FeClMW 884.0
N
N N
NFF
F
F F F F
F F
FFF
F
FF
Fe
Cl[M-Cl]+
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Loss of Counterion
500 600 700 800 900 1000 1100 1200m/z
849.3
45 850m/z
849.3
850.3
851.3847.3
Richard Decreau
[C37H8F15N4Fe]+
MW 849.0
C37H8F15N4FeClMW 884.0
N
N N
NFF
F
F F F F
F F
FFF
F
FF
Fe
Cl[M-Cl]+
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FePd-Porphyrin
1400 1500 1600 1700 1800 1900 2000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
1558.0
1601.9
Richard Decreau
NN
NN
HN
NHFe
O
N N ON
NNH
NNO
N
F3C
MeMeMe
Pd
HNN
O
ClCl
C77H55O4N16F3FePdCl2MW 1558.0
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[M]+
0 1600 1700 1800 1900 2000m/z
1558.0
1601.9
Richard Decreau
NN
NN
HN
NHFe
O
N N ON
NNH
NNO
N
F3C
MeMeMe
Pd
HNN
O
ClCl
C77H55O4N16F3FePdCl2MW 1558.0
155 2 15 5 4 15 5 6 15 58 15 6 0 15 6 2 15 6 4 15 6 6m / z
15 58 .0
155 6 .1
15 57 .1 15 6 0 .0
15 55 .0 15 6 1.0
15 6 1.9
15 5 4 .1 15 6 3 .0
15 6 4 .115 5 3 .0
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Fe(II) Oxidized to Fe(III)
0 1600 1700 1800 1900 2000m/z
1558.0
1601.9
Richard Decreau
NN
NN
HN
NHFe
O
N N ON
NNH
NNO
N
F3C
MeMeMe
Pd
HNN
O
ClCl
C77H55O4N16F3FePdCl2MW 1558.0
155 2 15 5 4 15 5 6 15 58 15 6 0 15 6 2 15 6 4 15 6 6m / z
15 58 .0
155 6 .1
15 57 .1 15 6 0 .0
15 55 .0 15 6 1.0
15 6 1.9
15 5 4 .1 15 6 3 .0
15 6 4 .115 5 3 .0
15 5 5 15 6 0 15 6 5m / z
15 5 8 . 2 0
15 5 6 . 2 015 5 9 . 2 0
15 6 1. 2 015 5 5 . 2 0
15 6 2 . 2 0
15 5 4 . 2 0 15 6 3 . 2 015 6 4 . 2 015 5 3 . 2 0 15
theoretical
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Exchangeable Hydrogens
220 222 224 226m /z
NH NH
NH
C13H21N3
MW 219.2
Xavi Ribas
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36
H/D Exchange Over Time
220 222 224 226m /z
223.3
C13H21N3MW 219.2C13H18D3N3
MW 222.2
Xavi Ribas
1 min
2 min
3 min
4 min
6 min
ND DN
ND
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37
H2N
NH
N
N
O
NH2N
O
O
PO
O
OH
NH
N
N
O
NH2N
O
O
PO
O
OH
NH
N
N
O
NH2N
O
O
PO
O
OH
O
O
PO
O
OH
NH
N
N
O
NH2N
O
OH
NH
N
N
O
NH2N
Oligonucleotides: -ESI
800 1000 1200 1400 1600m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
1581
.6
790.
580
1.4
1603
.616
25.6
C50H62N26O27P4
MW 1582.3
Greg Miller
GGGGG
-ESI
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38
Doubly Charged Ion [M-2H]2-
790 795 800 805 810 815m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
790.
579
0.9
801.
480
1.9
802.
2
812.
381
2.7
H2N
NH
N
N
O
NH2N
O
O
PO
O
OH
NH
N
N
O
NH2N
O
O
PO
O
OH
NH
N
N
O
NH2N
O
O
PO
O
OH
O
O
PO
O
OH
NH
N
N
O
NH2N
O
OH
NH
N
N
O
NH2N
C50H62N26O27P4
MW 1582.3
Greg Miller
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39
Peptide Charge States
400 600 800 1000 1200 1400 1600m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
819.2
1228.2
710.1 1050.2615.2
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40
Peptide MW = 2455 Da
400 600 800 1000 1200 1400 1600m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
819.2
1228.2
710.1 1050.2615.2
[M+2H]2+
[M+3H]3+
[M+4H]4+
Neutral mass = (819.2 * 3)-3
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41
Proteins: Multiple charge states031009_12216' # 1042-1147 RT: 27.33-30.13 A V: 106 NL: 2.73E7T: + p ESI Full ms [ 400.00-2000.00]
600 800 1000 1200 1400 1600 1800 2000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
948.0903.1
972.5
997.9842.9 1115.1
1148.91263.8 1353.9824.8
774.2 1404.0 1579.3743.9
1648.2729.7 1723.1
1806.3 1893.3690.5 1995.1
Ian Suydam
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42
Deconvoluted Protein Mass
20000 25000 30000 35000 40000 45000 50000 55000mass
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
37884.0
50629.0 54668.047473.041979.025315.0 33798.028480.020990.0
Ian Suydam
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43
Deconvoluted Protein Mass
20000 25000 30000 35000 40000 45000 50000 55000mass
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
37884.0
50629.0 54668.047473.041979.025315.0 33798.028480.020990.0
Ian Suydam
38000mass
37884.0
38059.0
37560.0 38632.0
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44
Charge State Series
700 800 900 1000 1100 1200 1300 1400m/z
+261458.2
+271404.0
+281353.9+29
1307.3
+301263.8
+311223.0
+321184.7
+331148.9
+351083.5
+361053.3
+38997.9
+39972.5
+41925.0
+43882.0
+45842.9+46
824.8+49774.2+51
743.9+54703.0
38000mass
37884.0
38059.0
560.0
37884
Ian Suydam
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45
Charge State Series
700 800 900 1000 1100 1200 1300 1400m/z
+261458.2
+271404.0
+281353.9+29
1307.3
+301263.8
+311223.0
+321184.7
+331148.9
+351083.5
+361053.3
+38997.9
+39972.5
+41925.0
+43882.0
+45842.9+46
824.8+49774.2+51
743.9+54703.0
850 900 950 1000 1050 1100 1150m/z
+331154.3+34
1120.4
+351088.5+36
1058.2
+371029.6
+381002.6
+39976.9+40
952.5+41929.3
+42907.3
+43886.1
+44866.1+45
846.7
+46828.4
38000mass
37884.0
38059.0
560.0
37884
38059
Ian Suydam
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46
75 kDa Protein Charge States011105_4139 #281-317 RT: 10.11-11.44 AV: 37 NL: 1.76E6T: + p ESI Full ms [ 550.00-2000.00]
1000 1200 1400 1600 1800 2000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
+381967.73
+391917.27
+401869.47
+421780.60
+431739.13
+451662.07
+481558.27
+511466.53
+521438.60
+551360.00
+561335.67
+601246.87
+621206.40
+661133.27
0.5 x 2 mm Michrom Peptide CapTrap
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47
75 kD Protein Deconvoluted# 1 RT: 0.00 P: + NL: 2.82E7T: + p ESI Full ms [ 550.00-2000.00]
65000 70000 75000 80000 85000
mass
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
74742 Da
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48
High-Resolution MS – Q-Tof
O O
OOO
OMeMe
H
Me Me
H
O
Me
NH2
O
M (neutral)C19H29NO8
MW 399.1893[M+Na]+
C19H29NO8NaMW 422.1791
421 422 423 424m/z0
100
%
031103_12403_AH 132 (2.257) Sm (SG, 2x3.00); Sb (5,40.00 ); 3.29e3422.1802
423.1803422.6927 424.1900
Andrew Hinman
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49
Centroided Spectrum
O O
OOO
OMeMe
H
Me Me
H
O
Me
NH2
O
M (neutral)C19H29NO8
MW 399.1893[M+Na]+
C19H29NO8NaMW 422.1791
421 422 423 424m/z0
100
%
0
100
%
031103_12403_AH 132 (2.257) AM (Cen,4, 80.00, Ar,5000.0,0.002.26e4422.1798
423.1862
422.6973 424.1897
031103_12403_AH 132 (2.257) Sm (SG, 2x3.00); Sb (5,40.00 ); 3.29e3422.1802
423.1803422.6927 424.1900
Andrew Hinman
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50
Elemental Composition Report
[M+Na]+C19H29NO8NaMW 422.1791
Andrew Hinman
O O
OOO
OMeMe
H
Me Me
H
O
Me
NH2
O
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51
Elemental Composition Report
[M+Na]+C19H29NO8NaMW 422.1791
Andrew Hinman
O O
OOO
OMeMe
H
Me Me
H
O
Me
NH2
O
422.1791 amu, 1.7 ppm, C19H29NO8Na
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52
ESI-MS of Epothilone C
Epothilone C from E. coli culture broth
[M+H]+ = 478.26[M+Na]+ = 500.24
[M+H]+
[M+Na]+
C28H39NO5SMW: 477.25
OHO
HO N
S
O
O
Chris Boddy
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Epothilone C Biosynthesis is Reconstituted in E. coli
14C Radio-TLC assay
1 2 31. EpoC standard2. 2 mM substrate3. Negative control
LC/MS analysisExtracted Ion Chromatogram of [M+H]+
epothilone C standard
E. coli with 2 mM substrate
Epothilone C is observed in metabolically engineered E. coli cultures Chris Boddy
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54
Characterization by Isotopic Derivatization
12C propionate feeding
12Cm/z = 478.3
13Cm/z = 481.3
SNAC
OH
OO
O
Na
**
* *OHO
HO
O
O
N
SN
S
* = 13C
EIC
Chris Boddy
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55
13C Propionate increases mass by 3 Da
13C propionate feeding
SNAC
OH
OO
O
Na
**
* *OHO
HO
O
O
N
SN
S
* = 13C
EIC
Chris Boddy
12Cm/z = 478.3
13Cm/z = 481.3
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Conclusion
ResourcesAcknowledgements
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57
http://mass-spec.stanford.edu
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58
http://mass-spec.stanford.edu
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59
Online Tools & Linkshttp://mass-spec.stanford.edu
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Acknowledgements• Wandless Lab
Hiroko Tanaka • Waymouth Lab
Ned BowdenKuo-Wei Huang
• Stack LabRussell PrattXavi Ribas
• Collman LabRichard Decreau
• Kool LabGreg Miller
• Boxer LabIan Suydam
• DuBois LabAndrew Hinman
• Khosla LabChris Boddy
SUMS:• Andrew Guzzetta• Michael Kitamura
MS Committee:• Pehr Harbury• Peter Jackson• Chaitan Khosla• Al Smith• Tom Wandless
Funding:• Stanford Bio-X Initiative• Vincent & Stella Coates
Foundation