High Resolution Mass Spectrometers role in small molecule studies TuKiet T. Lam, PhD Chem 395:...
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Transcript of High Resolution Mass Spectrometers role in small molecule studies TuKiet T. Lam, PhD Chem 395:...
1
High Resolution Mass Spectrometers role in small molecule studies
TuKiet T. Lam, PhDChem 395: Bioanalytical Chemistry
April 12, 2011
2
Instrumentations, Fundamental Principles, and Advantages
Various Forms of MS instruments
Aebersold and Mann (2003) Nature 422, 198-207
Thermo Fisher Scientific nano-UPLC ESI LTQ-Orbitrap MS system
ABI 4800 MALDI TOF/TOF Tandem MS
System
Bruker APEX 9.4 Tesla ESI FT-ICR MS System
ABI nano-UPLC ESI QTRAP-4000 MS system
ABI ESI QSTAR Elite MS System
ABI API QTRAP 5500
Waters CapLC-ESI QTOF Micro MS System
Mass Spectrometers
FT-ICR MS Ion Optics
--Apollo II Source
--Improved sensitivity (>10 x)--Very robust,--Less source Maintenance
Quadrupole
CollisionCell
TransferOptics
ECD
IRMPD
HeatedGlass
Capillary
Apollo II ESI source
LTQ-FT
LTQ-FT specs• Resolution
– 100 000 resolution at m/z 400 at 1 Hz repetition rate– >500 000 resolution broadband mode
• Mass Range– m/z 50-4000 (standard range)– 1-order-magnitude in single scan (e.g. m/z 400-4000)
• Mass Accuracy– 2 ppm RMS, external mass calibration– <1 ppm RMS, internal mass calibration
• Dynamic Range – >500 000 between mass spectra
5000 within mass spectrum• IRMPD• ECD
Courtesy from David C. Muddiman (Currently at Department of Chemistry at NCSU)
Why FT-ICR MS?
A.G. Marshall, C.L. Hendrickson, and G.S. Jackson. Mass Spectrometry Reviews, 1998, 17, 1-35.
zm
Bων o
7c
c
10535611.1
2
-B
v
v
Bvq Bvq
yz
x
B q B m=
Once the ion is trapped,
the magnet bends it into
a circular path.
We measure the frequency
We know theMagnetic Field
So we can calculate the mass of the ion
m
“Light” Ions have a High frequency
“Heavy” Ions have a Low frequency
Differential Amplifier
Time (ms)8007006005004003002001000
0.050.040.030.020.010
-0.01-0.02
-0.03-0.04-0.05
Time-Domain Transient
Image Current
As the spiraling ion gets neara detect plate, it induces a current that is detected by our instrument.
The signal is recorded fora period of time and then displayed by the software
Frequency (kHz)300250200150100500
FT
Frequency Spectrum
mz
A= + B
2
m/z
14001300120011001000900800700600500
Mass SpectrumTime (ms)
8007006005004003002001000
0.050.040.030.020.010
-0.01-0.02
-0.03-0.04-0.05
Time-Domain Transient
Image CurrentA Fourier Transform thenconverts the “time” domainsignal into all the frequenciesthat compose the “time” signal
We know how frequency relatesto mass, so we convert to the“Mass Spectrum”
Ion Energy
Ion Trapping TimeUpper Mass LimitNumber of Ions
Mass Resolving PowerScan Speed (LC/MS)
Highest Non-Coalesced Mass
00B0 (tesla)
2525B0 (tesla)
9.4 T
9.4 T
7 T7 T
25 T 25 T
Our experiments get easierat higher magnetic fieldsLinear increases
Increase as B2
14.5 T
14.5 T
Once we make an ion, we move it into the center of the Magnet.Then, we trap it before it can escape.
ION+
Electrostatic Barrier
“Gate” shut before the ion escapes
Ion is now trapped in the magnet.
Ion sees barrierand is turned back
From Primer 1998 Marshall.
• Robust Accurate Mass– 5 ppm rms external calibration– 2 ppm rms internal calibration
• High Resolution– 60,000 at m/z 400 with a scan repetition
rate of 1 Hz– Maximum Resolution >100,000
• Mass Range– 50-2000; 200-4000
• Sub-fmol Sensitivity (LC/MS)• MS/MS and MSn
• High Dynamic Range– >2,500 within mass spectrum
LTQ Orbitrap Operation Principle1. Ions are stored in the Linear Trap2. …. are axially ejected3. …. and trapped in the C-trap
4. …. they are squeezed into a small cloud and injected into the Orbitrap5. …. where they are electrostatically trapped, while rotating around the central electrode and performing axial oscillation
The oscillating ions induce an image current into the two outer halves of the orbitrap, which can be detected using a differential amplifier
Ions of only one mass generate a sine wave signal
Ion Motion in Orbitrap• Only an axial frequency does
not depend on initial energy, angle, and position of ions, so it can be used for mass analysis
• The axial oscillation frequency follows the formula
zm
k
/
w = oscillation frequencyk = instrumental const.m/z = …. what we want!
A.A. Makarov, Anal. Chem. 2000, 72: 1156-1162.A.A. Makarov et al., Anal. Chem. 2006, 78: 2113-2120.
Ions of Different m/z in Orbitrap• Large ion capacity - stacking
the rings • Fourier transform needed to
obtain individual frequencies of ions of different m/z
z
φ
r
Korsunskii M.I., Basakutsa V.A. Sov. Physics-Tech. Phys. 1958; 3: 1396.Knight R.D. Appl.Phys.Lett. 1981, 38: 221.Gall L.N.,Golikov Y.K.,Aleksandrov M.L.,Pechalina Y.E.,Holin N.A. SU Pat. 1247973, 1986.
Electrostatic Field Based Mass Analyser
APGC APPI, APCInanoFlowTMESI ESI/APCI, ESCi(r)
Physical Components of Instrument SYNAPT G2 HDMS
Internal Component of SYNAPT G2 HDMS
1 sec
MSE Alternating High/Low Energy Acquisition
MSPrecursor
MSE
Fragments
Retention Time
High Definition UPLC/MSE analysisTime Aligned Parallel (TAP)
fragmentation
CID CIDIMS
Ionization Methods
John B. Fenn
Koichi Tanaka
Electrospray Ionization
Matrix Assisted Laser Desorption Ionization
(MALDI)
(Nobel, e-museum)
Nobel Prize in Chemistry 2002
Resolution Mass Accuracy
Fragmentation Capabilities
Fourier Transform Ion Cyclotron Resonance (FT-ICR) MS
H2N C C N
OR1
C C N
ORn-1
C C
ORn
OH
m+nHn+
y1
bn-1
z1·
cn-1
...
ECD
IRMPDCID
Retention of labile modificationsNo X-P cleavage
Facile loss of H3PO4
X-P cleavage preferred
m/z 434432430428
429.22623
Deuterated (D)
Protonated (P)
430.22990
431.23346
429.22657
430.22835
430.23262
431.23617 432.23963
PDD
D
P
P
P
P
220 260 300 340 380 m/z
263 264 265 266 267m/z
265.04689 (Exp.)
Zoom
265.04713 (Cal.)
0.00024 (Diff.)- 0.9 ppm (Error)
(m/z)max(m/z)minm/z
Peak Capacity = m50%
(m/z)max - (m/z)min
m50%
Ultra-high Resolving Power
Separation Method
Maximum # of Components
MaximumPeak Capacity
TheoreticalPlates
HP-TLC 6 25 1,000Isocratic LC 12 100 15,000
Gradient LC 17 200 60,000HPLC 37 1,000 1,500,000CE 37 1,000 1,500,000Open Tubular GC 37 1,000 1,500,000
ESI FT-ICR MS 525 200,000 60,000,000,000
m/m50% > 200,000200 < m/z < 1,000
maverage +/- 0.25 Da Skip Prior Chemical Separationand Identify Components by MS!
9.4T Bruker Qe FT-ICR MS 26
274.1860 386.2585 477.2301
609.2821
716.4519
040208_Cerno_32K-64K_000004.d: +MS
0.00
0.25
0.50
0.75
6x10Intens.
200 300 400 500 600 700 800 900 m/z
274.1874 386.2558477.2305
609.2817
716.4601
040208_Cerno_64K-128K_000002.d: +MS
0
1
2
3
6x10
200 300 400 500 600 700 800 900 m/z
386.2556 477.2313
609.2811
716.4596
040208_Cerno_128K-256K_000002.d: +MS
0
1
2
3
6x10
200 300 400 500 600 700 800 900 m/z
393.0840477.2312
609.2811
716.4590
040208_Cerno_512K-1M_000002.d: +MS
0
1
2
7x10
200 300 400 500 600 700 800 900 m/z
386.2557 477.2312
609.2814
716.4591
040208_Cerno_1M-2M_000002.d: +MS
0
2
4
7x10
200 300 400 500 600 700 800 900 m/z
386.2557477.2312
609.2818
716.4594
040208_Cerno_2M-4M_000002.d: +MS
0
2
4
6
7x10
200 300 400 500 600 700 800 900 m/z
Zoom
5,682
22,621
45,094
93,767
Resolving Power (m/z at 609)
609.2821
610.2754 611.2755
607 608 609 610 611 612 613 m/z
609.2817
610.2825611.2790
607 608 609 610 611 612 613 m/z
609.2811
610.2840611.2865
607 608 609 610 611 612 613 m/z
609.2811
610.2847611.2877
607 608 609 610 611 612 613 m/z
609.2814
610.2850611.2877
607 608 609 610 611 612 613 m/z
609.2818
610.2854611.2890
608 609 610 611 612 613 m/z
2,840
1,396
Resolving Power vs Cycle Time
785.0 785.2 785.4 785.6 785.8 786.0 786.2 786.4 786.6 786.8 787.0 787.2 787.4 787.6 787.8 788.0 788.2m/z
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
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80
100
Rela
tive
Abun
danc
e
0
20
40
60
80
100
785.8419R=5901 786.3435
R=5900
786.8447R=5900
787.3463R=6000 787.8453
R=5800785.5934R=6200
785.8421R=23801
786.3434R=23900
786.8446R=24000 787.3457
R=24100 787.8471R=15600
785.5992R=24300
785.8419R=48101 786.3435
R=47700
786.8446R=48200 787.3458
R=47500787.8477R=42000
785.5994R=47100
785.8413R=94801 786.3428
R=95200
786.8442R=93600
787.3458R=98000785.5989
R=95800787.8477R=89200
0.9 s
1.6 s
RP 75000.2 s
RP 300000.5 s
RP 60000
RP 100000
Improvement in performance using a 240-core GPU compared with a quad-core CPU for processing LD/MSE data files of varying file size from different chromatographic Separation.
Computing Enhancement with GPU for more complex data set
m/z800750700650600550500450400350300250
*
*
* Internal Calibrant
420400380360340320300
Measured Theoretical Assignment Error
361.23485 361.23548 C20H34O4Na -1.7 ppm
361.27361.23361.19361.14361.10
#
# Peaks of interest
375.28375.24375.19375.15375.11
# 375.21416 375.21474 C20H32O5Na -1.6 ppm
*
Johnston, Murray
Bryostatin 2 (+ ion)Quad Select 885 (+1) peak, then IRMPD at 12W 90ms
Parent
900750600450300150
- 44 - 44- 44- 44 - 88- 176- 191 - 38 - 32
- 18
m/z 1,5001,200900600300 m/z 1,5001,200900600300
* Internal Calibrants
**
[M+Na]+ = Exp. 885.4257 ± 0.9 ppm Theo. 885.4249
Broadband with int. cal.
Quad Select 885 (+1) peak
*
Manning, Thomas, … Lam, TuKiet, et al., Natural Product Research, 19, 467, (2005).
1
10
100
1000
10000
100000
100 1000 10000 100000 1000000 10000000
Target value, ions
S/B
m/z 1522
m/z 524
m/z 195
Dynamic Range in a Single Spectrum
(0.75 sec Acquisition)
Orifice to FT-ICR MS
384-nozzle nanoESI chip
TriVersa NanoMate
ControlB3a #4869 RT: 41.56 AV: 1 NL: 7.39E6T: FTMS + p NSI Full ms [465.00-1600.00]
500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
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75
80
85
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95
100
Re
lativ
e A
bu
nd
an
ce
600.9776
804.3450
558.7548
532.2505
649.9460
699.3472897.9816
716.0311956.8159
849.8573 974.9185 1116.5020
Parallel Detection in Orbitrap and Linear Ion Trap
• Total cycle is 2.4 seconds• 1 High resolution scan with accuracies < 5 ppm• External calibration • 5 ion trap MS/MS in
parallel
RT: 41.56High resolutionFull scan # 4869
High resolution full scan in Orbitrap and 5 MS/MS in linear ion
trap
ControlB3a #4870 RT: 41.57 AV: 1 NL: 7.16E3T: ITMS + c NSI d Full ms2 [email protected] [150.00-1810.00]
200 400 600 800 1000 1200 1400 1600 1800
m/z
0
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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
437.9462
542.7487
590.2733
983.4816
776.4982
623.5060
301.24471084.6279
1171.8290
RT: 41.57MS/MS of m/z 598.6Scan # 4870
ControlB3a #4874 RT: 41.60 AV: 1 NL: 3.86E2T: ITMS + c NSI d Full ms2 [email protected] [295.00-1130.00]
300 400 500 600 700 800 900 1000 1100
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
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100
Re
lativ
e A
bu
nd
an
ce
1098.4486921.5529
1018.6340
480.2985
680.4445
805.3505
637.2200361.1457
952.3358
784.3491
514.2266
853.4705706.2417459.1983 588.2148871.4709
445.2212333.3748
ControlB3a #4873 RT: 41.59 AV: 1 NL: 1.54E3T: ITMS + c NSI d Full ms2 [email protected] [255.00-1960.00]
400 600 800 1000 1200 1400 1600 1800
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
1092.6033
1409.7291
856.3868
539.2245
1294.7877965.7724
1223.7373
654.2495 757.5266 1801.9797
1513.5245436.2499
1674.7556393.1896
ControlB3a #4871 RT: 41.58 AV: 1 NL: 4.17E3T: ITMS + c NSI d Full ms2 [email protected] [140.00-1655.00]
200 400 600 800 1000 1200 1400 1600
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
535.5252
690.1100
490.3550
575.8568
450.8616361.2963
747.4839
330.2767262.1056
900.6165 1022.6853234.2242
1088.7388
RT: 41.58MS/MS of m/z 547.3Scan # 4871
RT: 41.58MS/MS of m/z 777.4Scan # 4872
RT: 41.59MS/MS of m/z 974.9Scan # 4873
RT: 41.60MS/MS of m/z 1116.5Scan # 4874
ControlB3a #4872 RT: 41.58 AV: 1 NL: 3.27E3T: ITMS + c NSI d Full ms2 [email protected] [200.00-790.00]
200 250 300 350 400 450 500 550 600 650 700 750
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
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95
100
Re
lativ
e A
bu
nd
an
ce
701.4880
592.5975
400.3238
729.5197
767.4117
654.3235
354.2529 683.1174371.1810
309.1429 547.4052512.5754469.5364252.0748
0.0 0.5 1.0 1.5 2.0 2..5
Time [sec]
34
Small Molecule Analyses
The mass spectrum is obtained for a surface sample from a PEG 4000 treated board on the Vasa’supper gun deck Each peak corresponds to a certain molecular mass. The difference between the major peaks is 44 mass units, which corresponds to one -CH2CH2O- entity (n ± 1) in the PEG chain. The three clusters of peaks with mean values of about 615, 1450 and 3920 mass units show that commercial compounds labelled PEG 600, PEG 1500, and PEG 4000 consist of a distribution of molecules, and that the PEG 600 from inside the board has penetrated into the PEG 4000 surface layer.
420.5
470.0
573.4
617.4
705.4
749.5
811.5
855.5
899.5
943.61031.6
1361.8
1725.0
2234.3
2425.4
0400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 m/z
PEG: Polyethylene glycol
811.5837.5
855.5
881.5
899.5
925.6
943.6
965.6
987.6
1009.6
1031.6
1053.6
1063.6
1075.7
1097.6
1108.6
1119.71141.7
1152.6
800 850 900 950 1000 1050 1100 m/z
PEG: Polyethylene glycol
943.6
946.1
947.5
949.5
953.6957.1
959.5962.0
963.5
965.6
967.6
969.6
975.5
979.1981.6984.1
987.6
991.6
993.6
0
940 945 950 955 960 965 970 975 980 985 990 m/z
PEG: Polyethylene glycol
9.4T Bruker Qe FT-ICR MS
483.1826
578.8010
656.7787
716.7460
898.9883
600 800 1000 1200 1400 1600m/z
*
**
*
*
*
*
*
*
*
Monoisotopic796.0344
795.4532
796.7201797.7598
798.9503
800.0326
800.9896802.0326
803.0275
804.0353
804.7449 806.0281
806.7413
807.9725
808.7395
796 798 800 802 804 806 808 m/z
Theoretical – 796.0330Experimental – 796.0344
Error – 1.6ppm
Theoretical isotopic distribution of Ruthenium containing compound
* - detectable isotope of molecule of interest
Zoom
W. McNamara; T. Lam; T. Voss
Resolving Power ~71,000
293.1755351.1336
429.1493
520.9085
609.3397656.8838
792.8607 -MS, 16.5-16.6min #(865-874)
300 400 500 600 700 800 900 m/z
349.1100
351.1336
352.1370353.1306
354.1338349 350 351 352 353 354 355 356 m/z
Zoom
351.1336
351.1627
351.06 351.10 351.14 351.18 351.22m/z
Zoom
Observed monoisotopic m/z
from Average MS of EIC
Predicted Elemental
Composition (GMF)
Theoretical monoisotopic m/z (-1 charge state)
Error (ppm)
251.0927 C6H15N6O3S1 251.09318 -1.9277.0061 C5H7N7O3S2 277.00573 1.3291.1996 C6H21N13O 291.19975 -0.5351.1336 C11H25N7S3 351.13390 -0.9351.1627 C10H25N9O1S2 351.16290 -0.6349.2046 C15H31N3O4S1 349.20408 1.5313.2379 C18H33O4 313.23843 -1.7289.1054 C12H21N2O2S2 289.10499 1.4315.2535 C18H35O4 315.25408 -1.8269.0778 C10H15N5S2 269.07744 1.3
McCarty, K; Lam, TT
9.4T Bruker Qe FT-ICR MS 41
Deuterated
Protonated
Mix
808.10563
811.12458
812.12800
0.00
0.25
0.50
0.75
1.00
1.25
7x10Intens.
807 808 809 810 811 812 813 m/z
808.10398
809.10860
0
1
2
3
4
5
6
7x10
807 808 809 810 811 812 813 m/z
808.10538
809.10891811.12406
0
1
2
3
4
5
7x10
807 808 809 810 811 812 813 m/z
D. Spiegel; T. Lam
9.4T Bruker Qe FT-ICR MS42
Resolution~666,500
Resolution~473,700
Deuterated
Protonated
Mix (Manual)
Peak Area18,999
Peak Area2,047
Peak Area62,633
Peak Area13,340
808.10563
0.00
0.25
0.50
0.75
1.00
1.25
6x10Intens.
808.04 808.08 808.12 808.16 m/z
808.10398
0
1
2
3
4
5
6
7x10
808.04 808.08 808.12 808.16 m/z
808.10538
0
1
2
3
4
5
7x10
808.04 808.08 808.12 808.16 m/z
811.12458
0.00
0.25
0.50
0.75
1.00
1.25
7x10Intens.
811.04 811.08 811.12 811.16 m/z
0.0
0.5
1.0
1.5
2.0
2.5
5x10
811.04 811.08 811.12 811.16 m/z
811.12406
0
2
4
6
86x10
811.04 811.08 811.12 811.16 m/z
D. Spiegel; T. Lam
9.4T Bruker Qe FT-ICR MS 43
269.09569
441.37237
1210.24578
110706_McCarty_3HBaP_std\4: +MS
0.0
0.5
1.0
1.5
7x10Intens.
200 400 600 800 1000 1200 1400 1600 1800 m/z
267.07990
535.16570
110706_McCarty_3HBaP_std\5: -MS
0.00
0.25
0.50
0.75
1.00
1.25
1.50
8x10
200 400 600 800 1000 1200 1400 1600 1800 m/z
265.96061
268.08787
269.09569
270.09905
270.60161
110706_McCarty_3HBaP_std\4: +MS
0.0
0.5
1.0
1.5
7x10Intens.
264 266 268 270 272 274 m/z
267.07990
268.08322
269.08666
110706_McCarty_3HBaP_std\5: -MS
0.00
0.25
0.50
0.75
1.00
1.25
1.50
8x10
264 266 268 270 272 274 m/z
A – Isotopic peaks of Compound 3-hydroxybenzo[a]pyreneB – Isotopic peaks of Compound 3-hydroxybenzo[a]pyrene + H+
A
A
BB
B
Positive Mode
Negative Mode
A
A
A
Zoom
Sample Formula (M)Theoretical Mono (M)
Experimental Mono (M)
Error (ppm)
Neutral C20H12O 268.088266 268.08787 1.5"+1" C20H13O 269.096091 269.09569 1.5"-1" C20H11O 267.080441 267.0799 2.0
K. McCarty; T. Lam
DHB_POS_10_M10.d: +MS DHB_POS_10_M11.d: +MS DHB_POS_10_M12.d: +MSDHB_POS_10_M13.d: +MS DHB_POS_10_M14.d: +MS DHB_POS_10_M15.d: +MSDHB_POS_10_M16.d: +MS DHB_POS_10_M17.d: +MS DHB_POS_10_M18.d: +MSDHB_POS_10_M19.d: +MS
701.40696
701.40701
701.40689
701.40701
701.40670
701.40695701.40695
701.40689
701.40705
701.40690
701.40 701.45 701.50 701.55 701.60 701.65m/z
Reproducibility of MALDI FTICR at 12T
459.24732
616.95886
770.98423
946.991011073.40991
1260.46798
DHB_POS_10_M19.d: +MS200 400 600 800 1000 1200 1400m/z
*
* = peak compared below
P. Mistry; M. Easterling; T. Lam
266.94300
459.24756
518.32084
701.40760
812.46106
1013.64937 1249.73056 1437.77929THAP_POS_8_A15.d: +MS
357.05897
547.08271737.10609 THAP_NEG_10_A15.d: -MS
200 400 600 800 1000 1200 1400 m/z
542.26098
544.33635545.30465
546.35200
547.35530
548.47723
550.62722
551.63059
552.88097554.31827
541.06590543.05142
545.06717
546.07041
547.08271
548.08614
552.03578
542 544 546 548 550 552 554m/z
Zoom
Comparison of Positive and negative MALDI FT-ICR MS of lipid/small molecule for a post treatment patient sera
P. Mistry; M. Easterling; T. Lam
Hierarchal cluster of Lipid/small molecule from sera of patients pre/post treatment analyzed with MALDI FTICR (THARP matrix)
Post-Treatment
Mass
P. Mistry; J. Lee; T. Lam
479.4T Bruker Qe FT-ICR MS
(Isolation and Fragmentation of m/z at 325)
93.02141
117.49194
142.99257
164.06702 182.97512 202.04189 227.51176
250.99233
272.97436
0
1
2
3
4
5
7x10Intens.
100 120 140 160 180 200 220 240 260 280 m/z
93.02141108.32685
142.99256
164.06712 202.04194 227.51170
250.99238
272.97453
0
2
4
6
7x10
100 120 140 160 180 200 220 240 260 280 m/z
93.02140
108.32687
142.99251
182.97500216.59026 239.59321
250.99232
272.97431
0
1
2
3
4
7x10
100 120 140 160 180 200 220 240 260 280 m/z
A. Nassar; T. Lam
48
NH
O N
N
S
O
O
Cl
SO
O
NH
ON
N
S
O
O
Cl
SO
O
ND
ON
N
S
O
O
Cl
SO
O
[M+NH4]+, m/z 325
-NH3
308
251
143
-N2
Rearrangement
-CH3SO2H
-CH3SO2
93
-CH3SO2H
63
-HCl10781
-C2H3Cl
[M+NH4]+, m/z 327
-NH3310
253
145
-N2
Rearrangement
-CH3SO2H
-CH3SO295
-CH3SO2H
65
-HCl109
*
*
[M+ND4]+, m/z 330
-ND3310
253
144
-N2
Rearrangement
-CH3SO2D
-CH3SO295
Asterisk indicate positions of the 13C-label
A. Nassar; T. Lam
510.3395
539.1089
585.2792
629.1546
780.5535
899.4229
987.1921 1046.2339
063010_Araujo_SL1_BB_000001.d: +MS500 600 700 800 900 1000 m/z
758.5718
780.5535
786.6029 808.5854828.5522
844.5264
760 770 780 790 800 810 820 830 840 m/z
I. Araujo; T. Lam; E. Voss
39(Δ1.86)
26(Δ1.64)
15(Δ1.33)11
(Δ1.02)
24 Da 24 Da 24 Da
I. Araujo; T. Lam; E. Voss
9.4T Bruker Qe FT-ICR MS
357.1005
359.0967
360.1011361.0000
061609_Buettner_KMBMannitolMKT406-09_000004.d: +MS
357.1015 358.1007
359.0969
360.1001
361.0937
061609_Buettner_KMBMannitolMKT406-09_000004.d: C 16 H 23 O 6 Ti 1 ,359.100.0
0.5
1.0
1.5
2.0
2.5
3.0
6x10Intens.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
6x10
357 358 359 360 361 m/z
269.1356
270.1392
C 11 H 16 N 7 Na 1
061609_Buettner_KMBMannitolMKT406-09_000004.d: +MS
269.1359
270.1393
061609_Buettner_KMBMannitolMKT406-09_000004.d: C 11 H 16 N 7 Na 1 ,269.140
2
4
6
8
8x10Intens.
0
2
4
6
8x10
269.00 269.25 269.50 269.75 270.00 270.25 270.50 270.75 271.00 m/zK. Buettner; T. Lam; E. Voss
459.14601+
601.29702+
672.32591+
922.45491+
1068.57452+
1201.59021+
'1359.67992+
600 800 1000 1200 1400 m/z
489.24082+
733.39211+
810.42001+
960.45141+
1109.47211+
'1260.60532+
'1444.71112+
487.77152+
606.83492+
733.39211+
810.42011+
'888.10693+
1138.53982+
'1444.71152+
468.29281+ 596.3535
1+
861.45121+
1039.49331+
1231.68331+
A
B
C
D
'1331.67852+
1336.57151+
'1341.61623+
'1367.17032+
1375.58411+
1330 1340 1350 1360 1370 1380 m/zZoom
*
'1444.71112+
1470.75311+
1521.68891+
'1606.41263+
1440 1460 1480 1500 1520 1540 1560 1580 1600 1620 m/z
*
Int. Calibrant Zoom
T. Biederer; T. Lam; E. Voss
N-Glycosylation at the SynCAM (Synaptic Cell Adhesion Molecule) Immunoglobulin Interface Modulates Synaptic Adhesion*
Adam I. Fogel‡1, Yue Li‡, Joanna Giza‡, Qing Wang‡2, TuKiet T. Lam§, Yorgo Modis‡, and Thomas Biederer‡3
From the ‡Department of Molecular Biophysics and Biochemistry and the §W. M. Keck Foundation Biotechnology ResourceLaboratory, Yale University, New Haven, Connecticut 06520
Received for publication, March 8, 2010, and in revised form, August 3, 2010 Published, JBC Papers in Press, August 25, 2010, DOI 10.1074/jbc.M110.120865
T. Biederer; T. Lam; E. Voss
54L. Leng; T. Lam; E. Voss
m/z 1,7501,5001,2501,000750500
** *
* Calibrants
1,7001,6001,5001,4001,3001,200
*m/z1,4101,4051,4001,395
F-DTXR fragment 30-115
nonF-DTXR fragment (~18 Da less)
F-DTXR Fragment 30-115:IAERLEQSGPTVSQTVARMERDGLVVVASDRSLQMTPTGRTLATAVMRKHRLAERLLTDIIGLDINKVHDEACRWEHVMSDEVERR
~93% Fluorinated
Tryptic digest of F-DTXR
Trypsin Fragment
7+
6+
7+
8+
Logan, T; Lam, TT
400 405 410 415 420 425 430 435 440 445 m/z
~
~10X
20X
Zoom
Conc. ~66 fmole/µL
TPP Standard
Ad2
Ad5
Ad12
Not TPP: m/z at 423.207
m/z at 423.030
m/z at 423.034
m/z at 423.033
P. Freimuth; T. Lam
25 Compounds mixture from Chemistry Department
S. Lai; T. Lam; E. Voss
Sample A
Sample B
Separation of lipid classes by Chromatographic Means
Low Energy
High Energy
RT
1
2 4
5
7
61
3
4
4
4
3 45 7
64
11 different precursors elute in 3 secondsLC-IMS-MSE analysis groups all ions by drift time
In normal LC-MSE analysis, all product ions would be shared
1
2
Separation of lipid classes by Ion Mobility (note similarity in RT)
9.4T Bruker Qe FT-ICR MS 61
'828.79899-
'932.52478-
'1069.02457-
050809_Lopalco_oligo-lipid_000002.d: -MS
0.0
0.5
1.0
1.5
7x10Intens.
600 800 1000 1200 1400 1600 1800 2000 2200 2400 m/z
554.14341- 635.3605
1-
'745.818810-
'828.79899-
'932.52478-
'1069.02457-
'1163.10567-
'1251.02656-
'1360.78896-
'1497.04125-
050809_Lopalco_oligo-lipid_000002.d: -MS
0.00
0.25
0.50
0.75
1.00
1.25
7x10Intens.
500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z
Zoom
M. Lopalco; T. Lam; E. Voss
9.4T Bruker Qe FT-ICR MS 62
'811.675610-
'828.79899-
843.19301-
859.51261- 869.5331
1-
'901.97399-
'932.52478-
050809_Lopalco_oligo-lipid_000002.d: -MS
0.00
0.25
0.50
0.75
1.00
1.25
7x10Intens.
800 820 840 860 880 900 920 940 m/z
M. Lopalco; T. Lam; E. Voss
9.4T Bruker Qe FT-ICR MS
'821.47149-
'823.91489-
'826.35689-
'828.79899-
'831.24159-
'833.68439-
'836.12619-
'838.56819-
050809_Lopalco_oligo-lipid_000002.d: -MS
0.00
0.25
0.50
0.75
1.00
1.25
7x10Intens.
822.5 825.0 827.5 830.0 832.5 835.0 837.5 840.0 m/z
'828.79899-
050809_Lopalco_oligo-lipid_000002.d: -MS
0.00
0.25
0.50
0.75
1.00
1.25
7x10Intens.
828.00 828.25 828.50 828.75 829.00 829.25 829.50 829.75 830.00 830.25 m/z
Zoom
M. Lopalco; T. Lam; E. Voss
Separation of Isomeric Compounds
Meta-, Ortho-, Para-hydroxylated Mobility (Drift Time separation)
Glycosylation Analysis
NIH SIG Application Submitted (March 2011): Synapt G2 Mass Spectrometer. PI: Tukiet Lam
Key Feature: Mobility separation by charge and shape – provides additional separation modality within the MS
Potential applications: – Lipids (e.g., separation of isomeric
lipids varying by position of cis/trans double bonds)
– Small molecule (e.g. metabolites)– Carbohydrate analysis with Mse
capability useful for mapping sites of glycosylation
oxonium ion annotation
carbohydrate annotation
MSE elevated energy fragment ion spectrum
YPED for routine accurate/exact mass analyses servicesSeparate module for Chemistry analyses Editable sample submission
form built into YPED
Schematic Workflow
452.1606
466.1763
460 480
452.1606
466.1763
460 480
452.1606
466.1763
480
00.3452.16060452.1606452.1606452.1606452.160483C27H21N3O4TTL_234 00.3452.16060452.1606452.1606452.1606452.160483C27H21N3O4TTL_234
STDError (ppm)
Average massTrial 3Trial 2Trial 1
Mono (M+Na)+
Mono (M+H)+Formula (M)Sample
ExperimentalTheoretical
STDError (ppm)
Average massTrial 3Trial 2Trial 1
Mono (M+Na)+
Mono (M+H)+Formula (M)Sample
ExperimentalTheoretical
pk of interest
452.1606
466.1763
460 480
452.1606
466.1763
460 480
452.1606
466.1763
480
00.3452.16060452.1606452.1606452.1606452.160483C27H21N3O4TTL_234 00.3452.16060452.1606452.1606452.1606452.160483C27H21N3O4TTL_234
STDError (ppm)
Average massTrial 3Trial 2Trial 1
Mono (M+Na)+
Mono (M+H)+Formula (M)Sample
ExperimentalTheoretical
STDError (ppm)
Average massTrial 3Trial 2Trial 1
Mono (M+Na)+
Mono (M+H)+Formula (M)Sample
ExperimentalTheoretical
452.1606
466.1763
460 480
452.1606
466.1763
460 480
452.1606
466.1763
480
452.1606
466.1763
460 480
452.1606
466.1763
460 480
452.1606
466.1763
480
00.3452.16060452.1606452.1606452.1606452.160483C27H21N3O4TTL_234 00.3452.16060452.1606452.1606452.1606452.160483C27H21N3O4TTL_234
STDError (ppm)
Average massTrial 3Trial 2Trial 1
Mono (M+Na)+
Mono (M+H)+Formula (M)Sample
ExperimentalTheoretical
STDError (ppm)
Average massTrial 3Trial 2Trial 1
Mono (M+Na)+
Mono (M+H)+Formula (M)Sample
ExperimentalTheoretical
pk of interest
Sample TTL_234
PowerPoint Slide MS Results
FT-ICR MS analysis
User submit sample & submission form
Samples analyzed based on services selected
Results reported onto PowerPoint slide
PP slides are upload onto YPED & stored on secure FTP site
Users can visualize & download results
Service charges uploaded onto FMP**
** Currently under construction.
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High End Fourier Transform ICR Mass Spectrometry for Protein and Small Molecule Applications
Uses• Exact/Accurate mass of small molecules, peptides, oligos
(RNA/DNA), lipids, and intact proteins, drugs, etc.• Structural Elucidation of small molecule• Protein Post Translational Modification• Protein Identification & Peptide sequencing• Comparative protein/peptide profiling.
Advantages• Ultra High Resolution for separation of molecular masses
less than 0.002 Da.• High Mass Accuracy (<3ppm with Ext. Calibration) for
elemental assignment• Multi-fragmentations capabilities for structural
elucidation and protein PTM analysis.
Impact• Since Feb2008, >1250 samples from 94+ Yale Chemistry
Faculties, Postdocs, Graduate Students, and As. Res. Scientist have been analyzed. Additionally 300+ analyses from 30+ investigator from Yale and non-Yale institutions.
H2N C C N
OR1
C C N
ORn-1
C C
ORn
OH
m+nHn+
y1
bn-1
z1·
cn-1
...
ECD
IRMPDCID
Retention of labile modificationsNo X-P cleavage
Facile loss of H3PO4
X-P cleavage preferred
220 260 300 340 380 m/z
263 264 265 266 267m/z
265.04689 (Exp.)
Zoom
265.04713 (Cal.)
0.00024 (Diff.)- 0.9 ppm (Error)
m/z434432430428
429.22623
Deuterated (D)
Protonated (P)
430.22990
431.23346
429.22657
430.22835
430.23262
431.23617 432.23963
PDD
D
P
P
P
P
Resolution (170,000)
67
AcknowledgementThe Keck Group
Ken Williams (The Boss)Kathy Stone (The Overseer)Erol Gulcicek (The Phospho Guy)Chris Colangelo (The MRM Guy)Terence Wu (The Gel Guy)Mary LoPresti (The SamplePrep Lady)Jean Kanyo (The MALDI Lady)Tom Abbott (The 2nd MRM Guy) Kathrin Wilczak-Havill (The iTRAQ Lady)Matt Berberich (The Velos Man)Ted Voss (The ICR Protector)
All collaborators and clients
Fundings(FT-ICR) NIH/NCRR 1 S10 RR17266-01
(NBC) Proteomics Core