High Resolution Mass Spectrometers role in small molecule studies TuKiet T. Lam, PhD Chem 395: Bioanalytical Chemistry April 12, 2011 1

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

60

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

70

75

80

85

90

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

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

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


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

90

95

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


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


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


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

90

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


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


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


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


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


(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


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)


'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-


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


'811.675610-

'828.79899-

843.19301-

859.51261- 869.5331

1-

'901.97399-

'932.52478-


0.00

0.25

0.50

0.75

1.00

1.25

7x10Intens.

800 820 840 860 880 900 920 940 m/z



'821.47149-

'823.91489-

'826.35689-

'828.79899-

'831.24159-

'833.68439-

'836.12619-

'838.56819-


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-


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


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)


Mono (M+Na)+



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)


Mono (M+Na)+



STDError (ppm)


Mono (M+Na)+



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)


Mono (M+Na)+



STDError (ppm)


Mono (M+Na)+



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

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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

Documents

High Resolution Mass Spectrometers role in small molecule studies TuKiet T. Lam, PhD Chem 395: Bioanalytical Chemistry April 12, 2011 1