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18th IMSC Conference A New Instrument for ... - Thermo Scientific

18 th IMSC

Conference

A New Instrument for High-speed

Proteomics: Orbitrap Mass

Analyzer Interfaced to a Dual

Pressure Linear Trap

Eduard Denisov

Eugen Damoc

Jean Jacques Dunyach

Jens Griep-Raming

Oliver Lange

Philip Remes

Jae Schwartz

Maurizio Splendore

Dennis Taylor

Alexander Makarov

Thomas Moehring

Eloy Wouters

Vlad Zabrouskov

Thermo Fisher Scientific

September 1, 2009

Velos

ETD

+++ -

-

-

HCD +

MALDI


Motivation for a new hybrid: why do we do it?

Cars

Records vs Real life

Motto→ “Only the best results matter” “All-round high performance”

• Racing on perfect surface,

weather, special roads, etc.

• Never mind noise, comfort,

emissions, costs, etc.

• High performance on all

roads, especially where no

help is expected

MS

M.Pettitt

• Analysis for optimum (not

too high, not too low)

concentrations/ samples/

m/z, simple mixtures, etc.

Chris5aw

• Analysis for very low and

very high concentrations/

m/z, complex samples, etc.

Instrument must provide

maximum number of IDs for

these conditions

Basing on these data, we do not

need to improve MS any more!

2

This is where we have a long

way to go…


What does it mean for hybrid instrumentation?

• Analysis for very low and

very high concentrations/

m/z, complex samples, etc.

Instrument must provide

maximum number of IDs for

these demanding conditions

•Low detection limit in presence of matrix

(sensitivity, resolving power, dynamic

range)

•High reliability of identification (accurate

m/z of precursors & fragments, MS n ,

multiple fragmentation techniques,

sensitivity)

•High dynamic range of ID (dynamic

range of detection and mass accuracy)

•MS/MS on maximum number of

precursors (sensitivity, spectra

acquisition rate)

•Quantitation (sensitivity, linearity,

dynamic range of quantitation)

3


New atmosphere-to-vacuum interface with an S-lens

• S-lens is used for RF focusing of ions in 2 mbar region

• S-lens is a set of stacked apertures with two IDs and variable spacing :

• larger ID to capture entire expansion from transfer tube

• smaller ID to focus ion beam through exit lens

• increasing spacing = increasing field penetration to focus ion beam

• small number of electrodes

• 2x shorter ion transfer tube than before

• Interface protection from droplets

SENKO M. W.; KOVTOUN V. V.;

ATHERTON P.R.; DUNYACH

J.J.; WOUTERS E. R.;

SPLENDORE M.; SIEBERT W.

US2009045062

SENKO M. W.; KOVTOUN V. V.

US7514673,

Implementation of a Progressively Spaced

Stacked Ring Ion Guide on a Linear Ion Trap

Mass Spectrometer

E. R. Wouters; M. Splendore; C. Mullen; J. C.

Schwartz; M. W. Senko; J.-J. Dunyach –

ASMS 2009, MPH 194

4


RF focusing works well even in the simple design

90

85

80

75

70

65

60

55

50

45

40

35

30

25

x5

10 0 NL: 1.07E3

m/z=

2 73 .50 -2 74 .50 +

95

Standard LTQ

Intensity:

1.1E3

280.50-281.50 M S

AlpStdLTQ100fg_0

1

NL: 1.07E3

m/z=

2 73 .50 -2 74 .50 +

280.50-281.50 M S

alpstdltq100fg_02

NL: 1.07E3

m/z=

2 73 .50 -2 74 .50 +

280.50-281.50 M S

alpstdltq100fg_03

NL: 1.07E3

m/z=

2 73 .50 -2 74 .50 +

280.50-281.50 M S

alpstdltq100fg_04

NL: 1.07E3

m/z=

2 73 .50 -2 74 .50 +

280.50-281.50 M S

alpstdltq100fg_05

NL: 1.07E3

m/z=

2 73 .50 -2 74 .50 +

280.50-281.50 M S

alpstdltq100fg_06

NL: 1.07E3

m/z=

2 73 .50 -2 74 .50 +

280.50-281.50 M S

AlpStdLTQ100fg_0

7

NL: 1.07E3

m/z=

2 73 .50 -2 74 .50 +

280.50-281.50 M S

alpstdltq100fg_08

Relative Abundance

10 0

95

90

85

80

75

70

65

60

55

50

45

40

35

30

25

x5

+S-Lens:

Intensity:

8.8E3

NL:

8.80E3

m/z=

273.50-274.50+

280.50-281.50

MS

A lpDualID7_5m

m tp6_2m m CL80t

h_06

NL:

8.80E3

m/z=

273.50-274.50+

280.50-281.50

MS

alpdualid7_5m m t

p6_2m m cl80th_0

7

NL:

8.80E3

m/z=

273.50-274.50+

280.50-281.50

MS

alpdualid7_5m m t

p6_2m m cl80th_0

8

NL:

8.80E3

m/z=

273.50-274.50+

280.50-281.50

MS

alpdualid7_5m m t

p6_2m m cl80th_0

9

NL:

8.80E3

m/z=

273.50-274.50+

280.50-281.50

MS

alpdualid7_5m m t

p6_2m m cl80th_1

0

20

20

15

15

10

10

5

5

0

0.0 0.2 0.4 0.6 0.8 1.0 1.2

Time (min)

0

0.0 0.2 0.4 0.6 0.8 1.0

Time (min)

Signal intensity:

x7…10 for SIM and MS/MS!

x>3…5 for wide m/z range

5 injections of Alprazolam

5


Dual pressure linear trap improves performance by separating functions

High Pressure Cell

High Trapping Efficiency

High Dissociation Efficiency

Faster trapping and activation

5.0 x10 -3 Torr He

Low Pressure Cell

Higher resolution at higher

scan rate

3.5x10 -4 Torr He

Jae C.

Schwartz

MPH 196: P. M. Remes; J. C. Schwartz.

“Comparison of a Dual Cell Linear Ion Trap with a

Four-Fold Symmetric Stretch versus a Two-Fold

Symmetric Stretch”

MPH 195: J. C. Schwartz; D. M. Taylor; P. M.

Remes. “Performance Improvements in High Mass

Range Modes on a Dual Cell Linear Ion Trap”

6


Low pressure cell: Resolution/Scan Rate

0.7

Effect of Scan Rate on Peak Width

(Resonance Ejection Optimized)

FWHM (amu)

0.6

0.5

0.4

0.3

0.2

0.1

0

Effect of

pressure

decrease

Std LTQ

Scan Rate

0 5000 10000 15000 20000 25000 30000 35000

Scan Rate (amu/sec)

2x Current

Scan Rate

Scan Rate Range 1

Scan Rate Range 2

Scan Rate Range 3

7


Cycle Time Improvement with Dual Pressure Ion Trap

(Full Scan MS/MS- real life!)

300

Time (ms)

250

200

150

100

Inter Scan Overhead

Intra Scan Overhead

Mass Analysis

Activation

Isolation

Injection Time

Prescan

50

0

LTQ XL

LTQ Velos

8


New combo-cell

• HCD cell with axial field is directly attached to the C-trap

• There is no gas supply to the C-trap anymore

• Gas leaking from the HCD cell (3-5 mbar) is used for ion

trapping and cooling in the C-trap

• Dedicated pump for HCD cell protects low pressure cell of

LT from gas overload

9


Ion Transfer into Collision Cell

HCD in long

quadrupole

Transfer

fragments

to C-trap

10


HCD spectrum of MRFA peptide at low target number

MRFA_HCD_7500_5e3#709 RT: 2.83 AV: 1 NL: 1.33E6

T: FTMS + p ESI Full ms2 524.26@hcd40.00 [80.00-600.00]

100

95

90

85

80

75

70

65

60

55

50

45

40

35

30

25

20

15

Relative Abundance

0

MRFA_HCD_7500_5e3 #709 RT: 2.83 AV: 1 NL: 2.83E6

T: FTMS + p ESI Full ms2 524.26@hcd40.00 [80.00-600.00]

104.05276 120.08071

R=17404 R=16604

C4H10NS C8H10N

-0.78525 ppm -0.53743 ppm

100

95

90

85

80

75

70

65

100 120 140 160 180 200 220 240

m/z

60

149.07414

R=14204

C5H13ON2S

-1.12584 ppm

229.10049

R=11704

C10H17O2N2S

-0.13368 ppm

271.12219

R=11004

C 11 H 19 O 2 N 4 S

-0.48202 ppm

5000 ions

1 amu isolation width

R=7500- as in routine experiments

Relative Abundance

55

50

45

40

120.08071

R=16604

C 8 H 10 N

-0.53743 ppm

35

30

25

20

15

10

5

149.07414

R=14204

C 5 H 13 ON 2 S

-1.12584 ppm

185.10271

R=13704

259.15570

R=11304

C 14 H 19 ON 4

1.40037 ppm

306.15878

R=10300

C 17 H 18 N 6

0.12026 ppm

376.19803

R=9104

C 18 H 26 O 4 N 5

0.25993 ppm

453.22781

R=8604

C 20 H 33 O 4 N 6 S

-0.08157 ppm

524.26489

R=8006

C 23 H 38 O 5 N 7 S

-0.13707 ppm

0

100 150 200 250 300 350 400 450 500

m/z

11


A “hotter” orbitrap

• High-temperature bake-out of the Orbitrap results in improved ultimate

vacuum (10 -11 torr)- for analysis of small- and medium-size proteins with

isotopic resolution

• Water-cooled pre-amplifier

CA +20

1000 2000

t, ms

+ Software:

• Non-linear mass calibration

•Predictive AGC

12


LC/MS analysis of intact carbonic anhydrase

CA_SIM_1200_1220

RT: 10.00000 - 49.00000

Relative Abundance

Relative Abundance

100

90

80

70

60

50

40

30

20

26.28860

1201.81873

1/16/2009 7:03:17 PM

26.98301

1201.79382

32.20549

1210.32910

42.55516

1201.82202

10

24.00312

14.95406 16.76379 21.69970

34.54685

10

47.29105

1203.37769

1201.64868 1200.26160 1202.57947

1210.45081

5

1210.86584

0

10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 0 42 44 46 48

Time (min)

100

50

0

100

50

0

measured

(6 x 5 μscans)

1209.49166

R=66511

z=?

simulated

Base Peak Chromatogram

1209.66090

R=55501

z=?

1209.74439

R=71894

z=?

1209.95331

R=71889

z=?

1210.16163

R=70596

1210.32848

R=72560

z=24 1210.49481

R=69563

z=24

z=24 1210.70446

R=49108

z=24

1210.16221

R=71876

z=?

1210.32933

R=71837

z=? 1210.49644

R=71831

z=?

1210.70531

R=71813

z=?

1210.91417

R=71790

z=?

1211.19973

R=59902

z=?

1211.16479

R=71765

z=?

1211.40849

R= 57133

z=1

1211.37368

R=71734

z=?

1209.2 1209.4 1209.6 1209.8 1210.0 1210.2 1210.4 1210.6 1210.8 1211.0 1211.2 1211.4 1211.6

m/z

CA_SIM_1200_1220-qb_XT_00001_M_ #2 RT: 2.00 AV: 1 NL: 6.84E2

F: FTMS + p ESI SIM ms [1200.00-1220.00]

Relative Abundance

100

95

90

85

80

75

70

65

60

55

50

45

40

35

30

25

20

15

Deconvolution

NL:

2.79E3

Base Peak

F: ms MS

CA_SIM_12

00_1220

-0.96 ppm

29003 29004 29005 29006 29007 29008 29009

m/z

NL:

1.58E3

CA_SIM_1200_1220#200-

205 RT: 32.07-32.76 AV: 6

T: FTMS + p ESI SIM ms

[1200.00-1220.00]

NL:

2.14E3

C 1312 H 2020 N 358 O 384 S 3 :

C 1312 H 2020 N 358 O 384 S 3

p (gss, s /p:40) Chrg 24

R: 72000 Res .Pwr . @FWHM

29006.65484

External mass calibration

13


LC-MS/MS analysis of intact carbonic anhydrase

HCD of the [M+34H] 34+ ion

CA_HCD_854_iw15_3e5_new_tune

1/16/2009 8:47:17 PM

RT: 9.41 - 46.27 SM: 5G

100

90

80

Relative Abundance

70

60

50

40

30

Base Peak

Chromatogram

32.33

NL:

8.53E3

Base Peak

MS

CA_HCD_8

54_iw15_3e

5_new_tun

e

20

10

24.43

38.47 41.13

9.76 12.65 14.65 15.54 17.76 19.10 21.76

27.99 29.76

37.35

44.00

10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46

Time (min)

CA_HCD_854_iw15_3e5_new_tune#145-156 RT: 31.99-33.64 AV: 12 NL: 3.02E3

T: FTMS + p ESI Full ms2 854.60@hcd14.00 [200.00-2000.00]

740.92102

R=96922

100

z=4

1007.40950

R=80875

90

z=7

Relative Abundance

80

70

60

50

40

30

20

10

0

337.18735

R=137339

z=1

539.28274

R=108646

z=1

592.93781

R=104297

z=5

627.85490

R=99916

z=4

951.01997

R=82073

z=8

881.60983

R=86322

z=8

836.80458

R=86704

z=?

1086.87941

R=77819

z=7

1175.14406

R=71184

z=6

1362.39623

R=69486

z=3

HCD(854.6)

(12 x 5 μscans)

1547.82364

R=65866

z=?

1708.82417

R=61194

z=?

1965.69783

R=65833

z=?

400 600 800 1000 1200 1400 1600 1800 2000

m/z

14


ProSight PTM Fragmentation Details

External mass calibration

15


HCD scan speed / injection times for a real sample

LTQ OT Velos

RT: 25.21729 - 25.46477

Relative Abundance

10

8

6

4

2

0

25.23390

217.13762

25.24955

442.95422

25.29739

442.95398

25.34755

442.95361

25.39639

442.95361

TOP10 HCD TOP10 HCD TOP10 HCD TOP10 HCD

2.87s 3.0s 2.93s 2.85s

25.25958

989.45386 25.26833

519.26385

25.31708

436.76416

25.33197

475.76273

25.35691

673.36365

25.38092

489.29245

25.41839

475.76254

25.43304

515.28351

25.44389

443.20313

25.45441

573.80988

25.22 25.24 25.26 25.28 25.30 25.32 25.34 25.36 25.38 25.40 25.42 25.44 25.46

Time (min)

NL: 2.75E8

TIC MS

ecoli_redalk_115mi

nrun_90mingrad_to

p10_hcd_sf100_50

0ng_ii

LTQ OT XL

HCD(403.21)

EColi_500ng_TOP7_HCD_1 #1726 RT: 20.08 AV: 1 NL: 4.26E5

T: FTMS + p NSI d Full ms2 403.21@hcd35.00 [100.00-1220.00]

80

60

40

20

0

Relative Abundance100

519.26190

5e4 ions, IT=246.4ms

143.11728

171.11238

258.14352 314.07578 399.23126 475.74728 568.79596

706.33539 820.37189 891.41278

1212.36926

100 200 300 400 500 600 700 800 900 1000 1100 1200

m/z

LTQ OT Velos

HCD(403.21)

ecoli_redalk_115minrun_90mingrad_top10_hcd_sf100_500ng_ii #3532 RT: 25.27 AV: 1 NL: 1.05E6

T: FTMS + p NSI d Full ms2 403.21@hcd35.00 [100.00-1220.00]

519.26385

80

60

40

20

0

Relative Abundance100

143.11804

5e4 ions, IT=26ms

171.11298 403.23132

475.74820 568.79834

258.14438 332.19357

636.34796 706.33167 820.37091 891.41516

990.51270 1110.35706

100 200 300 400 500 600 700 800 900 1000 1100 1200

m/z

Example: 500 ng E.coli digest, 90 min gradient

16


NanoLC MS/MS analysis of 100ng E. Coli digest

ecoli_redalk_115minrun_90mingrad_top2...

3/22/2009 11:15:42 AM

RT: 0.00000 - 119.98852

Relative Abundance

100

80

60

40

20

0

536

13.58847

403.55136

10620

50.84714

601.96875

14152

62.70613

696.37006

0 10 20 30 40 50 60 70 80 90 100 110

Time (min)

RT: 67.29512 - 67.33349

Relative Abundance

10

8

6

4

2

0

15528

67.29668

502.89407

1885

19.18529

401.24530

15529

67.29960

1389.90320

3135

23.79309

442.95309

15530

67.30279

842.41577

7249

39.38719

688.82196

1.58s

15531

67.31146

358.36810 15533

67.31631

643.10107

15701

67.86813

747.38892

15534

67.31886

790.55511

21098

85.99689

594.32062

20638

84.56251

533.62384

23404

100.23787

536.16541

21140

86.11696

594.32098 23364 23491

15535

67.32150

638.38196

15536

67.32412

400.42453

99.15263

391.28442

102.65589

352.33987

23511

103.22128

690.50726

23528

103.78664

391.28452

15538

67.32915

963.67200

67.300 67.305 67.310 67.315 67.320 67.325 67.330

Time (min)

ecoli_redalk_115minrun_90mingrad_top20_CID_SF1_100ng_II_locked_100k #15530 RT: 67.30 AV: 1 NL: 2.46E5

T: FTMS + p NSI Full ms [350.00-1800.00]

842.41577

R=92601

100

z=2

791.88892

R=96001

80

z=2

Relative Abundance

60

40

20

0

429.08856

R=129701

z=1

536.29193

R=116101

z=3

TIC

1x OT full scan (R=100k) + 8x parallel ddITMS2

593.82440

R=109301

z=2

724.85583

R=99500

z=4

1

2

3 4 5 6

871.41907

R=86400

z=3

967.47815

R=84400

z=2

FTMS, 1e6, R=100,000

1035.90845

R=60804

z=?

1126.90845

R=69004

z=?

1200.55200

R=78600

z=2

24000

119.45536

536.16534

15539

67.33189

775.99420

1263.40918

R=68604

z=?

NL: 8.70E7

TIC MS

ecoli_redalk_115mi

nrun_90mingrad_to

p20_CID_SF1_100

ng_II_locked_100k

NL: 1.32E7

TIC MS

ecoli_redalk_115mi

nrun_90mingrad_to

p20_CID_SF1_100

ng_II_locked_100k

400 500 600 700 800 900 1000 1100 1200 1300 1400

m/z

7

8

1403.11755

R=65304

z=?

17


LTQ OT Velos: E. Coli digest (100ng load)

10000

9000

8000

Peptide IDs (< 0.57% FDR), 2 runs

7784 7862

Experimental conditions:

• 100ng E. coli digest on column

(75um x 10cm)

• 90 min gradient at 300nl/min

• Top20 Method: OT fullscan @

100,000 resolving power + 20 DD

CID MSMS (LT detection)

• Top10 Method: OT fullscan @

30,000 resolving power + 10 DD

HCD MSMS (OT detection)

• Data Processing by Proteome

Discoverer

7000

6000

5987

6221

5000

4000

3000

2000

Best prior art data

reported using

latest Orbitrap or

Q-TOF technology

1000

0

#1 #2 #1 #2

"High/Low" "High/Low" "High/High" "High/High"

TOP20 CID method

TOP10 HCD method

CID

HCD

• Improved quality of

MS/MS spectra

• 1 spectrum is enough

for ID (no summing)

18


LTQ OT Velos: E. Coli digest (100ng load)

Protein IDs (< 0.57% FDR), 2 runs

• increased

coverage

1000

900

800

828 835

796 789

700

600

500

400

Best prior art data

reported using

latest Orbitrap or

Q-TOF technology

300

200

100

0

#1 #2 #1 #2

"High/Low" "High/Low" "High/High" "High/High"

TOP20 CID method

CID

TOP10 HCD method

HCD

What about more complex samples?

19


LTQ OT Velos vs. LTQ OT XL: C. Elegans digest (1μg load)

Unique Peptide IDs at 40% ACN in 150min

75’ & 150’ values: mean of 2

20


Conclusion

• Increase of signal intensity + higher scan rate= increase of number of

identifications in real-life samples and their reliability.

New analytical methods are made possible by the new technology.

• We are entering the era of high-speed proteomics, with 5-10 MS/MS

per second for real-life samples!

Back to the car analogy:

from 10…15 mph…

If 1 MS/MS ~ 1 turn of the

car wheels…

Chris5aw

…to 30…40 mphnot

bad for such

roads!

21


Acknowledgements

LTQ Velos team

San Jose, CA:

LTQ Orbitrap Velos

team Bremen, DE:

Development

teams:

• LTQ

• LTQ FT

• MALDI

• ETD

• Exactive

J. Schwartz

P. Remes

M. Splendore

E. Hemenway

M. Antonczak

M. Senko

J. Syka

T. Second

B. Tehlirian

N. Izgarian

P. Fong

J. Gabel

S. Fenske

P. Cardenas

M. Konicek

R. Hermezian

W. Dewey

B. Siebert

P. Atherton

M. Ahrens

J. Sklenar

S. Zanon

W. Wang

J. Phillips

J. Horner

J. Blethrow

J. J. Dunyach

D. Taylor

E. Wouters

V. Zabrouskov

E. Damoc

E. Denisov

J. Griep-Raming

O. Lange

A. Venckus

W. Balschun

S. Horning

O. Hengelbrock

D. Nolting

R. Malek

H. Kuipers

A. Makarov

T. Moehring

22

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