Q ExactiveTM: A True Qual-Quanapps.thermoscientific.com/media/SID/LSMS/PDF/...Exactive Q-Exactive...

Q ExactiveTM: A True Qual-Quan HR/AM Mass Spectrometer for

Routine Proteomics Applications

Yi Zhang, Ph.D. ThermoFisher Scientific

2

Outline

• Introduction of Q Exactive

• Performance in Discovery Proteomics

• Flexible Targeted Quantification

• Mass Measurement of Intact mAb

• Summary

3

Q ExactiveTM - Innovations and Features

First Benchtop Quadrupole-Orbitrap Mass Spectrometer

ORBITRAP Resolution: 140,000

Mass Accuracy: < 2 ppm

QUADRUPOLE MASS FILTER Mass Range: 50 – 4000 amu Prec Isolation: 0.4 – 10 amu

HCD/C-Trap Combo Cell

ION SOURCE S-Lens

4

High Sensitivity with Quadrupole-based SIM Scan

0

1000

2000

3000

4000

5000

6000

195.082 195.084 195.086 195.088 195.09 195.092 195.094

S/N

(spe

ctru

m)

S/N (FMS) S/N (SIM10)

Gain in sensitivity (7x)

Caffeine

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

0

20

40

60

80

100

195.0876 N=248402.81

195.0877 N=20741.58

Full MS S/N = 745

SIM (10amu) S/N = 5400

Sensitivity Gain: 5 – 10 folds

5

Improved Resolution – Advanced Signal Processing

0

20000

40000

60000

80000

100000

120000

140000

160000

0 5 10 15

Reso

luti

on

Scan Speed [Hz]

Exactive

Q-Exactive

Resolution at m/z 200

Resolution at m/z 400

Max. scan speed (Hz)

17,500 12,500 12 35,000 25,000 7 70,000 50,000 3 140,000 100,000 1.5

• Increases resolution by 2X at the same transient length

6

Scan Innovations

• Parallel Filling and Detection

• Spectrum Multiplexing

7

Outline



• Peptide/Protein Identification

• TMT based Quantification



• Summary

8

Discovery Proteomics – 10Hz HCD Scan

256ms 64ms

• Parallel Filling and Detection • Fast Orbitrap Scanning • pAGC

10 HR/AM HCD scans in 1s

9

1 Full MS +10 HR/AM HCD Scans in One Second

1 2

3 7

5 6

4 8

9

10

1 2 5 6 7 8 9 10 4 3

∆ = 0.0181 min = 1.086 sec

Ecoli_50min_newcolumn_Top10_15cm_reje... 2/17/2011 3:39:42 PM

RT: 15.8279 - 51.4773

16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 Time (min)

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

41.5779 41.6221

25.9001 16.0095 33.0433 47.5177 27.4920 30.4806 17.9024 25.7411 22.3863 27.1739

39.1805 35.5786 30.9771 38.7325

23.9072 28.4175 49.3818 20.3567 18.7207 41.1998 37.5942 34.8784 25.4406 43.4203 21.9561 34.7425 41.0942 46.5441 43.5925 49.1418

49.6742

RT: 32.2438 - 32.2701

32.244 32.246 32.248 32.250 32.252 32.254 32.256 32.258 32.260 32.262 32.264 32.266 32.268 32.270 Time (min)

0.0

0.1

0.2

0.3

0.0

0.1

0.2

0.3

Rel

ativ

e A

bund

ance

32.2464 32.2645

32.2538 32.2564 32.2577 32.2551 32.2590 32.2603 32.2629 32.2694 32.2525 32.2512 32.2616 32.2447

Ecoli_50min_newcolumn_Top10_15cm_reject_1 # 12365 RT: 32.25 AV: 1 NL: 1.58E8 T: FTMS + p NSI Full ms [300.00-2000.00]

350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

536.7949 z=2

575.8452 z=2

664.3292 z=2

392.7555 z=2

484.2459 z=3

382.9046 z=3

581.9907 z=3

711.3712 z=3

445.7585 z=2

505.2733 z=2 419.2133

z=3

329.2184 z=2

1150.6826 z=1

551.8184 z=2

950.9638 z=2

784.5037 z=?

1072.5819 z=1

630.3730 z=2

725.8647 z=2 700.3623

z=2 902.8879

z=2 1008.5358

z=1 871.9796

z=2 835.3868

z=2 1096.3795

z=? 1182.6012

z=?

x2 x5

10

High Quality HCD Spectra

y₅⁺589.29150

b₂⁺247.14401

y₁⁺175.11888

y₆⁺688.36139

a₂⁺219.14905

y₂⁺288.20270

a₁⁺120.08099

100 200 300 400 500 600 700 800 900

m/z

0

2

4

6

8

10

12

Inte

nsity

[cou

nts]

(10^

6)

Extracted from: C:\Xcalibur\data\Kai\Ecoli_50min_newcolumn_Top10_15cm_reject_1.raw #12410 RT: 32.33 FTMS, HCD, z=+2, Mono m/z=467.75339 Da, MH+=934.49950 Da, Match Tol.=20 mmu

y₂⁺260.19653

y₄⁺388.25467

y₁⁺-H₂O129.10219

a₁⁺120.08088

100 200 300 400 500 600 700

m/z

0

1

2

3

4

Inte

nsity

[cou

nts]

(10^

6)


y₅⁺475.28632

y₁₀⁺931.52252

y₄⁺388.25351

y₆⁺532.31097

y₁₀⁺-NH₃914.49854

y₈⁺702.41449

y₉⁺803.45911

a₉²⁺-H₂O398.24179b₃⁺

272.15991

y₇⁺589.32959

b₂⁺171.11258

b₃⁺-H₂O254.14955

200 400 600 800 1000 1200

m/z

0

1

2

3

4

5

Inte

nsity

[cou

nts]

(10^

6)


a₅⁺438.30667 y₇⁺

685.38513

a₇³⁺-NH₃197.12845 y₆⁺

588.33417

b₃⁺, a₆²⁺268.16522

y₅⁺517.29755

b₅⁺466.30200

b₄⁺367.23297

y₁⁺-H₂O129.10220

y₂⁺246.18097

200 400 600 800 1000 1200

m/z

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Inte

nsity

[cou

nts]

(10^

6)


a₁₀⁺-H₂O, y₉⁺929.44843

y₁₀⁺1044.47485

y₄⁺440.26059

y₈⁺800.40826

y₇⁺729.36896

y₅⁺527.29431

a₆³⁺, b₂⁺, a₄²⁺201.12317

y₆⁺598.33002

a₂⁺173.12833

200 400 600 800 1000 1200 1400

m/z

0.0

0.5

1.0

1.5

2.0

2.5

Inte

nsity

[cou

nts]

(10^

6)


b₇⁺692.37195

a₇²⁺332.69171

y₄⁺430.23016

y₆⁺676.36572

y₁₁²⁺592.30835

y₅⁺577.29816

y₈⁺846.47083

y₉²⁺492.26865

y₂⁺244.16534

a₂⁺157.13342

200 400 600 800 1000 1200 1400

m/z

0

1

2

3

4

Inte

nsity

[cou

nts]

(10^

6)


b₆²⁺, b₃⁺342.20215

y₅⁺618.30170

y₆⁺731.38538

y₇⁺860.42780

b₂⁺213.15939

y₄⁺487.26202

a₂⁺185.16463

100 200 300 400 500 600 700 800 900 1000 1100

m/z

0

10

20

30

40

Inte

nsity

[cou

nts]

(10^

6)


y₁⁺175.11887 y₁₁⁺

1132.57153y₅⁺

620.31427y₉⁺

934.47302

y₆⁺707.34601

a₁⁺120.08097

200 400 600 800 1000 1200 1400

m/z

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Inte

nsity

[cou

nts]

(10^

6)


b₅⁺620.26434

y₁₁⁺1165.69214

y₁⁺147.11258

b₄⁺521.19879

y₇⁺755.46484

b₃⁺393.13989

a₂⁺250.11826

200 400 600 800 1000 1200 1400 1600 1800

m/z

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Inte

nsity

[cou

nts]

(10^

6)


b₅⁺-H₂O528.19189

b₄⁺-H₂O457.15570

y₈⁺909.54944

y₃³⁺129.10216

y₂⁺272.17108

b₃⁺-H₂O328.11536

b₂⁺-H₂O241.08124

a₂⁺231.09689

y₁₇²⁺926.00580

y₁₀²⁺568.84778

y₁₄²⁺782.45026

y₁₂²⁺653.90033

y₁₃²⁺717.92920

200 400 600 800 1000 1200

m/z

0

50

100

150

200

250

300

Inte

nsity

[cou

nts]

(10^

3)


11

Peptide/Protein Identification

*About 4500 proteins are expressed in yeast during log-phase growth

2452

6975

8353

1361

3539

4767

0

2000

4000

6000

8000

10000

10ng 100ng 1000ng

Q Exactive TripTOF 5600

691

1321 1395

357

724

1017

0

250

500

750

1000

1250

1500

10ng 100ng 1000ng

Number of Unique Peptides Number of Unique Protein Groups

Yeast Tryptic Digest (10, 100, 1000ng)

Column: C18, 75µmx15cm LC: 350nL/min, 90min

Q Exactive: Top10

TripleTOF 5600: Top20

12

0

20

40

60

80

100

120

140

160 Q Exactive

TripleTOF 5600

Peptide/Protein Identification

Protein identified from 10 ng yeast digest

*Yeast cellular protein copy numbers are from Weissman and co-workers, Nature, 2003, 16, 737-7341.

Same sample, Same LC column, Same gradient, 1% FDR

Num

ber o

f Pro

tein

s Id

entif

ied

Cellular copy number

4x more ID

13

Peptide/Protein Identification - Larger Dynamic Range on Q Exactive

Same Sample, Same LC column, Same Gradient, 1% FDR

Most Abundant Least Abundant

10 ng load

1000 ng load

Precursor intensity of identified peptides (normalized to the most intense peak)

TripleTof 5600

Q Exactive

14

Peptide of YOR020C, 149 copy number, identified from 10 ng yeast digest

b₅⁺-NH₃498.17874

y₆⁺-H₂O758.45410

y₉⁺1120.55273

y₂⁺322.18576

y₈⁺1006.51752

y₆⁺776.46594

y₁₀⁺1177.57886

y₁⁺175.11826

y₇⁺891.48932

y₅⁺647.42157

y₃⁺435.26990

y₄⁺548.35284

200 400 600 800 1000 1200

m/z

0

10

20

30

40

50

Inte

nsity

[cou

nts]

(10^

3)

Extracted from: C:\Xcalibur\data\Zhiqi\ID_QE\20110321_10ngYeastDigest_Top10_140min_01.raw #23048 RT: 77.42 FTMS, HCD, z=+2, Mono m/z=645.83948 Da, MH+=1290.67168 Da, Match Tol.=20 mmuL G N D D E V I L F RL G N D D E V I L F R MASCOT IonScore: 90

Exp Value: 3.8 E-9

Peptide/Protein Identification – High Quality HCD Spectrum

High confidence identification from 10 ng of Yeast Digest

15

Discovery Quan (TMT)

Num

ber o

f pep

tides

at 1

% F

DR

Amount of E. coli digest

2736

3888 4376

5252

6527

3245

4850

5537

7180

9298

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

20ng 40ng 80ng 200ng 500ng

unique peptide total IDed MSMS spectra quantifiable MSMS spectra

TMT 6-plex labeled E. coli digest

Quantification Rate: >97%

16

Discovery Quan (TMT) – Accuracy and Precision

1.04 1.01 1.02 1.05 1.03

0

0.2

0.4

0.6

0.8

1

1.2

1.4

127/126 128/126 129/126 130/126 131/126

observed expected

TMT

repo

rter i

on ra

tio

Result from 80 ng E. Coli digest

Quantification Error <5%, CV<13%

17

Outline




• Targeted msx SIM

• Targeted HCD


• Summary

18

HR/AM Targeted Quantification on the Q Exactive

HR/AM Targeted Quantification

Quantification

Targeted msx SIM Targeted HCD

LC Peak Area of Precursor

LC Peak Area of MS/MS Fragments

Instrument method

Selectivity, Speed, Sensitivity

19

Quantification with Targeted msx SIM

492.2665

492.2495

492.2634

R: 35K R: 140K

m/z

492.2661

492.2511

R: 70K

30ppm

High Resolution (140K) Accurate Mass (<5ppm) Selectivity

Spectrum Multiplexing Speed

20

Quantification with Targeted msx SIM

Quadrupole-Based SIM Sensitivity

21

17.0 17.2 17.4 17.6 17.8 18.0 18.2 18.4 18.6 Time (min)

0

20

40

60

80

100 0

20

40

60

80

100

Rel

ativ

e A

bund

ance

0

20

40

60

80

100 18.12

18.11

18.10

Quantification with Targeted msx SIM in Medium Complex Background

12 Heavy Peptides (10amol – 100fmol)

In 10ng Yeast Digest

msx tSIM Resolution: 140K SIM: 4amu msx: 4

GISNEGQNASIK 5ppm XIC

100fmol

1fmol

10amol

0.E+00

1.E+09

2.E+09

3.E+09

4.E+09

0 50 100 Sample Amount (fmole)

Pea

k A

rea

SSAAPPPPPR

GISNEGQNASIK

DIPVPKPK

LOD: 10amol LOQ: 50amol (CV <10%) Linear Dynamic Range: 4 orders

R2: 0.9991-0.9999

22

• Full MS/MS Spectra – All Transitions

• Selectivity • MS/MS • High Resolution: 17,500 • Accurate Mass: <5ppm

• Speed

• 12 Hz

• Sensitivity • High quality HCD scan at low amol level

Quantification with Targeted HCD

23

Quantification with Targeted HCD in Complex Matrix RT: 14.97 - 21.09

15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0Time (min)

0

20

40

60

80

100

Relat

ive A

bund

ance

0

20

40

60

80

100

Relat

ive A

bund

ance

0

20

40

60

80

100

Relat

ive A

bund

ance

15.74

15.82

15.7215.67

15.8515.88

15.96

18.6218.67

18.60

18.57

18.6918.55

20.6420.6120.58

20.65

20.5720.67

20.54

GPGEDFR394.193-633.3051

VAHTVAYLGK355.877-324.2350

YFQGYAR457.732-594.2989

11 Heavy Peptides (10amol – 100fmol)

In 250ng CSF Digest

y = 3820.1x + 578349R² = 0.9999

0

50000000

100000000

150000000

200000000

250000000

300000000

350000000

400000000

450000000

0 20000 40000 60000 80000 100000 120000

Peak

are

a

Sample amount (amole)

1000

10000

100000

1000000

10000000

100000000

1E+09

1 10 100 1000 10000 100000

YFQGYAR

tHCD Resolution: 17,500

LOD: 10-100amol Linear Dynamic Range: 3-4 Orders

10amol

24

1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 m/z

0 5

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95

100

Rel

ativ

e Ab

unda

nce

2745.7720

2851.3544

2907.2595 2556.4844

2965.3764 2471.3405

3025.8632 2391.6288

-7 ppm G0F+G1F

G0F+G0F G0F+G2F (or 2G1F)

G1F+G2F G2F+G2F G0+G0F

G0+G0 2xMan5

G1F+G2F+SA

5.0 ppm

-0.7 ppm -8.5 ppm

-0.9 ppm

2680 2700 2720 2740 2760 2780 2800 2820 m/z

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

2745.7720

2797.5697 2695.8919

Mass Measurement of Intact Monoclonal Antibody

Protein Deconvolution

R: 17.5K

25

Summary

• Q Exactive is the most competitive instrument for routine discovery proteomics analysis, with its 10Hz duty cycle, high dynamic range and high quality HCD scans.

• Q Exactive offers flexible HR/AM quantification approaches, targeted msx SIM and targeted HCD, with improved throughput, higher sensitivity and 4-order linear dynamic range.

• Q Exactive enables smooth transition from discovery proteomics to targeted quantification.

• Q Exactive accurately and reproducibly measures the mass of intact antibody to ~7ppm, and provides a robust QC solution for biopharmaceutical products.

26

Acknowledgement

S. Horning I. Mylchreest A. Guiller R.A.Purrmann F. Grosse-Coosmann A. Kuehn T. Rietpietsch E. Denisov R. Malek M. Biel C. Henrich M. Mueller A. Venckus F. Gebrehewit F. Afroz O. Hengelbrock M. Antonczak S. Moehring S. Nimkar M. Kosak K. Scheffler S. Daniels Y. Xun

D. Cho X. He Y. Huang A. Kholomeev K. Persson F. Paffen B. Rose A. Boegehold J. Grote W. Huels A. Schumbera S. Simmel P. Bennett K. Cook A. Huhmer Z. Hao S. Peterman Y. Zhang K. Comstock T. Stratton D. Ghosh C. Yang M. Blackburn

Q ExactiveTM: A True Qual-Quanapps.thermoscientific.com/media/SID/LSMS/PDF/...Exactive Q-Exactive...

Documents

Transcript of Q ExactiveTM: A True Qual-Quanapps.thermoscientific.com/media/SID/LSMS/PDF/...Exactive Q-Exactive...