introduction to the q exactive hf-x for proteomics · 2017-12-11 · hela digest, 3 technical...

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October 2017 Josh Nicklay

Introduction to the Thermo Scientific Q Exactive HF-X MS for Proteomics

2

• Revolutionizing insights, from discovery to verification

• Enhance your productivity

• Achieve faster than ever scan speed

• Confirm with greater confidence

• Superior consistency in quantitative accuracy, sensitivity and reproducibility

Pushing the leading edge in protein analysis

Thermo Scientific Q Exactive HF-X Hybrid Quadrupole-Orbitrap MS

3

Novel architecture with a high capacity transfer tube and electrodynamic ion funnel

Ultra-high field Orbitrap analyzer

• 240,000 resolution at m/z 200

• 40 Hz data acquisition speed @ 7,500 resolution

Advanced Peak Determination (APD)

BioPharma Option for intact proteins


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Ultra-High Field Orbitrap

Mass Analyzer

HCD Cell

C-Trap

HyperQuad Mass Filter with Advanced Quadrupole Technology

(AQT)

High Capacity Transfer Tube

(HCTT)

Electrodynamic Ion Funnel

Advanced Active Beam Guide

(AABG)

Thermo Scientific Q Exactive HF-X MS – New Architecture

Brighter ion beam More sensitive

Dedicated transient for TMT 10plex

Optimized Scan Matrix with accelerated HCD

40 Hz MS/MS

Advanced DDA (APD) for bottom-up and top-down

5

Key Technologies of Thermo Scientific Q Exactive HF-X MS

High Capacity Transfer Tube Electrodynamic Ion Funnel

Ultra-High Field Orbitrap Optimized Scan Matrix and new Transient lengths

16 ms

16 ms

maxIT 32 ms 32 ms

28 Hz

40 Hz

6

Optimized Scan Matrix

32 ms maxIT

32 ms

MS/MS Q Exactive HF

18-22 Hz • Brighter ion beam, reduced

scan overhead, and accelerated HCD (aHCD) is boosting acquisition speed

• Advantage for both MS and MS/MS mode

• Fast and high quality MS/MS acquisition up the 40 Hz with new 16 msec transient (7,500 resolution setting)

Orbitrap detection

Maximum fill time with precursor ions

Inter and intra scan overheads

15k

Q Exactive HF-X Longer maxIT Reduced scan overhead aHCD

maxIT 32 ms 32 ms

28 Hz

15k

Similar maxIT Reduced scan overhead aHDC

16 ms

16 ms

40 Hz

7.5k

7

MS/MS Scan Rate

30

35

40

45

50

5517

21

25

29

33

37

0 5 10 15 20

Scan

tim

e [m

s]

Inject time [ms]

Scan

rate

[Hz]

• MS/MS scan rate at resolution setting 7,500 @ m/z 200 for Q Exactive HF-X with respect to the ion injection time is displayed.

• Sample: Calmix

• 40 Hz MS/MS scan rate is reached with a maximum injection time (maxIT) of 10 ms

ASMS’17: TP 389, T.N. Arrey et al. New innovations implemented on the Thermo Scientific™ Q Exactive™ HF MS.

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Ultra Fast MS/MS Scan Speed > 40 Hz

39.48 39.49 39.50 39.51 39.52 39.53 39.54Time (min)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

Relat

ive A

bund

ance

7477639.5424

7473539.5242

7469439.5072

7461239.4725

7465339.4900

7471539.5162

7466539.4960

7462939.4801

7465539.4925 74672

39.4983

7469839.5103

7462539.4787

7467839.5004

7471939.5176 74749

39.530974761

39.535874632

39.4813 7468039.5011

MS MS

Duty cycle: 1.05 s Scan rate (1 FS + 40 MS2): 38.1Hz 40 MS2: 45.1 Hz

Duty cycle: 1.09 s Scan rate (1 FS + 40 MS2): 36.6 Hz 40 MS2: 42.8 Hz

Duty cycle: 1.02 s Scan rate (1 FS + 40 MS2): 39.2 Hz 40 MS2: 46.1 Hz

Duty cycle: 1.03 s Scan rate(1 FS + 40 MS2): 38.8 Hz 40 MS2: 45.8 Hz

MS MS MS

1 full scan (60,000 @ m/z 200) and 40 MS2 scans 7,500@ m/z 200) at LC time scale in 1 second. 30 min gradient, MS2 max IT: 11 ms


9

Calculated Scan Rate Across the LC Gradient for 1 µg HeLa Digest

0

5

10

15

20

25

30

35

40

45

50

0 10 20 30 40 50 60 70

Scan

Fre

quen

cy [H

z]

Retention Time [min]

Scan Frequency [Hz]

30min_7.5k, 35ms, Top20

30min_7.5k, 25ms, Top20

30min_7.5k, 11ms, Top40 30 min gradients:

• Top40 method: Scan rates of 38 Hz are obtainable at high ion flux with 11 ms max. inj. times applied

• Top20 method: Scan rates of > 25 Hz are obtained with longer max. inj. times (25 ms and 35 ms, resp.)

30 min_7.5k, 11 ms, Top40

30 min_7.5k, 35 ms, Top20

30 min_7.5k, 25 ms, Top20


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Protein Identification Faster than Ever

• Maximizing protein identifications • Same protein identifications in half the

analysis time • Faster, with same high quality results

1 µg HeLa, nano LC, 120k res MS1, DDA Top 20/40, minimum injection time 45/25 msec

3954 3535

Q Exactive HF, 60 min Q Exactive HF-X, 30 min

Protein groups Q Exactive HF, 60 minQ Exactive HF-X, 30 min

Similar data, half time

24083 25336

Q Exactive HF, 60 min Q Exactive HF-X, 30 min

Unique Peptides Q Exactive HF, 60 minQ Exactive HF-X, 30 min

Similar data, half time


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Deeper Dive into Proteome - More Productivity with Thermo Scientific Q Exactive HF-X MS

Maximizing peptide identifications

• Highest peptide coverage • Deep proteome analysis • Spectral library building • 2x productivity increase vs.

Q Exactive HF

• 4x productivity increase vs. Q Exactive Plus

• Sample: 1 ug Pierce HeLa digest

16455

24083

34780

Peptide Groups - 60min

Q Exactive Plus MSQ Exactive HF MSQ Exactive HF-X MS

increased peptide ID efficiency 22707

24083 25336

Peptide Groups

Q Exactive Plus MS 120 minQ Exactive HF MS 60 minQ Exactive HF-X MS 30 min

10Hz 18Hz 40Hz

MS/MS speed


12

Dilution Series Pierce HeLa Digest

60 min gradients each. Method set-up adapted to according to different sample load on column from 0.5 ng … 2000 ng

Reproducible and consistent identifications across a large range of sample load on column

0.5ng 1ng 5ng 10ng 50ng 100ng 200ng 500ng 1000ng 2000ng363 462 926 1151 1684 2168 2686 3719 4142 4612

0

1000

2000

3000

4000

5000

Aver

age

no o

f pro

tein

gro

ups

0.5ng 1ng 5ng 10ng 50ng 100ng 200ng 500ng 1000ng 2000ng1475 1739 5097 6180 12762 16874 21081 28885 33683 37372

0

5000

10000

15000

20000

25000

30000

35000

40000

Aver

age

no o

f uni

que

pept

ides

13

Rapid Proteomics - More than 1,000 Peptides Identified per Minute

Data with courtesy from J. Olsen, Novo Nordisk Foundation, Center for Protein Research, University of Copenhagen.

50% increased rate of peptide identifications when ion flux is max Up to ~ 1100 peptides identified per minute Sample: 1 µg HeLa

0

200

400

600

800

1000

1200

7.5 15 30 60

num

ber o

f uni

que

pept

ides

per

min

ute

gradient length (min, log scale)

Q Exactive HF (15k)

Q Exactive HF - X (7.5k)• Short transient time of

16 ms combined with accelerated HCD allows for scan rates up to 40 Hz in data dependent and targeted analyses

• Benefit is increased productivity, expressed by more peptide IDs per time.

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Proteome Perspectives: Rapid and Deep Proteome Sequencing in Less than Half the Acquisition Time

New data from Thermo Scientific™ Q Exactive™ HF-X MS overlaid with published data

100 µg fractionated into 12 concentrated fractions 30 min gradient, 15,000 res. method on Q Exactive HF-X

Data with courtesy from J. Olsen, Novo Nordisk Foundation, Center for Protein Research, University of Copenhagen

J. Proteome Res. 2014, 13(12), pp6187-6195

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New 96 msec Transient – Ideal for TMT 10plex

130.13 130.14 130.15 m/z

R=66,702 at R=66,202 at m/z 130.13504

R=87,702

m/z 130.14114

R=86,902

Optimal separation of TMT reporter ions

• A dedicated resolution setting of 45,000 at m/z 200 (FWHM)

• Optimally resolves reporter ions of the Thermo Scientific™ TMT10plex™ Isobaric Mass Tag Labeling Kit

• Frees up scan time to quantify 10–20% more peptides in discovery experiments

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29.16 29.18 29.20 29.22 29.24 29.26 29.28 29.30Time (min)

0

5

10

15

20

250

5

10

15

20

25

Rel

ativ

e A

bund

ance

29.1556 29.2051 29.2543 29.3045

29.1541 29.1923 29.2304 29.2685 29.3066

MSMSMS

scan cycle: 3.0 sScan freq (1FS+20MS2): 6.8Hz

Duty cycle: 2.3 sScan freq (1FS+20MS2): 8.7Hz

Scan cycle: 3.0 sScan freq (1FS+20MS2): 6.6Hz

MS

Scan cycle:2.9sScan freq (1FS+20MS2): 6.8Hz

MSMSMS


scan cycle: 2.3 sScan freq (1FS+20MS2): 8.7Hz

MS


MS

60k

45k

Scan Cycle Comparison

Scan cycle comparison between

• 60k resolution setting, Thermo Scientific™ Q Exactive™ HF (top)

• Novel 45k resolution setting

Thermo Scientific™ Q Exactive™ HF-X (bottom)

Method: FullMS (120k res), dd Top 20 MS/MS (60k or 45k) scans

Acquisition speed increase by ca. 25% allowing quantitation of more peptides per unit time with TMT 10plex.

60k

45k

17

60k on HF vs. 45k on HF-X

TMT11-plex labeled HeLa 1 µg on column, ~90 min gradient 120/86 ms max inject on Thermo Scientific™ Q Exactive™ HF/HF-X MS

>90% quantified >90% quantified

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Robust DIA Proteome Profiling using CapLC with Thermo Scientific QE HF-X MS VALUE: Highest depth of coverage and robust quantitative analysis

ASMS’17: ThP 237 Y. Xuan et al. Revolutionary Proteome Profiling and Quantitation without Compromising Speed, Sensitivity, and Selectivity.

HeLa digest, 3 technical replicates each, 60 mins total run time

HR-DIA data processed with Spectronaut software, Biognosys AG, Switzerland

Similar data on HF-X with 2 µg as HF with 4 µg Greater proportion of peptides with CVs < 10% on HF-X

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Top Down Proteomics - A Novel Workflow Brings Intact Proteins Into Focus

Thermo Scientific™ Q Exactive™ HF-X MS Full scan acquired in Protein Mode at a resolution setting of 7,500, detecting multiple charge envelopes.

Base peak chromatogram of E. coli ribosomal proteins separated in a 30 min gradient.

0 5 10 15 20 25 30 35 40 Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

17.36 820.89

36.47 413.27

15.49 783.14 25.00

1110.48 20.48

818.99

24.06 1014.51 9.45

699.92 1.15

574.92 5.36 644.72

22.37 897.37

RT 13.52

Full MS @ RT 13.52 min

On-the-fly deconvolution based on charge envelope to select a single charge state of each dominant proteoform

500 600 700 800 900 1000 1100 1200 1300 1400 m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

742.8 z=30

696.5 z=32

795.9 z=28

857.0 z=26

891.3 z=25 673.0

z=19

631.5 z=41

968.7 z=23 1012.7

z=22 1172.3

z=19 588.5 z=44 1237.5

z=18 1310.3 z=17

20

500 600 700 800 900 1000 1100 1200 1300 1400 m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

696.5 z=32

857.0 z=26

891.3 z=25 673.0

z=19

631.5 z=41

968.7 z=23 1012.7

z=22 1172.3

z=19 588.5 z=44 1237.5

z=18 1310.3 z=17

Top Down Proteomics - A Novel Workflow Brings Intact Proteins Into Focus On-the-fly deconvolution based on charge envelope to select a single charge state of each dominant proteoform.

MS/MS analysis of each proteoform fragmented with optimal collision energy and detected at a resolution setting of 120,000.

742.8 z=30

752.1 z=17

T: FTMS + p ESI d Full ms2 [email protected] [200.0000-8000.0000]

500 600 700 800 900 m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

798.11 R=68306

z=15

748.29 R=70906

z=16

735.41 R=70506

z=16 870.66

R=63806 z=11

811.58 R=65806

z=15

508.63 R=85806

z=3 703.33

R=70506 z=17

905.11 R=60806

z=8

MS/MS Proteoform 1

MS/MS Proteoform 2


600 700 800 900 1000 m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

687.55 R=76106

z=7

756.72 R=72306

z=7

754.29 R=72306

z=7 801.98 R=70406

z=6

872.47 R=66906

z=8 677.41 R=75806

z=7 902.74

R=64406 z=8 997.11

R=59906 z=7

MS/MS Proteoform 3


500 600 700 800 900 m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

711.67 R=72506

z=15 762.44

R=70806 z=14

691.57 R=73206

z=22 601.60 R=80806

z=4 813.39 R=65106

z=13 559.08

R=78506 z=4

667.20 R=73106

z=16 495.69

R=83406 z=5

699.7 z=37

Deconvolution with Respect

0

20

40

60

80

100 22256.3

25850.8

12767.8

15000 20000 25000 Mass

Reö

ativ

e In

tens

ity

post acquisition

21

• Revolutionizing insights, from discovery to verification

• Enhance your productivity

• Achieve faster than ever scan speed

• Confirm with greater confidence

• Superior consistency in quantitative accuracy, sensitivity and reproducibility

Pushing the leading edge in protein analysis


22

Acknowledgement

Eric Couzijn Oliver Lange Christian Thoeing Dirk Nolting Jens Grote Matthias Mueller Anastassios Giannakopulos Alexander Makarov Andreas Boegehold Nicole Zehethofer Bjorn Rose Dennie Kemper Patrick Huesing Matthias Vollrath Sebastian Kanngiesser Sascha Moehring Stefanie Raffelhueschen

Eloy Wouters Dean Dumitresku Franz-Josef Paffen Karsten Goepel Tabiwang Arrey Martin Zeller Christian Klaas Eugen Damoc Markus Kellmann Kerstin Strupat Yue Xuan Thomas Moehring Alexander Harder Maciej Bromirski Keeley M. Murphy Aaron O. Bailey Jonathan Josephs Aaron Gajadhar Michael Krawitzky Andreas Huhmer

Brenda Kesler Tony Ziberna Rick Carberry Albar Martucci Claire Dauly Gary Woffendin Glenn Damkroeger Jenny Ho Wilfried Voorhorst Jocelyn Dupuy Kai Scheffler Olaf Scheibner Lin Tang Goh Jing Li Kentaro Takahara Eunyoung Lim Ruby Ong Darren Jones Michael Mariani

LSMS team (Bremen & San Jose): Sales Advisory board

Collaborators: Jesper V. Olsen, Christian D. Kelstrup, Dorte B. Bekker-Jensen Albert J. R. Heck, Kyle L. Fort, Michiel van de Waterbeemd, Sem Tamara

introduction to the q exactive hf-x for proteomics · 2017-12-11 · hela digest, 3 technical...

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