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Thermo Scientific™ Q Exactive™ UHMR Mass Spectrometer

2

Market Trend & Driving the Need for the Thermo Scientific™ Q Exactive™ UHMR MS

Customer type Structural BiologistsUniversity research institute

Functional ProteomicsUniversity research institute

Functional ProteomicsUniversity research institute

Structural BiologistsUniversity research institute

BioPharma industryResearch

Goals Native MS analysis of megaDalton nucleic acids-protein complexes (i.e ribosomal particles, virus particles)

Native MS and native top-down analysis of membrane protein complexes

Integrative Structural Biology, native MS

Integrative Structural Biology, Cryo-EM

Characterization of complex bio-therapeutics using native MS

Needs • Efficient desolvation in the source region of the mass spectrometer

• Sensitivity in ultra-high mass range

• Resolution in ultra-high mass range

• Efficient removal of detergent micelles in the source region of the mass spectrometer

• Resolution

• Sensitivity

• Structure determination of protein complexes using native MS and native top-down analysis

• Need to obtain complementary structural information to facilitate downstream modeling

• Fast screening and optimization of Cryo-EM samples

• Need to obtain complementary structural information (i.e. subunit composition and subunit stoichiometry) to facilitate Cryo-EM data interpretation

• Sensitivity in ultra-high mass range

• Resolution in ultra-high mass range

Value DriverHelp me SUCCEED

CollaboratorHelp me COLLABORATE

Science StrategistHelp me ADVANCE

InitiatorHelp me be BETTER

Value DriverHelp me be FASTER

Utrecht University Oxford University Krogan’s lab/UCSF David Agard’s lab/UCSF Genentech

3

Introducing a New Member to the Thermo Scientific™ Exactive™ Family

Unmatched Native MS and Native Top-Down Performance

• Innovate in Structural Biology & BioPharma research

• Accelerate native protein structure analysis

• Study protein interactions for deeper understanding of biological processes

• Achieve accurate characterization of non-covalent protein complexes

• Determine ligand biomolecular interactions under native conditions

4

Product at a Glance | Unique Value to our Customers

FeaturesFeatures BenefitsBenefits

Unprecedented resolution and orders of magnitude enhanced sensitivity at high m/z

In-source trapping capability that enables improved transmission and controllable desolvation and fragmentation.

High mass quadrupole selection and higher HCD fragmentation efficiency for native top-down analysis

Analyze intact MegaDalton assemblies and resolve small differences in masses that reveal key ligands, modifications and interactions

Gain detailed structural insights for deeper understanding of biological processes

Quickly verify sample quality prior to analysis by cryo-electron microscopy (cryo-EM), and determine sample composition and homogeneity to assure successful cryo-EM analysis

5

Current Exactive Series Product Portfolio for Native MS Market

Incr

ease

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Exactive Plus EMR

• Orbitrap analyzer (D30)• Mass Range EMR m/z 350 - 20,000• Mass Accuracy: <1ppm (internal)• Mass Resolution >140,000• Scan Speed up to 12 Hz• Octapole transfer for MS

• Orbitrap analyzer (D30)• Mass Range m/z 350 – 80,000• Mass Accuracy <1ppm (internal) and

<3ppm (external) for CsI cluster ions under defined conditions

• Max. Mass Resolution 200,000 at m/z 400

• Scan speed up to 12Hz at resolution of 12,500 @ m/z 400

• Quadrupole selection up to m/z 25,000 (SIM, MS/MS & pseudo-MS3)

• Full Scan, AIF, SIM and ddHCD (TopN)

Q Exactive UHMR

Characterize large proteins & protein assemblies

Ribonucleoprotein, membrane protein, protein assembly

High resolution, High sensitivity, Ultra‐High Mass Range

Characterize proteins in intact form, high res native MS

2013

2018New

Very Large molecules

D30Orbitrap

6

Thermo Scientific™ Q Exactive™ UHMR Hybrid Quadrupole-Orbitrap™ MS

Performance CharacteristicsMax resolution 200,000 at m/z 400

Mass range m/z 350 to 80,000

Scan rate 12 Hz at resolution setting of 12,500 @ m/z 400

Mass Accuracy* Internal: < 1 ppm RMSExternal: < 3 ppm RMS

Quadrupole Selection

Up to m/z 25,000 (SIM, MS/MS, pseudo-MS3)

Dissociation Source CID, In-source trapping, HCD

Analyzer Orbitrap

Scan Functions

FS: Full ScanAIF: All Ion FragmentationSIM: Selected Ion MonitoringddHCD: data dependent HCD (Top N)

*For CsI cluster ions under defined conditions

7

Schematic of the Q Exactive UHMR Mass Spectrometer

HCD Cell

C-Trap

Orbitrap Mass Analyzer

HyperQuad Mass Filter with Advanced Quadrupole

Technology (AQT)

RF-Lens

Advanced Active Beam Guide

(AABG)

4

8

9

7

5

6

Transport multipole

2

Injection Flatapole

3

Inter-Flatapole Lens

1

1 RF-lens stacked-ring ion guide captures and efficiently focuses the ions into a tight beam. The RF-lens exit aperturewith reduced diameter acts as the entrance lens to the ion trapping region.

2, 3 The injection flatapole is pulsed down to a negative voltage to improve desolvation of large protein complexes, while the inter-flatapole lens is maintained at a high positive potential to prevent ions from eluting out. Trapping is followed by restoration of the voltage levels, allowing low-energy elution of ions into the bent flatapole (Advanced Active Beam Guide).

4 The bent flatapole guides and focuses ions using an axial DC field and a focusing RF field, enhancing sensitivity.

2, 4–8 The RF frequencies of all ion routing multipoles—the injection and bent flatapoles, quadrupole, transport multipole, C-Trap and HCD cell—are reduced to improve ion transmission.

9 High mass ions are efficiently injected into the Orbitrap mass analyzer by adjusting the slew rate of the high-voltage pulse that captures ions in the analyzer.

M. Belov, US2015340213 (2015), US9887074 (2018)

In-source trapping

8

• Front-end

• Thermo Scientific™ Vanquish™ (F & H) UHPLC

• Thermo Scientific™ UltiMate™ 3000 HPLC & Thermo Scientific™ UltiMate™ 3000 RSLC

• Thermo Scientific™ Easy-nLC™ & Thermo Scientific™ Easy-nLC™ 1200 systems

• Sources

• Thermo Scientific™ H-ESI II™ ion source

• Thermo Scientific™ Nanospray Flex™ ion source

• Triversa NanoMate, Advion

• ZipChipTM, 908devices

NPI Product Compatibility

• Software

• Thermo Scientific™ Xcalibur™ SW

• Thermo Scientific™ BioPharma Finder™ SW

• Thermo Scientific™ Respect™ Deconvolution SW

• Thermo Scientific™ ProSightPC™ SW

9

• Native MS analysis of intact megaDalton protein-nucleic acid complexes • 70S, 30S and 50S E. coli ribosomal particles • 9.3MDa Flock House Virus, 3MDa and 4MDa hepatitis B virus capsids

• Native top-down analysis of soluble protein complexes • GroEL protein complex• 20S proteasome complex

• Native top-down analysis of membrane protein complexes• AmtB membrane protein complex

Application Suitability & Proof Points

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Native MS Analysis of E. coli 70S, 30S and 50S Ribosomal Particles

30000 35000 40000 45000m/z

10000 15000 20000 25000 30000 35000 40000m/z

30S ribosome70S ribosome

50S ribosome

Mg2+ removal

Nat Methods. 2017 Mar;14(3):283-286. doi: 10.1038/nmeth.4147

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High-fidelity Native MS Analysis Unveils Heterogeneity in Intact Ribosomal Particles

24000 26000 28000 30000 32000 34000m/z

50S – RL10(RL7/12)4 1,389,667 +/- 151 Da

50S – (RL7/12)2 1,431,517 +/- 645 Da

20000 22000 24000 26000 28000m/z

30S – RS1 788,594 +/- 82 Da

30S 850,091 +/- 99 Da

30S – RS1 + SRA 793,748 +/- 78 Da

30S + SRA 855,256 +/- 48 Da

Nat Methods. 2017 Mar;14(3):283-286. doi: 10.1038/nmeth.4147

12

9.3MDa Flock House Virus (left) and 3MDa and 4MDa Hepatitis B Virus Capsids (right)

Nat Methods. 2017 Mar;14(3):283-286. doi: 10.1038/nmeth.4147ASMS 2018, MOF am 09:50, Tobias Woerner, Utrecht University

Native MS analysis under charge reducing conditions

13

Native MS and MS/MS Analysis of Hepatitis B Virus Particles

10000 20000 30000 40000 50000 60000 70000 80000m/z

0

50

1000

50

100

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bund

ance

0

50

100

Quadrupole selection

MS2 (HCD, 300eV)

66000 68000 70000 72000m/z

4 MDa

3 MDa55+57+

53+ 52+

56+

54+

‐ 14 x‐ 15 x‐ 16 x‐ 17 xMS1

Nat Methods. 2017 Mar;14(3):283-286. doi: 10.1038/nmeth.4147

14

0

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100

Rel

ative

Inte

nsity

800746.38

400454.25

500000 1000000Mass

Native Top-down Pseudo-MS3 Analysis of the GroEL Complex

5000 10000 15000 20000 25000m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

5361.9769

2696.3860

13970.33329051.7368

15069.929612460.5581 18327.5454 21500.2037 25558.2598

500 1000 1500 2000 2500 3000 3500 4000m/z

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

1406.7993z=3 2682.5039

z=21046.5243z=1

1789.0018z=3 2753.4817

z=22109.6936z=2

2604.7175z=2

3019.5930z=3

2330.5786z=6

885.3068z=1 3801.2073

z=2

5000 10000 15000 20000 25000 30000 35000 40000 45000m/z

0

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40

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1000

20

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60

80

100

Rel

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0

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MS1

MS3 (HCD)

14‐mer

14‐mer

13‐mer

12‐merMonomer

Quadrupole selection

MS3 (deconvoluted)

…

ReSpectdeconv.

21% Residue Cleavages

MS2 (In‐source trapping)

15

Native MS and MS/MS Analysis of the Rabbit 20S Proteasome complex

5000 10000 15000 20000 25000 30000 35000 40000m/z

0

20

40

60

80

1000

20

40

60

80

1000

20

40

60

80

100R

elat

ive

Abun

danc

e0

20

40

60

80

100 11753.0811564.85

11950.97

11377.08 12156.19

12364.7411204.7210907.21 12596.14

11948.55

12153.0811561.65

12358.9516818.7011377.61

19150.8811212.251707.8317235.091821.70 17674.7716416.89

19144.571951.71 16033.5928778.451986.32

26492.6412069.762151.73 24523.83 31506.942347.21 11681.79

2868.71 33098.0011173.28

1821.68

18621.8927584.5925476.9317233.2912069.88 30080.09

11680.0133078.4211171.74

- 6, - 2

- 6

717023.187

200000 400000600000 800000 1000000

Mass

ReSpect deconvolution

MS2 (HCD, 160eV)

- 6, - 2, - 5

MS1

MS2 (HCD, 200eV)

MS2 (HCD, 220eV)1000 1500 2000 2500 3000 3500m/z

0

5

10

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30

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40

45

50

55

60

65

70

75

80

85

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95

100

Rel

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1821.68

1951.72

1707.88

1986.34

2151.76

1607.48

2347.24

2581.851518.17

2868.671462.20 2644.63 3227.07

2450.53

2938.40 3305.651323.00

16

ID: Rabbit 20S Proteasome α-6 Subunit with Top-down Pseudo-MS3 Analysis

20180420_20sproteasome1707_1804231404... 04/23/18 15:36:51 This file is created by QualbrowserFor more information see StatusLog20180420_20sproteasome1707_180423140424-_1000_2000qb #1 RT: 23.19 AV: 1 NL: 1.72E2T: FTMS + p NSI Full ms [1000.0000-50000.0000]

1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

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100

Rel

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1607.4614R=70704

z=17

1707.8005R=70404

z=161518.2137R=67104

z=18 1821.6521R=71704

z=151295.5345R=120306

z=1

1343.4070R=117906

z=11475.4489R=112506

z=1 1931.5190R=95906

z=2

1739.5308R=102206

z=11844.4469R=70104

z=14

1643.5110R=105006

z=1

1568.7012R=108406

z=1

1289.6433R=119906

z=1

20180420_20sproteasome1707_180423140424-_1000_2000qb_XT_00001_M_ #2 RT: 2.00 AV: 1 NL: 4.67E2T: FTMS + p NSI Full ms [1000.00-50000.00]

21000 22000 23000 24000 25000 26000 27000 28000 29000 30000 31000m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

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bund

ance

27292.69226

25793.19448 27746.47770

26420.12217 28392.81042 29375.9862121356.57294 31006.88560

-1.8ppm

-3.7ppm -5.6ppm

-3.6ppm

Quad selection

MS2 (deconv.)

-3.2ppm

MS2 (In‐source trapping)

P

17

Native MS Analysis of the AmtB Membrane Protein Complex

5000 6000 7000 8000 9000 10000 11000m/z

0

10

20

30

40

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60

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100

Rel

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7925.46

8453.76

7459.36

9057.547044.85

17+

16+

15+

18+ 14+

+ 2.9ppm

AmtB sample was provided by A. Laganowsky

18

Native Top-down Analysis of the AmtB Trimer

1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000m/z

0

20

40

60

80

100

Rel

ativ

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unda

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4051.0059R=51604

z=7

3079.0283R=38504

z=5 4982.4927R=31706

z=27926.6577

R=17102z=16

2432.2810R=41706

z=31299.6334R=58506

z=27459.8511

R=21202z=17

5759.0264R=29404

z=3 8454.66895R=10102

z=157043.8652

R=21102z=18

5000 10000 15000 20000 25000m/z

0

20

40

60

80

100

Rel

ativ

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unda

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15381.0053

2614.2637 17264.0994

9956.95611 22444.8745

7474.870728331.0084

6650.5527

14907.96247 25621.7640011056.70154 20675.85380

MS2

MS2 (deconvoluted)

19

Testimonials

“In the analysis of large protein assemblies like viruses, ribosomes, and proteasomes, the Q Exactive UHMR mass spectrometer has made things possible that we couldn’t do before. The major benefits of the system are the substantial increase in sensitivity and resolution that we get for very large protein assemblies, together with the ability to do MS/MS experiments.”

Prof. Dr. Albert J. R. Heck, University of Utrecht, Scientific Director Netherlands Proteomics Centre and Scientific Director Utrecht Institute for Pharmaceutical Sciences

“Thermo Fisher Scientific has made a major contribution to native mass spectrometry in recent years. In the past we couldn’t get resolution of very large protein complexes with few charges. This was a real stumbling block for us. We have benefited from recent developments in native high resolution mass spectrometry. We didn’t realize what we weren’t seeing before. It gives us a new view of our molecules and this is an exciting transformation. I’m very excited where it will take us in the future.”Professor Dame Carol Robinson, University of Oxford, Founder and Chief Scientific Consultant, OMass Technologies

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Brochure

Specifications sheet

Lockout Specifications

Product video

Product images

Planet Orbitrap content

Product page TF.com

Customer facing presentation

eBlast NPI announcement

Press release

3 talks, 5 posters at ASMS 2018

2 Poster notes (after ASMS)

3 Publications

Sales Tools & Support

Case Study Publications

21

• Q Exactive UHMR MS is a unique solution to the pain points encountered by research and industrial customers in structural biology and biopharma research.

• It’s designed for those scientists who need higher resolution and sensitivity in the ultra-high mass range and the ability to perform highest quality native MS and native top-down experiments.

• Its unique combination of high resolution, high sensitivity and MS2/pseudo-MS³ capabilities makes it ideal for these applications.

• It’s the perfect solution for native top-down characterization of protein complexes, enabling new insights into native protein structure and protein interactions.

• It offers the unparalleled ability to resolve and characterize co-occurring assemblies, to resolve small differences in masses that reveal key ligands, modifications and interactions.

• Competitor Q-ToF systems have much lower resolution and sensitivity, and cannot resolve/see these small differences.

Take Home Message

Structural Biology and Pharma/Biopharma Research

Highest quality native MS & native top‐down analysis

Pseudo‐MS³ capabilities 

High res native MS analysis of large and heterogeneous protein assemblies