Higher-Quality Data, Faster Than Ever

Speed • Productivity • Confidence

Thermo Scientific Q Exactive HF Orbitrap LC-MS/MS System

2

• Ultra-High-Field Orbitrap (HF) mass analyzer for faster scan speed and higher resolution

• Advanced Quadrupole Technology (AQT) improves precursor selection and transmission for more accurate quantitation of low abundant analytes in complex matrices

• Sophisticated Data-Independent Acquisition (DIA) and Parallel Reaction Monitoring (PRM) deliver reproducible quantitation with complete qualitative confidence

• The Advanced Active Beam Guide (AABG) reduces noise and improves instrument robustness

• Optional intact Protein Mode provides superior trapping of large molecules for improved analysis of intact proteins

The Thermo Scientific™ Q Exactive™ HF hybrid quadrupole-Orbitrap mass spectrometer features

an ultra-high-field Orbitrap analyzer which doubles its speed and resolution – more sample

runs and amazing data quality. Built on the proven performance of the Q Exactive Plus platform,

the Q Exactive HF mass spectrometer utilizes the same Active Beam Guide technology and

Advanced Quadrupole design for very stable system operation and exceptional analytical

performance. The Q Exactive HF mass spectrometer is designed for your toughest discovery

and quantitation challenges where the combination of speed and highest performance are

essential for the highest quality scientific results.

Transforming discovery quantitation

HCD Cell

Ultra-High-Field Orbitrap (HF) mass analyzer

Quadrupole Mass Filter with Advanced Quadrupole

Technology (AQT)

RF LensAdvanced Active Beam Guide (AABG)

3

� Comparable number of protein IDs in half the time Base peak chromatogram (BPC) of a 120 min gradient on the Q Exactive Plus MS (red) and a 60 min gradient on the Q Exactive HF MS (blue) showing one half the analysis time.

� Improved productivity with Q Exactive HF MS

The Q Exactive HF MS system completes 1 full MS scan at 60,000 resolution and 20 HCD MS/MS scans at 15,000

resolution in 1.13 sec, 37% shorter compared to the Q Exactive Plus MS.

Faster protein identification The highest quality protein identification from

complex biological samples over the widest

dynamic range is an absolute requirement for

all proteomics labs. The Q Exactive HF system

delivers the mass accuracy and resolution you

expect combined with far greater speed than

ever before, enabling protein identification that

is faster and deeper than ever, while maintaining

the resolution and accuracy required for

confidence in your results. Under the same

LC gradient conditions, the Q Exactive HF MS

identifies approximately 30% more unique

peptides and 20% more protein groups than the

Q Exactive Plus MS system. The speed of the

system enables gradient runs that are twice as

fast (steeper gradients) while maintaining the

ability to identify roughly the same number of

unique peptides and protein groups.

The faster scan speed of the Q Exactive HF

MS results in higher number of precursor

ions triggered for MS/MS—synonymous

with improved productivity.

Analyze complex mixtures faster than ever!

The Q Exactive HF mass spectrometer, with an ultra-high-field Thermo Scientific™ Orbitrap™ detector, provides new

levels of speed and resolution for every scan while maintaining high spectral quality for sensitive detection and

confident quantitation of peptides, lipids, metabolites, contaminants and other important compound classes.

Rela

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A) Q Exactive Plus MS

0 20 40 60 80

100 31.88848

31.85865

31.82908

31.79956

31.77800

B) Q Exactive HF MS

31.78 31.80 31.82 31.84 31.86 31.88 31.90 Time (min)

0 20 40 60 80

100 31.91221

31.89353

31.87478

31.85611

31.83719

31.81816

31.79957 31.78061

31.840 31.844 31.848 31.852

Time (min)

0

20

40

60

80

100 31.84339 31.84016

31.84259

31.84422 31.85078

31.83939 31.84504 31.84669

31.84181 31.84750 31.85157 31.85404 31.84831 31.84585 31.84997 31.84099

31.85240 Rela

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1.77 sec

20 MS/MS in 1.13 sec

1.13 sec

Q Exactive Plus MS, 120 min gradient

Q Exactive HF MS, 60 min gradient

4

� Improved chromatographic sampling Extracted ion chromatograms (XIC) of the same peptide showing (A) only 4 points across the chromatographic peaks, and (B) 8 points across the peak. The short cycle time on the Q Exactive HF system samples the peak more frequently, ensuring the best quality quantitation.

� Improved quantitation linearity and reproducibility with PRM As a result of the improved selectivity, better quantitation over a large linear dynamic range is obtained. This demonstrates great reproducibility for very low amounts of target peptide in a complex background, in this example, 200 ng HeLa digest. The 2 amu isolation window clearly shows higher variability at the 0.01 fmol level vs. the narrower isolation width of 0.7 amu.

Parallel Reaction Monitoring – the new paradigm for high-throughput targeted quantitation The high resolution accurate mass (HR/AM) capability of Orbitrap MS enables a new mode of quantitation – Parallel Reaction Monitoring (PRM).

PRM relies on HR/AM to provide exquisite selectivity, high sensitivity, accurate and high throughput quantitation of analyte targets, and excellent

MS/MS data to confirm target identification.

Achieving quantitative precision The best precision in quantitation is achieved with the highest possible

ion transmission and rapid data collection to correctly define analytical

peak shape. The ultra-high-field Orbitrap analyzer of the Q Exactive HF

system scans fast enough to accurately define narrow UHPLC peaks

for optimal quantitative results.

Enabling quantitative accuracy Quantitative accuracy is achieved by removing interferences from

co-eluting analytes. The unique Advanced Active Beam Guide

technology offers the ability to narrow the quadrupole isolation

width to as little as 0.4 amu, significantly reducing the possibility

of signal interference that can decrease selectivity and accuracy.

For low level peptide quantitation by PRM, the extreme sensitivity

of the Q Exactive HF system enables routine quantitation down

to 0.01 fmole and provides extremely high precision using 0.7 amu

isolation —as demonstrated by the very low coefficients of variation

(CV) at the lowest levels of quantitation.

Quantify with the highest confidence

With its unique spectrum-multiplexing capabilities and faster scan speed, the Q Exactive HF mass

spectrometer provides the speed necessary for precise quantitation even for narrow ultra-high-performance

liquid chromatography (UHPLC). Industry leading high resolution accurate mass (HR/AM) MS provides you

with a more complete quantitative picture.

98 99 100 101 102 Retention Time

0.0

0.5

1.0

1.5

2.0

Inte

nsity

10^

6

102.5 103.0 103.5 104.0 Retention Time

0.0

0.5

1.0

1.5

2.0

Inte

nsity

10^

6

A B

Inte

grat

ed In

tens

ity

Peptide Concentration (fmol)0.005

1E10

1E9

1E8

1E7

1E6

1E5

1E40.01 0.05 0.1 0.5 1 5 10 50 100

R² = 0.9992

R² = 0.9994

Peptide

GISNEGQNASIK (2 amu)

GISNEGQNASIK (0.7 amu)

CV (0.05 fmol)

2.19%

2.09%

CV (0.01 fmol)

9.70%

2.03%

5

� Comparison of replicate DIA quantitation results While replicate 4 exhibits a good correlation to the spectral library (0.97), replicate 3 is only moderately correlated (0.73). However, data can be confirmed at the MS1 level with 0.4 ppm mass error and with isotopic dot products greater than 0.9. If one replicate is questionable the excellent mass accuracy of the MS1 peak can be used to match the peptide and “save or recover” a poor replicate, so quantitation is performed on the MS1 precursor.

Orbitrap-based HR/AM reduces false positives The Q Exactive HF MS provides high resolution and practical, experimental mass

accuracies of less than 3 ppm for both precursor and fragment ions enabling very

narrow mass windows to be used for data extraction and comparison to library

spectra, greatly reducing the risk of false positive identifications using the

BASIC-DIA method, relative to standard SWATH™ methods.

High quality data to address complex samples The high resolution capability of the Q Exactive HF MS is a key

attribute in separating legitimate signal from potential interferences.

With resolutions up to 240,000 FWHM @ 200 m/z and low-ppm MS

and MS/MS mass accuracy, the Q Exactive HF MS system provides

superior selectivity to resolve isobaric fragments—significantly better

than Q-ToF systems with their lower resolution, and limited accuracies.

� Selectivity through mass accuracy Comparison of selectivity obtained with 50 ppm mass accuracy (typical of ToF systems) vs. 5 ppm mass accuracy in MS/MS analyses, and the selectivity it brings to DIA analyses-only obtainable on Orbitrap-based systems.

Targeted quantitative methods deliver the highest sensitivity, best precision, and lowest detection limits, but require a priori

knowledge of the precursor ions (target molecules) and significant time and effort for SRM or PRM method development

and optimization.

In a true discovery quantitation environment, the targets may not be known and relative quantitation may be sufficient. In such

cases, data independent acquisition, is a viable quantitative approach. The Q Exactive HF MS enables several methods for DIA

data acquisition, including BASIC-DIA, pSMART-DIA, and MSx-DIA. These DIA methods fragment all ions present in the sample

to create a high-quality, permanent digital data archive of a sample that can be reanalyzed to look for new targets.

A digital data archive – New levels of DIA capability

62.5

Inte

nsity

(10^

3)

Inte

nsity

(10^

3)

Retention TimeRetention Time

Retention TimeRetention Time

Inte

nsity

(10^

6)

Inte

nsity

(10^

6)

63.0 63.5 64.0 64.5 62.0 62.5 63.0 63.5 64.0 64.5

62.5 63.0 63.5 64.0 64.5 62.0 62.5 63.0 63.5 64.0 64.5

2.0

1.5

1.0

0.5

0.0

80

70

60

50

40

30

20

10

0

400

350

300

250

200

150

100

50

0

2.5

2.0

1.5

1.0

0.5

0.0

Replicate 3 Replicate 4

MS1 MS1

MS2 MS2

50 ppm

5 ppm

Rela

tive

Inte

nsity

(3.4

04e+

5)

61.3 61.8 62.3 62.9 63.4 63.9 64.4 65 65.5 66 66.5

Rela

tive

Inte

nsity

(2.5

10e+

5)

100

90

80

70

60

50

40

30

20

10

0

61.3 61.8 62.3 62.9 63.4 63.9 64.4 65 65.5 66 66.5

100

90

80

70

60

50

40

30

20

10

0

The analysis of intact proteins presents a different set of challenges in which maximum resolution of the

spectral features can help to distinguish major protein isoforms from lower abundant species. Particularly

in biopharmaceutical applications, the analysis of heterogeneous protein mixtures requires the highest

resolving capabilities combined with the high mass accuracy provided only with Orbitrap technology.

The Q Exactive HF MS, with the optional Intact Protein Mode, enables high resolution analysis of intact

proteins, allowing detailed characterization of larger, more highly charged protein species such as intact

antibodies, as well as the individual heavy and light chains. With the additional resolving power of the

Q Exactive HF MS, proteins as large as 50 kDa can be isotopically resolved. This improved resolution

allows routine, detailed characterization of proteins and biopharmaceuticals.

Superior intact protein characterization

� � Analysis of monoclonal antibody, Herceptin Direct infusion of the mAb Herceptin. (A) Full MS spectrum of the intact mAb charge envelopes of the monomer (orange) and the dimer (green). Insets show one charge state each with different glycoforms. (B) Full MS spectrum of a reduced mixture of Herceptin light and heavy chain. (C) Deconvoluted spectrum of mAb Herceptin from (A). Peaks represent the various glycoforms. Obtained mass accuracies for the six most abundant glycoforms are between 1 and 10 ppm.

6

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100

90

80

70

60

50

40

30

20

10

0

3706.5 3530.0

3447.9 3369.6

3294.8 3223.2

3154.6

5490.6

3880 3900 3920 m/z

0 20 40 60 80

100

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3901.5 3905.8 3897.2

3910.0 3893.3

3914.3

5020 5040 m/z

0 20 40 60 80

100

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5028.2 5022.8

5030.7 5020.1 5033.4

5017.5 5038.8

3901.5 4235.8 3801.5

4360.4

4492.4

4632.9

4782.3 4941.7

4118.2

2000 3000 4000 5000m/z

6000

B. Reduced mAb Herceptin Light chain Heavy chain

A. Intact mAb Herceptin

147500 148000 148500

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100

90

80

70

50

30

60

40

20

10

0147485.712

G0/G0

Man5/Man5147600.712

G0/G0F147911.359

G0F/G0F148058.141

(G0F/G2F) / (G1F/G1F)148380.328

148219.719 G0F/G1F

G1F/G2F148542.734

G2F/G2F148704.212

148833.312

C. Deconvoluted spectrum of intact mAb Herceptin

149000Mass

800 1000 1200 1400m/z

1600 1800 2000

1303.0

1379.7

1465.9

1563.5

1675.2

1954.2

1803.9

1234.5

1205.6 1100.9

1077.5

1055.1

1033.5

1012.9

993.1

974.0

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100

90

80

70

60

50

40

30

20

10

0

7

The analysis of lipids poses a unique analytical challenge

in research, as the closely related molecular structure of

many lipid species produce multiple isobaric species within

a relatively narrow mass range. This creates the risk of

ambiguous lipid structure assignments. The high resolving

power of the Q Exactive HF system greatly reduces this

risk compared to instrumentation with lower resolving power.

The Q Exactive HF MS is ideal for both infusion and LC-based

workflows. Fast acquisition speed allows the system to keep

pace with demanding lipid separations from complex samples.

Increased productivity and confidence for lipidomics

� Identification of more lipid species at varying concentrations While the LC gradient for the Q Exactive HF system was 33% shorter (steeper LC gradient), the speed and resolution of the system still allowed identification of 20% more lipid species from a bovine heart lipid extract. LC method, column and gradients for the Q Exactive Plus MS and for the Q Exactive HF MS were identical.

� Increased resolution improves identification/quantitation The effect of resolving power for the identification of lipid species is illustrated for two Lyso phospholipid species, 18:1 LPE (lysophosphoethanolamine) and 16:0p LPC (lysophosphocholine). Identification of minor lipid species is challenging without sufficient mass resolution, leading to fewer identifications. In order to baseline-separate these two lipid species, a minimum of 60,000 resolving power (or higher) is required, easily achievable using high resolution Orbitrap technology.

# of

iden

tifie

d lip

ids

700

27%

25%

20%600

500

400

300

200

100

0100ng

300

382

Load

Lipids found or # of lipids

Lipids found or # of lipids

500ng

419

527

2500ng

522

608

Q Exactive Plus MS (top 15)

Q Exactive HF MS (top 20)

m/z 480.3 480.4

480.3463

480.3436

480.3444

480.3100

480.3085

480.3445

R=15000

R=30000

R=60000

R=120000

LPC 16:0p C24 H51 NO6 P

LPE 18:1 C23 H47 NO7 P

Lyso PE 18:1 (9Z)

Lyso PC 16p

BR64052-EN 08/16S

For Research Use Only. Not for use in diagnostic procedures. www.thermofisher.com©2016 Thermo Fisher Scientific Inc. All rights reserved. PepFinder incorporates MassAnalyzer software, copyright © 2012 by Amgen Inc. Protein Deconvolution incorporates ReSpect deconvolution algorithm, © Copyright 2013 by Positive Probability Ltd. SWATH is a registered trademark of AB SCIEX Pte. Ltd. LipidSearch is a trademark of Mitsui Knowledge Industry Co., LTD. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.

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