Thermo Scientific Q Exactive HF
Orbitrap LC-MS/MS System
Higher-Quality Data,
Faster Than Ever
Speed • Productivity • Confidence
Transforming discovery quantitation
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.
Advanced Active Beam
Guide (AABG)
Quadrupole Mass Filter with
Advanced Quadrupole
Technology (AQT)
Ultra-High-Field Orbitrap (HF)
mass analyzer
HCD Cell
• 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
2
RF Lens
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.
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.
Q Exactive Plus MS, 120 min gradient
Q Exactive HF MS, 60 min gradient

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.
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.
100
31.88848
A) Q Exactive Plus MS
31.85865
80
31.82908
60
1.77 sec
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.
31.79956
20
0
100
31.77800
31.91221
B) Q Exactive HF MS
31.89353
80
31.87478
60
31.85611
31.83719
40
20
0
31.81816
31.78061
31.79957
31.78
31.80
31.82
1.13 sec
31.84
Time (min)
31.86
31.88
31.90
100
31.84339
31.84016
31.84259
80
20 MS/MS in 1.13 sec
Relative Abundance
„
Relative Abundance
40
31.84422
31.85078
60
31.83939
31.84504
40
20
0
31.84669
31.84181
31.84750
31.84585
31.84099
31.840
31.84831
31.85157
31.85404
31.84997
31.85240
31.844
31.848
31.852
Time (min)
3
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.
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.
Enabling quantitative accuracy
Achieving quantitative precision
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.
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.
Integrated Intensity
1E9
R² = 0.9994
1E8
4
1.5
1.5
1.0
1.0
0.5
0.5
1E7
1E6
0.0
98
1E5
1E4
0.005 0.01
0.05 0.1
0.5
1
5
Peptide Concentration (fmol)
10
50
100
Peptide
CV (0.05 fmol)
CV (0.01 fmol)
GISNEGQNASIK (2 amu)
2.19%
9.70%
GISNEGQNASIK (0.7 amu)
2.09%
2.03%

B
Intensity 10^6
R² = 0.9992
A
Intensity 10^6
1E10
2.0
2.0
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.

99
100
101
Retention Time
102
0.0
102.5
103.0
103.5
Retention Time
104.0
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.
A digital data archive – New levels of DIA capability
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.
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.
2.5
MS1
1.0
0.5
63.0
63.5
64.0
Retention Time
0.0
64.5
400
MS2
350
60
Intensity (10^3)
Intensity (10^3)
70
1.0
0.5
62.5
50
40
30
0
0
Replicate 3
60
50
40
30
10
MS2
0
61.3 61.8 62.3 62.9 63.4 63.9 64.4 65 65.5 66 66.5
100
90
5 ppm
80
150
50
70
20
200
10
64.5
62.0 62.5 63.0 63.5 64.0 64.5
Retention Time
250
100
63.0
63.5
64.0
Retention Time
80
300
20
62.5
90
50 ppm
1.5
Relative Intensity (2.510e+5)
80
100
Relative Intensity (3.404e+5)
1.5
0.0
MS1
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.
2.0
Intensity (10^6)
Intensity (10^6)
2.0
High quality data to address
complex samples
62.0 62.5 63.0 63.5 64.0 64.5
Retention Time
70
60
50
40
30
20
10
Replicate 4
0
61.3 61.8 62.3 62.9 63.4 63.9 64.4 65 65.5 66 66.5

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.

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.
5
Superior intact protein characterization
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.
A. Intact mAb Herceptin
90
4235.8
3801.5
3706.5
3530.0
80
3880
3223.2
4632.9
4782.3
4941.7
30
80
60
40
G0F/G0F
Relative Intensity
80
6
5000
5038.8
5020
5040
m/z
6000
1234.5
1379.7
1100.9 1205.6
1465.9
1077.5
1563.5
1055.1
1033.5
1675.2
1012.9
1803.9
993.1
1954.2
974.0
800
1000

ƒ
(G0F/G2F) / (G1F/G1F)
148058.141
148380.328
70
C. Deconvoluted spectrum
60
of intact mAb Herceptin
50
G1F/G2F
40
148542.734
30
0
5033.4
G0F/G1F
90
10
5020.1
5017.5
1303.0
100
90
80
70
60
30
50
20
40
10
0
1200
1400
m/z
1600
1800
2000
148219.719
100
20
5030.7
0
10
4000
m/z
5022.8
20
20
3000
3920
5028.2
5490.6
0
2000
3914.3
3900
100
4492.4
3154.6
40
3910.0
3893.3
20
m/z
3294.8
50
40
4360.4
Relative Abundance
60
Light chain
Heavy chain
60
0
3447.9
3369.6
70
3905.8
3897.2
80
Relative Abundance
100
Relative Abundance
Relative Abundance
4118.2
3901.5
B. Reduced mAb Herceptin
3901.5
100
G0/G0F
Man5/Man5
147911.359
G2F/G2F
147600.712
148704.212
148833.312
G0/G0
147485.712
147500
148000
Mass
148500
149000
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.
Increased productivity and confidence for lipidomics
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
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.
power of the Q Exactive HF system greatly reduces this
Q Exactive Plus MS (top 15)
Q Exactive HF MS (top 20)
ƒ
700
20%
# of identified lipids
600
25%
500
27%
400
300
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.
200
100
0
100ng
500ng
2500ng
Lipids found or # of lipids
300
419
522
Lipids found or # of lipids
382
527
608
Load
LPC 16:0p
C24 H51 NO6 P
480.3463
Lyso PE 18:1 (9Z)
480.3436
ƒ
R=15000
Lyso PC 16p
480.3444
R=30000
LPE 18:1
C23 H47 NO7 P
480.3445
480.3100
R=60000
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.
480.3085
R=120000
480.3
m/z
480.4
7
Accelerating productivity with
software solutions
Proteome Discoverer Software
Thermo Scientific™ Proteome Discoverer™ 1.4 software is a comprehensive and flexible data analysis
platform for qualitative and quantitative proteomics research, offering a wide array of bio-software
tools and customizable workflows for every proteomics experiment.
Pinpoint Software
Thermo Scientific™ Pinpoint™ 1.4 software is a targeted proteomics quantitation software supporting the
analysis of proteins, peptides, and now glycoproteins. Automated workflows facilitate all experimental
aspects, from method development and acquisition to data verification and reporting.
PepFinder Software
Thermo Scientific™ PepFinder™ 1.0 software is a new peptide mapping tool for the characterization
of biotherapeutic proteins, including relative quantitation of PTM’s and amino acid substitutions,
and location of disulfide bonds. A novel fragmentation prediction and scoring provides fast,
comprehensive and confident assignments.
Protein Deconvolution Software
Thermo Scientific Protein Deconvolution 3.0 software provides accurate mass measurement
and isoform quantification for intact proteins such as biotherapeutics. Now includes enhanced
deconvolution algorithms, and mirror plots for comparison of samples.
LipidSearch Software
Thermo Scientific LipidSearch software processes high-resolution accurate-mass data, to provide
accurate lipid identification. It automatically integrates complex data into reports and dramatically
reduces data analysis time.
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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