Performance Evaluation of the Q Exactive HF Hybrid Quadrupole-Orbitrap
Mass Spectrometer for High-Throughput Top-Down Proteomics
Eugen Damoc1, Ping Yip2, Leena Valmu3, Alexander Cherkassky2, Bernard Delanghe1, Eduard Denisov1, Helene Cardasis2,
Jason Neil2, Alexander Makarov1, Jim Stephenson2
1Thermo Fisher Scientific, Bremen, Germany; 2Thermo Fisher Scientific, Cambridge, MA, USA; 3Thermo Fisher Scientific,
Vantaa, Finland
Overview
Purpose: Evaluation of the Thermo Scientific™ Q
Exactive™ HF hybrid quadrupole-Orbitrap mass
spectrometer for high-throughput top-down proteomics.
Methods: Top-down analysis of an Escherichia coli extract
using the data dependent “TopN” method with and without
chromatographic separation.
Results: We demonstrate utility and applicability of the Q
Exactive HF mass spectrometer to perform highthroughput top-down proteome analysis.
Introduction
Figure 3 is retrieved from a second experiment, where an
MS/MS scan with higher-energy collisional dissociation
(HCD) of the charge state 34+ at a collision energy of 20
eV was performed. The AGC target value was 1e6 at a
resolving power setting of 120,000 (FWHM at m/z 200)
with 4 µscans in 1 second acquisition time. 36 b-type and
28 y-type fragment ions were identified using ProSightPC
3.0 software.
02/13/14 08:50:0 8
Relative Abundance
CAII_new_FullMS_512ms_2uscan #13-14 RT: 0.22-0.24 AV: 2 NL: 1.30E6
T: FTMS + p ESI Full ms [600.00-2000.00]
908.02685
854.64358 R=106098
968.49527
R=107344
z=32
785.4029 7
R=102251 1037.60117
100
z=34
R=112707
z=30
R=104386 1117.30146
z=37
R=100247
z=28
708.94888
z=26
R=120449
50
z=43
1210.32591
R=97036
z=24
LC Gradient
Total
Proteoforms
1320.30795
R=8995 8
z=22
0
600
700
800
900
1000
m/z
1100
1200
1300
5 min
15 min
30 min
60 min
722
996
1395
1964
1400
B
Relative Abundance
CAII_new_FullMS_512ms_2uscan #13-14 RT: 0.22-0.24 AV: 2 NL: 1.30E6
T: FTMS + p ESI Full ms [600.00-2000.00]
908.0 2685
937.2857 7
854.64358 880.45134
R=106098
R=106687
807.16403 830.2 5366 R=107344 R=108256
968.49527
1001.78765
z=32
z=31
z=33
R=102251
100 785.40297 R=113896 R=105699
z=34
R=104716
103 7.60117
R=112707
z=35
z=36
z=30
z=29
R=104386
z=37
z=28
845.86465 860.37466 893.61247
50
920.83455
R=1092 32 R=108093 R=121506
9 51.01686
1013.53716
981.80336
R=128747
z=?
R=114021
R=116553
R=117881
z=1
z=?
z=?
z=?
z=?
z=?
0
800
850
900
950
1000
1050
m/z
Relative Abundance
CAII_new_FullMS_512ms_2uscan #13-14 RT: 0.22-0.24 AV: 2 NL: 1.30E6
T: FTMS + p ESI Full ms [600.00-2000.00]
90 8.02685
907.93401
R=106098 908.12036
R=106073
R=109360
z=32
100
z=32
z=32
907.83958
908.24597
R=99357
R=103997
50 907.4969 8
z=3
2
9 07.68229
908.337 56
R=104826
z=32
R=110185
R=107714
z=1
z=32
z=32
0
907.5
907.6
907.7
907.8
907.9
908.0
908.1
908.2
908.3
908.4
m/z
908.49585
R=99319
z=1
908.5
908.65183
R=106399
z=1
908.6
FIGURE 3. Top: HCD fragmentation spectrum of
carbonic anhydrase II (4 µscans @ 120k res. pwr.
acq. time: 1 second). Bottom: Deconvoluted HCD
spectrum and ProSightPC results.
NPQV_CAII_HCD_854_120k_4us_fm235_1e6_20eV #15 RT: 0.26 AV: 1 NL: 1.18E6
T: FTMS + p ESI Full ms2 [email protected] [235.00-2000.00]
1007.41455
740.92365
R=62306
R=73506
z=7
z=4
100
FIGURE 6. Example of top-down protein identification
using ProSightPC: Glutamine-binding periplasmic
protein (MW: 24.9 kDa)
Relative Abundance
Base Peak Chromatogram
592.93958
R=80806
z=5
80
836.80676 923.84979
R=64606
R=67106
z=5
z=18
60
40
20
Methods
337.18826
539.28418
R=108306
R=83206
z=1
z=1
436.22073
R=85502
z=?
1086.88464 1177.88574
R=59406
R=49902
z=7
z=4
667.51404
R=75306
z=5
1267.86475
R=52506 1362.40320
R=50606
z=6
z=3
Full MS (5 µscans)
1548.16895
R=43306
z=3
0
300
HyperQuad Mass Filter with Advanced
Quadrupole Technology (AQT)
Advanced Active
Beam Guide
(AABG)
600
700
800
900
m/z
1000
1100
1200
1300
1400
1500
1600
Deconvoluted Full MS
90
80
7040.84443
70
HCD (3 µscans)
60
50
7597.10751
40
2506.39649 3642.68365
4612.20330
20 538.27690
30
10
14964.25442
10352.15006
6305.42165
8748.66680
0
2000
4000
6000
8000
m/z
12375.07862
10000
12000
14000
The increased performance in high-throughput top-down
proteomics experiments was evaluated using a complex
E. coli protein extract. Intact proteins from E. coli were
analyzed by direct static nanospray or LC-MS/MS. Without
chromatographic separation, 66 unique proteoforms
(Figure 4), could be unambiguously identified in less than
2 minutes acquisition time by using a “Top-N” “high-high”
method. ProSightPC analysis results are shown in Table 1.
In this case, an E. coli sample solution of 1 µg/µl was
directly infused at a flow rate of about 140 nl/min.
Furthermore, with chromatographic separation, we
demonstrate that the number of proteoforms identified
grow linearly with LC gradient duration (Figures 5a and
5b). For 5, 15, 30, and 60 min gradients we were able to
identify 722, 996, 1395, and 1964 proteoforms,
respectively. ProSightPC analysis was carried out for each
LC data set. Figure 6 shows top-down identification of
glutamine-binding periplasmic protein from the E. coli
extract separated by using a 5 min LC gradient.
RF Lens
Deconvoluted Full MS spectrum
66 Proteoforms
T: FTMS + p NSI Full ms [500.00-2000.00]
Ultra High Field Orbitrap
Mass Analyzer
795.5246
R=67806
z=12
100
90
Results
With the implementation of the compact ultra-high field
Thermo Scientific™ Orbitrap™ analyzer on the Q
Exactive HF instrument (see Figure 1), the resolving
power has been increased by 1.8 fold over that of the
previous Orbitrap detector. This enables high-resolution
analysis at high detection speed which makes the HF
instrument more suitable for top-down analysis at LC time
scale. The novel Intact Protein Mode allows adjustment of
the trapping gas pressure and optimizes the control logic
of the instrument to analyze intact proteins with masses
up to 50 kDa with isotopic resolution. Carbonic anhydrase
II with a molecular mass of 29 kDa was used to evaluate
the ability of the Q Exactive HF instrument to perform topdown analysis. Figure 2 shows results of an experiment,
where full MS scans were recorded at a resolving power
setting of 240,000 (FWHM at m/z 200) and AGC target
value of 3e6. The figure shows an averaged spectrum
over 2 seconds, where the isotopes are baseline resolved
and the charge states are properly assigned.
Relative Abundance
70
50
1004.0665
R=58906
z=15
626.3673
R=76406
z=10
30
1406.8544
R=45406
z=9
10
80
50
60
1167.1721
R=100302
30
1166.8297
R=89622
20
20
10
NL: 8.02E5
m/z=
1167.79-1167.80 MS
intact_enolase_20plex
_tsim_lower_z_1ms_1
41218145947
15.60
1167.80
1168.0944
R=92347
60
40
04/20/15 12:28:52
1167.9700
R=108154
1167.5973
R=88312
100
1168.2201
R=96502 1168.6950
R=98049
40
1168.9955
R=93236
15.07
1167.80
0
0
1167.0
6
1167.5
1168.0
m/z
8
1168.5
10
12
1169.0
15.96
18.19
1167.80 1167.79
14
16
Time (min)
18
20
22
24
intact_enolase_20plex_tsim_lower_z_1ms_141218145947 #175 RT: 15.60 AV: 1 NL: 1.58E6
T: FTMS + p NSI SIM msx ms [1413.83-1416.83, 1457.90-1460.90, 1504.98-1507.98, 1555.27-1558.27, 1608.84-1611.84, 1372.24-1375.24, 1332.96-1335.9 ...
1139.3145
R=95304
100
1086.3466
1197.6643
R=104600
R=110104 1262.2942
1373.6721
1015.5188
80
R=101104
R=99900 1459.4631 1506.5414
R=90600
60
1610.1993
R=90700
R=93700
R=95500 1667.8857
40
R=80504
1712.4989
20
R=87900
0
1000
1100
1200
1300
1400
1500
1600
1700
m/z
1000
46642.4072
100
80
60
40
20
0
1200
m/z
1400
40000
41000
42000
43000
m/z
44000
45000
46000
Conclusion
1672.8867
1887.3022
R=38402
R=38900
z=?
z=?
0
800
70
39000
1186.2389
R=53106
z=13
20
600
90
80
intact_enolase_20plex_tsim_lower_z_1ms_141218145947_XT_00001_M_ #2 RT: 2.00 AV: 1 NL: 2.24E6
T: FTMS + p NSI SIM msx ms [1413.83-1416.83, 1457.90-1460.90, 1504.98-1507.98, 1555.27-1558.27, 1608.84-1611.84, 1372.24-1375.24, 1332.96-1335.9 ...
954.4283
R=60906
z=10
60
40
1167.7208
R=102609
intact_enolase_20plex_tsim_lower_z_1m...
100
RT: 4.89 - 25.14
Relative Abundance
734.4077
R=69606
z=13
80
FIGURE 7. Multiplex SIM spectrum of 20 consecutive
charge states of intact enolase (10 µscans @ 240k res.
pwr.
acq. time: ~ 5 seconds).
Relative Abundance
FIGURE 4. Full MS spectrum of the purified E. coli
sample, obtained by averaging eighty microscans in
direct static nanospray mode.
Most of the proteins identified using “Top-N” “high-high”
method have molecular weights < 35 kDa, which is why a
multiplex SIM approach was tested to see whether the
mass range of isotopically resolved proteins can be
extended beyond this limit. With this approach, different
charge states of a protein can be selected using the
quadrupole, then trapped in the HCD cell, and detected all
together with the Orbitrap analyzer. Using intact enolase
we could demonstrate that proteins up to about 50 kDa
can be analyzed with isotopic resolution at LC time scale
(see Figure 7).
Relative Abun d ance
C-Trap
500
Relative Abundance
FIGURE 1. The Q Exactive HF instrument layout.
400
NPQV_CAII_HCD_854_120k_4us_fm235_1e6_20eV_XT_00001_M_ #2 RT: 2.00 AV: 1 NL: 2.26E6
T: FTMS + p ESI Full ms2 [email protected] [235.00-2000.00]
2958.65930
100
Relative Abundance
Direct infusion experiments using intact carbonic
anhydrase II were carried out to evaluate the ability of the
Q Exactive HF instrument to perform top-down analysis.
Also, top-down microbial proteome analysis was
performed by LC-MS/MS or direct static nanospray
utilizing an E. coli extract. 1–2 µg of protein sample was
loaded onto a Thermo Scientific™ PepSwift™ Monolithic
PS-DVB (200 µm × 25 cm) EASY-Spray™ column, and
four different LC gradients (5, 15, 30, and 60 min) were
run on a Thermo Scientific™ EASY-nLC™ 1000 system. A
data-dependent “Top-N” method using the “high-high”
approach was employed to deliver high resolution and
high mass accuracy in both MS and MS/MS modes, using
the Q Exactive HF hybrid quadrupole-Orbitrap mass
spectrometer. Proteoforms were identified using a new
charge assignment and protein deconvolution algorithm.
Furthermore, the high-throughput top-down proteomics
data was analyzed using Thermo Scientific™ ProSightPC
3.0 software. Multiplexed SIM experiments were
performed using LC/MS analysis of intact enolase.
HCD Cell
A
FIGURE 2. Full-MS spectrum of intact carbonic
anhydrase II (2 × 2 µscans @ 240k res. pwr.
acq.
time: 2 seconds) with baseline resolution of the
isotopic pattern.
CAII_new_FullMS_512ms_2uscan
Major goals in every top-down proteomics experiment are
protein identification and characterization. The strategy
used to achieve these goals involves high-resolution mass
measurement of intact protein ions followed by their
fragmentation and analysis in the mass spectrometer. In
spite of enormous improvements in terms of speed and
sensitivity in FTMS instrumentation over the last few
years, top-down LC-MS/MS in large scale proteome
analyses will further benefit if high resolution analysis at
higher detection speed would be possible. Furthermore,
improvement to the current generation of charge
assignment and protein deconvolution algorithms to
handle complex top-down data will lead to more efficient,
complete, and accurate protein identification. Here we
demonstrate the improved performance of the Q Exactive
HF hybrid quadrupole-Orbitrap mass spectrometer in a
series of high-throughput top-down proteomics
experiments in conjunction with a new algorithm for
charge assignment and protein deconvolution.
Furthermore, a multiplex SIM approach to isotopically
resolve multiple charge states of proteins up to 50 kDa at
LC timescale is presented.
FIGURE 5. Proteoform MW distributions (A) and
cumulative distributions (B) for 5, 15, 30, and 60 min
LC gradients.
1600
1800
2000
TABLE 1. List of top 30 proteins by E-value identified
in the C4 purified E. coli sample using the direct static
nanospray and data dependent “TopN” method.
Q Exactive HF mass spectrometer with its Intact Protein
Mode and 1.8 fold increase in resolving power enables
high-res analysis at high detection speed which makes it
more suitable for high throughput top-down analysis.
Aided by a new charge assignment/deconvolution
algorithm, Q Exactive HF MS provides significant
proteoform and protein coverage, even from a single
direct infusion spectra.
A multiplex SIM approach allows analysis of intact
proteins up to about
50 kDa with isotopic resolution at LC timescale.
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