q exactive a true qual-quan hr/am mass spectrometer for ... 498.17874 ¢ , 758.45410 ¢ 1120.55273...
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b₅₊-NH₃
498.17874
y₆₊-H₂O
758.45410
y₉₊
1120.55273
y₂₊
322.18576
y₈₊
1006.51752
y₆₊
776.46594
y₁₀₊
1177.57886
y₁₊
175.11826
y₇₊
891.48932
y₅₊
647.42157
y₃₊
435.26990
y₄₊
548.35284
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m/z
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ity [co
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Extracted from: C:\Xcalibur\data\Zhiqi\ID_QE\20110321_10ngYeastDigest_Top10_140min_01.raw #23048 RT: 77.42 FTMS, HCD, z=+2, Mono m/z=645.83948 Da, MH+=1290.67168 Da, Match Tol.=20 mmu
Q Exactive – A True Qual-Quan HR/AM Mass Spectrometer for Routine Discovery and Target Quantification in Proteomics
Yi Zhang1, Zhiqi Hao1, Rosa Viner1, Shannon Eliuk1, Justin D. Blethrow1, Vlad Zabrouskov1, Markus Kellmann2 and Andreas F. Huhmer1
Thermo Fisher Scientific, San Jose, CA 95134, USA1; Thermo Fisher Scientific, Hanna-Kunath-Str. 11, 28199 Bremen, Germany2
Conclusion The Q Exactive is well suited for routine in-depth proteome analysis with its five
orders of magnitude dynamic range, fast high-resolution MS/MS scan rate of
12Hz, and high quality HR/AM HCD spectrum.
The Q Exactive generates accurate quantification data with high precision
(CV<15%) in TMT based quantitative discovery analyses.
The quadruple-based high-resolution SIM scans combined with the unique
spectrum multiplexing functionality enable high throughput HR/AM quantification
with high selectivity and sensitivity in target verification/quantification studies.
The Q Exactive is a true Qual-Quan mass spectrometer, which allows seamless
transition from discovery to target verification/quantification.
References1. Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, Dephoure N, O’Shea EK, Weissman JS.
Nature 2003,16, 737–7341.
AcknowledgementsWe would like to thank Professor Gygi from Harvard University for supplying the
purified yeast digest sample.
OverviewPurpose: The performance of a novel Q Exactive mass spectrometer was evaluated in
both routine peptide identification and targeted peptide quantification.
Methods: The peptide identification capability was investigated through in-depth
analysis of the yeast proteome. Discovery-based quantification accuracy and precision
were evaluated using TMT labeled E. coli digests. The sensitivity and linear dynamic
range for HR/AM targeted quantification were investigated with peptide standards
spiked into a complex yeast digest background.
Results: The Q Exactive identified twice as many peptides/proteins as the AB Sciex
TripleTOF 5600 in a well controlled head-to-head comparison. High precision
(CV<13%) was routinely achieved for TMT-based quantification experiments on the Q
Exactive. The quadrupole based SIM and ultra-high resolution of 140K resulted in
accurate and sensitive HR/AM targeted quantification. A LOD of 10 amol was obtained
on most peptide targets with low background.
FIGURE 1. Schematic of the Q Exactive
MethodsSamples: heavy isotope labeled peptide retention standards (Thermo Fisher Scientific, Cat #:
88321), yeast tryptic digest, TMT 6-plex labeled E. coli tryptic digest.
LC: Split-free EASY-nLC from Proxeon, Solvent A: 0.1% formic acid in H2O, Solvent B: 0.1%
formic acid in acetonitrile.
Head-to-Head Comparison of the Q Exactive against the AB SciexTripleTOF 5600 for
Peptide Identification: Yeast tryptic digests at 10 ng, 100 ng and 1000 ng were analyzed with
the same nano LC column (Dionex AcclaimPepMap100 C18, 75µm x 15cm, 3µm particle,
100Å pore size) and the same 60 min gradient on both the Q Exactive and TripleTOF 5600.
The instrument parameters on the TripleTOF 5600 were set at values recommended by AB
Sciex: 250 ms full scan with 30K resolution, top 20 MS/MS with 15K resolution and 50 ms
beam time, MS2 trigger threshold of 100 cps, dynamic exclusion of 30 s. On the Q Exactive,
the resolution was 70K for full scan and 35K for MS2, the AGC target was 1E6 for full scan and
1E5 for MS2, the maximum IT was 100 ms for both full scan and MS2 scan, top 10 HCD was
selected with MS2 trigger threshold of 5,000 and dynamic exclusion of 60s.
TMT Quantification Evaluation: E. coli tryptic digests were labeled with TMT 6-plex reagents
at a ratio of 1:1:1:1:1:1. Labeled peptides (20, 40, 80, 200, 500 ng) were separated over a
Michrom Magic C18 nano LC column (75µm x 20cm, 3µm particle) with a two-hour gradient,
analyzed with a data dependent top 10 HCD method on the Q Exactive. The resolution was
70K for full scan and 17.5K for MS2. The maximum IT was 250 ms for both full scan and MS2
scan, top 10 HCD was selected with MS2 trigger threshold of 1E5 and dynamic exclusion of
80s. The AGC targets were the same as above.
HR/AM Targeted Quantification: Heavy isotope labeled peptide retention standards at 0, 10
amol, 50 amol, 100 amol, 1fmol, 10 fmol, and 100 fmol were spiked into either 10 ng or 1000
ng of yeast tryptic digest. Each sample was analyzed three times with a 60 min LC gradient
over a Michrom Magic C18 nano LC column (75µm x 15cm, 3µm particle) and a full-msx tSIM
method on the Q Exactive. The resolution was set at 140k for both scan types. The AGC
target was 1E6 for full scan and 5E5 for SIM scan. The maximum ion injection time was 100
ms for full scan and 500 ms for SIM scan. The isolation width for SIM scan was 4 amu. The
peptide retention standards were monitored over a 4 min window. Multiplexing level was set at
4, which allowed isolation and accumulation of up to four peptide targets in the c-trap before
they were transferred to Orbitrap for detection (Figure 7)
Data Analysis Peptide identification and TMT quantification data were processed and
searched against their respective databases with Mascot using standard search parameter
settings in Proteome Discoverer 1.3. Targeted quantification using LC peak areas of heavy
peptide standards with ±5 ppm mass windows were automatically calculated for both full scan
and SIM scan using Quan browser in Xcalibur 2.2. Linear regression fittings in both normal
scale and log scale were performed to estimate LOD and LOQ.
Capillary
S-lens
(Improved Sensitivity)
Bent
Flatapole
Quadrupole Mass Filter
(dd HCD, SIM)
HCD/C-Trap Combo Cell
Spectrum Multiplexing
Orbitrap with
Enhance Resolution
Discovery- Protein Identification
A Head-to-Head Comparison against TripleTOF 5600
Same sample, Same column, Same gradient
Discovery-based HR/AM Qual/Quan
High Precision, High Accuracy TMT Quantification
HR/AM Target Verification/Quantification
High Selectivity, High Sensitivity, High Throughput
Q Exactive TripleTOF 5600
Most Intense Least Intense
10 ng load
TripleTOF 5600
1000 ng load
Q Exactive
Normalized Precursor Intensity of Identified Yeast Peptides
Figure 2: Q Exactive has 10x broader dynamic range of
identification
1000ng 100ng 10ng
Figure 3: Q Exactive identified 2x unique peptides and
proteins and 4x lower abundant proteins than the
TripleTOF due to its broader dynamic range, fast scan
rate, and high quality HCD spectra.
# o
f U
niq
ue
Pe
pti
de
s#
of
Pro
tein
Gro
up
s
# o
f P
rote
in G
rou
ps
Copy Number of Yeast Proteins1
149 copy/cell in 10 ng sample
MASCOT Score: 90
L G N D D E V I L F RL G N D D E V I L F R
High Quality HCD Spectrum identified with
the Q Exactive only
In-Depth Proteome Analysis
Figure 4: TMT 6-plex Quantitative Results on Q Exactive
Amount of TMT 6-plex labeled E. coli digest
A. Quantifiable MS2 spectra account for 97%
of the total identified MS2.
B. Measured ratios are within 5% of
expected values, with CV<13%.
Ratio
1.04 1.02 1.03 1.05 1.03
TMT channels for Quantification (80ng)
Figure 5: High resolution ensures accurate target selection
30 ppm
Figure 6: High sensitivity with quadrupole based SIM scan
Figure 7: Spectrum multiplexing and concurrent
injection/detection provides high throughput analyses
Orbitrap FTMS acquisition scan 2
A
G
COrbitrap FTMS acquisition scan 1
A
G
C
Collecting ions for scan 2 Collecting ions for scan 3
Figure 8: LOD of 10 amol or 100 amol is routinely
achieved with HR/AM targeted quantification in
medium or strong background, respectively
• Low amol detection
• CV < 10% at 50 amol
• 4 orders of linearity
Sample amount (fmol)
Pe
ak a
rea
SSAAPPPPPR*
GISNEGQNASIK*
DIPVPKPK*
R2: 0.9991-0.9999
Log
10(P
eak A
rea)
Log10(Sample Amount)
10amole 1fmole 100fmole
S/N: >40
Pe
ak a
rea
R2: 0.9997-1.0000
NGFILDGFPR*
GLILVGGYGTR*
Log
10(P
eak A
rea)
Log10(Sample Amount) 10amole 1fmole 100fmole
S/N: 2.5-6
• 100 amol - 1fmol detection
• 3-4 orders of linearity
Sample amount (fmol)
A. Sensitivity and linear dynamic range of peptide targets in 10 ng yeast digest
B. Sensitivity and linear dynamic range of peptide targets in 1000 ng yeast digest
Discovery- Protein Identification
High Dynamic Range, High Sensitivity, High Duty Cycle
Amount of Yeast Tryptic Digest Loaded
1000ng 100ng 10ng