Q Exactive - A True Qual-Quan HR/AM Mass Spectrometer for ......Q Exactive – A True Qual-Quan...
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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 M. 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, Germany
OverviewPurpose: The performance of a novel Q Exactive mass spectrometer was evaluated inboth routine peptides identification and targeted peptide quantification.
Methods: Peptide identification capability was investigated through in-depth analysis ofyeast proteome. Quantitation accuracy and precision was evaluated using TMT labeledE coli digest. HR/AM targeted quantitation was investigated with peptides standard incomplex yeast digest background.
Results: Q Exactive identified twice as many peptides/proteins as Triple ToF 5600 in awell controlled head-to-head comparison. High precision (CV<13%) was routinelyachieved on TMT based quantitation. The quadrupole based SIM and ultra-highresolution of 140K results in accurate and sensitive HR/AM targeted quantitation. ALOD of 10amol was obtained on most peptide targets with low background.
IntroductionMass Spectrometry has become an essential tool in obtaining global understanding ofbiological systems, such as proteome dynamics and signaling regulation.1 Whencoupled with multi-dimensional LC, modern mass spectrometers, such as the LTQOrbitrap Velos, are capable of identifying and quantifying thousands of proteins andpost-translation modifications on a routine bases, and play an increasingly importantrole in early discovery phase. Relative quantification through incorporation of heavyisotope, such as SILAC2 and TMT3, enables the identification and quantification ofseveral hundred potential protein candidates, including many novel ones, from acomplex biological system. Targeted MS approach, in particular multiple reactionmonitoring (MRM) on a triple stage quadrupole mass spectrometer, has become thepreferred platform for quantitatively analyzing tens to hundreds of peptidecandidates.4,5 However, the transition from discovery to target verification andquantification is still slow and cumbersome, mainly due to the dramatic differences
Discovery- Protein Identification
Comparison Study Using Q Exactive and TripleToF 5600
Same sample, Same LC, Same gradient
Quantitative Discovery
High Precision, High Accuracy TMT Quantitation
HR/AM Target Verification/Quantification
High Selectivity, High Sensitivity, High Throughput
492.2665
492.2495
492.2634
R: 35K R: 140K
m/z
492.2661
492.2511
R: 70K
1.0E-61.0E-51.0E-41.0E-31.0E-21.0E-11.0E+0
Most Abundant Least Abundant
10 ng load
TripleTOF 5600
1000 ng load
Q Exactive
Normalized Precursor Intensity of Identified Peptides
Figure 2: Q Exactive has 10x broader dynamic range. Figure 4: TMT-6plex Quantitative Result on Q Exactive.
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Amount of TMT 6-plex labeled E coli digest
A. Quantifiable MS2 spectra account for 97%
of total identified MS2.
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observed expected
B. Measured ratios are within 5% of
expected values with CV<13%.
Rat
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TMT channels for Quantitation (80ng)
Figure 5: High resolution ensures accurate target selection
30ppm
Figure 6: High sensitivity with quadrupole based SIM scan
558.3256Scan #: 5809Full Scan: (300-1000amu)100
Figure 8: LOD of 10amole or 100amol is routinely
achieved with HR/AM targeted quantitation in medium or strong background, respectively.
0E+0
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2E+9
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• Low amole detection• CV < 10% at 50amole
• 4 orders of linearity
Sample amount (fmole)
Pea
k ar
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SSAAPPPPPR*
GISNEGQNASIK*
DIPVPKPK*
R2: 0.9991-0.9999
Log 1
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Log10(Sample Amount) 10amole 1fmole 100fmole
S/N: >40
5E+8
6E+8
(Pea
k A
rea)
S/N: 2.5-6
• 50 amole-1fmole detection• 3-4 orders of linearity
A. Sensitivity and Linear dynamic range of peptide targets in 10ng yeast digest
B. Sensitivity and Linear dynamic range of peptide targets in 1000ng yeast digest
Conclusion� Q Exactive is well suited for routine in-depth proteome analysis with its five order
dynamic range, fast scan rate of 12Hz, and high quality HR/AM HCD spectrum.
� Q Exactive generates accurate quantitation with high precision (CV<15%) in TMT based quantitative discovery analysis.
� Quadruple-based high-resolution SIM scan and unique spectrum multiplexing function enable HR/AM quantitation with high selectivity, high sensitivity and high throughput in target verification/quantification study.
� Q Exactive is a true Qual-Quan mass spectrometer, which allows seem-less transition from discovery to target verification/quantification.
References1. Aebersold R, Mann M. Nature, 2003, 422,198–207.2. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M. Mol Cell Proteomics,
2002, 1, 376-386.3. Dayon L, Hainard A, Licker V, Turck N, Kuhn K, Hochstrasser DF, Burkhard PR, Sanchez JC. Anal Chem.
2008, 80, 2921-2931.4. Addona TA et. al. Nat Biotechnol. 2009, 27, 633-641.5. Makawita S, Diamandis EP, Clin Chem. 2010, 56, 212-222. 6. 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.
quantification is still slow and cumbersome, mainly due to the dramatic differencesbetween mass spectrometer platforms.
Here we introduce a newly developed Thermo Scientific Q Exactive, a Bench TopQuadrupole-Orbitrap Mass Spectrometer (Figure 1), which enables the seamlesstransition from discovery to target verification on a single platform. The incorporation ofa quadrupole mass filter affords precursor ion selection, thus data-dependent MS/MS.Advanced signal processing algorithm results in ultra-high resolution of 140,000 andhigh scan speed up to 12 Hz. In-depth analysis of complex proteome is achievedroutinely due to high dynamic range of detection and high quality HCD spectrum.Peptide candidates in the hundreds can be quantified immediately using HR/AMtargeted quantitation approach. The quadrupole based high resolution SIM scanensures accurate target selection and high sensitivity. Multiplexing SIM and concurrention injection and detection greatly improve duty cycle without sacrificing sensitivity andresolution.
FIGURE 1. Schematics of Q Exactive
MethodsSamples: heavy isotope labeled peptide retention standards (ThermoFisher Scientific, Cat #:88321), yeast tryptic digest, C elegans tryptic digest, TMT-6plex 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 against Triple ToF 5600 on Peptide Identification: Yeasttryptic digests at 10ng, 100ng and 1000ng were analyzed with the same nano LC column(Dionex AcclaimPepMap100 C18, 75µmx15cm, 3µm particle, 100Å pore size ), same 60 mingradient using a on both Q Exactive and Triple ToF 5600. The instrument parameters on TripleToF 5600 were set at values recommended by AB Sciex: 250ms full scan with 35K resolution,Top 20 MS/MS at resolution of 15K, MS2 trigger threshold of 50, dynamic exclusion of 60s. OnQ Exactive, the resolution was 70K for full scan and 35K for MS2, the AGC target was 1E6 forfull scan and 1E5 for MS2, the maximum IT was 100ms for both full scan and MS2 scan, top10 HCD was selected with MS2 trigger threshold of 5,000 and dynamic exclusion of 60s.
TMT Quantitation Evaluation: E coli tryptic peptides were labeled with TMT-6plex reagent atratio of 1:1:1:1:1:1. Labeled peptides of 500ng were separated over a Michrom Magic C18nano LC column (75µm x 20cm, 3µm C18 partical) with a two-hour gradient, analyzed withdata dependent top 10 HCD method on Q Exactive. The resolution was 70K for full scan and17.5K for MS2. The maximum IT was 250ms for both full scan and MS2 scan, Top 10 HCD wasselected with MS2 trigger threshold of 1E5 and dynamic exclusion of 80s. The AGC targetswere the same as above.
HR/AM Targeted Quantification: Heavy isotope labeled peptide retention standards at 0,10amole, 50amole, 100amol, 1fmole, 10fmole, 100fmole were spiked into either 10ng or1000ng of yeast tryptic digest. Each sample was analyzed three times with 60 min LC gradientover a Michrom Magic C18 nano LC column (75µm x 15cm, 3µm C18 partical) and a full-msxtSIM method on Q Exactive. The resolution was set at 140k for both scan types. The AGCtarget was 1E6 for full scan and 5E5 for SIM scan. The maximum ion injection time was100msfor full scan and 500ms for SIM scan. The isolation width for SIM scan was 4amu. Thepeptides rentention standards were monitored over a 4 min window. Multiplexing level was setat 4.
Data Analysis Peptide identification and TMT quantification data were processed andsearched against the proper databases with Mascot using standard search parameter settingsin Proteome Discoverer 1.3. LC peak areas of heavy peptide standards with ±5ppm masswindow were automatically calculated for both full scan and SIM scan using Quan browser inXcalibur 2.2. Linear regression fittings in both normal scale and log scale were performed toestimate LOD and LOQ.
Capillary
S-lens(Improved Sensitivity)
Bent Flatapole
Quadrupole Mass Filter(dd HCD, SIM)
HCD/C-Trap Combo CellSpectrum Multiplexing
Orbitrap withEnhance Resolution
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Q Exactive TripleToF 5600
1000ng 100ng 10ng 1000ng 100ng 10ng
Figure 3: Q Exactive identified 2x unique peptides and
proteins, 4x lower abundant proteins due to its broader dynamic range, fast scan rate and high quality HCD.
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Copy Number of Yeast Proteins6
Amount of Yeast Tryptic Digest Loaded Amount of Yeast Tryptic Digest Loaded
149 copy/cell in 10 ng sample
MASCOT Score: 90
b₅⁺ -NH₃
498.17874
y₆⁺-H₂O
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y₉⁺
1120.55273
y₂⁺
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y₈⁺
1006.51752
y₆⁺
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y₁₀ ⁺
1177.57886
y₁⁺
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y₇⁺
891.48932
y₅⁺
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y₃⁺
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y₄⁺
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m/z
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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 In-Depth Proteome Analysis
558.4 558.8
558.3256(S/N≈1)Scan #: 5809
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m/z
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558.3261(S/N=48) 558.8287
(S/N=22)
Figure 7: Spectrum multiplexing and concurrent
injection/detection results in high throughput
Orbitrap FTMS acquisition scan 2
AGC
Orbitrap FTMS acquisition scan 1
AGC
Collecting ions for scan 2 Collecting ions for scan 3
0E+0
1E+8
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Pea
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R2: 0.9997-1.0000
NGFILDGFPR*
GLILVGGYGTR*
Log 1
0(P
eak
Are
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Log10(Sample Amount) 10amole 1fmole 100fmole
Sample amount (fmole)