1 2 1 MSMS Func 1 2 1 - Waters Corporation...28.00 29.00 30.00 % 0 100 100amol Mix1 HDC ON Time...
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TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2013 Waters Corporation WIDEBANDENHANCEMENTCALIBRATION1.raw : 1 INTRODUCTION The average duty cycle of Time of Flight analysers is dependant on the m/z data acquisition range but the integration of travelling wave ion mobility separation (TWIMS) devices in QToF geometries can afford significant duty cycle improvements. When Ion Mobility is utilised in the Synapt G2-Si Mass Spectrometer (Figure 1), the instrument can be operated in Wideband Enhancement (WE) mode (Figure 2), where the pusher is synchronised with the pusher delay time, this being related to the drift time of species passing through the TWIMS. The Mass Spectrometer can be programmed to optimise this parameter for a specific charge state over the entire mass range. The Wideband Enhancement mode of operation can give five to ten times increase in sensitivity over the entire mass range for the chosen charge state, resulting from a duty cycle improvement from an average of 15% to approximately 89%. In this poster, we describe the use of Wideband Enhancement mode applied to peptide fragment ions in both non-targeted and targeted proteomics experiments. The increase in sensitivity leads to significant improvements in MSMS data quality and hence protein identification rates. In addition, the increased sensitivity extends the limit of detection in ToF MRM experiments. TRAVELLING WAVE ION MOBILITY ASSISTED DUTY CYCLE ENHANCMENTS FOR NON TARGETED AND TARGETED PROTEOMICS EXPERIMENTS Matthew Kennedy 1 , Chris Hughes 1 ,Johannes PC Vissers 1 , Keith Richardson 1 , Jason Wildgoose 1 , Arkadiusz Grzyb 2 and James I Langridge 1 1 Waters Corporation, Manchester, United Kingdom, 2 Inquiry Software, Bialogard, Poland METHODS Samples MPDS E. Coli and Mixture 1 (equimolar protein digest of ADH, Enolase, Phosphorylase B and BSA; Waters Corporation). Liquid Chromatography NanoAcquity (Waters Corporation) configured with Trap and Analytical columns. Trapping Column: 5μm Symmetry C18, 180μm x 20mm. Analytical Column: 1.85μm HSST3, 75μm x 250mm. Solvent A: 0.1% aqueous formic acid. Solvent B: 0.1% formic acid in acetonitrile. Trapping Phase: 5μL/min for 3min. Analytical Phase: 300nL/min gradient from 1% to 40% B in 30 or 120min (depending on sample complexity) followed by a column wash and re-equilibration. Mass Spectrometry Synapt G2-Si (Waters Corporation) Resolution: >20k FWHM Calibration: NaI/CsI mixture to 2000Da Wideband Enhancement Calibration: Infusion and fragmentation of Glu[1]-Fibrinopeptide B (GFP). IMS Data Directed Acquisition Mode: Survey 0.2s, MSMS 0.1s, Max 15 components selected for MSMS in each survey scan. Quad window set to transmit 1Da window. Lock Mass sampled every 60s. IMS MRM Mode: Dwell 0.1s for each analyte. Data Processing Wideband Enhancement Calibration: Driftscope DDA: PLGS 3.1 (Waters Corporation) and Mascot Distiller (Matrix Science). CONCLUSION Ion mobility enabled Q ToF operated in Wideband Enhancement mode improves the duty cycle of the instrument to approximately 89%. The increase in signal intensity thus afforded by this duty cycle improvement leads to an increase in confident protein identifications in non targeted experiments. Good overlap of HD-DDA protein identifications is found when compared to equivalent loads of ToF-DDA and HDMS E experiments. The use of the Wideband Enhancement mode in ToF MRM experiments extends the limit of detection. Acknowledgements The authors wish to acknowledge the useful discussions with Dominic Helm and Bernhard Kuster from Technical University Munich. We also wish to acknowledge the assistance in acquiring the data from Joanne Connolly and Ingvar Betner of Waters Corporation. Figure 4. Effect of enabling Wideband Enhancement on the intensity of ions produced during the Trap fragmentation of infused GFP, 30s of combined data. Vertical axes are linked. Figure 6. PLGS search result from 500ng E.Coli digest. A total of 957 proteins and 5316 peptides were identified. Figure 5. Effect of Wideband Enhancement from the untargeted DDA analysis of 500ng E.Coli digest. The combined TIC for each MSMS function shows significant increase for WE ON (RED) compared to WE OFF (BLUE). Inset is an MS2 spectrum from function 16, the least intense precursor ion selected for MS/MS at any given time. Figure 7. Venn intersection of protein identifications from the injections of 500ng E.Coli in three acquisition modes — HD-DDA, ToF only DDA and HDMS E .DDA data were searched with Mascot and HDMS E with PLGS. RESULTS Figure 1. Schematic of the Synapt G2-Si Ion Mobility Enabled Mass Spectrometer. Ion Mobility Separation is performed in the TriWave region of the instrument. Figure 9. Dilution series for the separation by a 30minute gradient of MPDS Mixture1 in E. Coli background matrix. The limit of detection was at least 5amol for the IGDYAGIK and EALDFFAR peptides, and higher for the less intense LVNELTEFAK peptide. OVERVIEW ToF analyser ‘Wideband Enhancement’ mode gives sensitivity increases of 5— 10 fold. Spectral data quality improvement in Ion Mobility Data Directed Analysis experiments leads to increased protein / peptide identification rates. Sensitivity increase extends the limit of detection in ToF MRM experiments. Figure 2a. Wideband Enhancement mode step 1. Ions (represented by the red and blue lines) leave the Trap T-Wave device and enter the Ion Mobility Separator, and are separated according to mass, shape and size. Figure 2b. Wideband Enhancement mode step 2. The Ions have a transit time through the Transfer T-Wave and optics towards the pusher region, whilst maintaining separation. Figure 2c. Wideband Enhancement mode step 3. Each of the ions are then subject to a synchronised pulse, leading to a duty cycle of approximately 89%, compared to around 15% in the deactivated case. Figure 3. Wideband Enhancement Calibration. M/z vs. drift time of data acquired from partial Trap fragmentation of GFP shows distinct bands of singly and multiply charged species. The singly charged band was selected for calibration. 1+ 2+, 3+ m/z 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 % 0 100 m/z 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 % 0 100 684.3 480.2 333.2 175.1 84.0 120.1 246.1 240.1 337.1 382.2 627.3 481.2 813.4 685.3 686.3 942.4 814.4 924.4 1056.4 943.4 1171.5 1057.4 1285.5 684.3 333.2 480.2 787.8 813.3 Wideband Enhancement OFF Wideband Enhancement ON Figure 8. Use of Wideband Enhancement in Targeted ToF MRM experiments, 100amol MPDS Mixture 1 separated by a 30min gradient. Shown are the extracted product ion chromatograms from peptides IGDYAGIK, LVNELTEFAK and EALDFFAR for WE ON (RED) and WE OFF (BLUE). Data are normalized to WE ON. LVNELTEFAK IGDYAGIK Time 28.00 29.00 30.00 % 0 100 Time 22.00 23.00 24.00 % 0 100 Time 30.00 31.00 32.00 33.00 % 0 100 Pusher Transfer T-WAVE Ion Guide Ion Mobility Separator Pusher Transfer T-WAVE Ion Guide Ion Mobility Separator Synchronised Pulse Synchronised Pulse m/z % m/z % EALDFFAR MSMS Func m/z 100 200 300 400 500 600 700 800 900 1000 1100 1200 % 0 100 230413_003 755 (56.795) 16: TOF MSMS 705.84ES+ 5.28e3 1035.5 136.1 86.1 851.4 249.2 137.1 736.3 608.3 277.1 567.8 447.2 376.2 349.2 551.3 705.8 609.3 737.4 964.5 852.4 853.4 965.4 966.4 1134.6 1036.5 1037.5 1135.5 1136.6 606 30 244 54 125 29 123 HD DDA HDMS E ToF DDA 606 30 244 54 125 29 123 606 30 244 54 125 29 123 HD DDA HDMS E ToF DDA 1.5 2 2.5 3 3.5 4 4.5 5 5.5 0.5 1 1.5 2 2.5 3 3.5 Log (amol on column) Log (Response) EALDFFAR IGDYAGIK LVNELTEFAK
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INTRODUCTION
The average duty cycle of Time of Flight analysers is dependant on the m/z data acquisition range but the integration of travelling wave ion mobility separation (TWIMS) devices in QToF geometries can afford significant duty cycle improvements.
When Ion Mobility is utilised in the Synapt G2-Si Mass Spectrometer (Figure 1), the instrument can be operated in Wideband Enhancement (WE) mode (Figure 2), where the pusher is synchronised with the pusher delay time, this being related to the drift time of species passing through the TWIMS.
The Mass Spectrometer can be programmed to optimise this parameter for a specific charge state over the entire mass range. The Wideband Enhancement mode of operation can give five to ten times increase in sensitivity over the entire mass range for the chosen charge state, resulting from a duty cycle improvement from an average of 15% to approximately 89%.
In this poster, we describe the use of Wideband Enhancement mode applied to peptide fragment ions in both non-targeted and targeted proteomics experiments. The increase in sensitivity leads to significant improvements in MSMS data quality and hence protein identification rates. In addition, the increased sensitivity extends the limit of detection in ToF MRM experiments.
TRAVELLING WAVE ION MOBILITY ASSISTED DUTY CYCLE ENHANCMENTS FOR NON TARGETED AND TARGETED PROTEOMICS EXPERIMENTS
Matthew Kennedy1, Chris Hughes1,Johannes PC Vissers1, Keith Richardson1, Jason Wildgoose1, Arkadiusz Grzyb2 and James I Langridge1
1Waters Corporation, Manchester, United Kingdom, 2 Inquiry Software, Bialogard, Poland
METHODS
Samples
MPDS E. Coli and Mixture 1 (equimolar protein digest of ADH, Enolase, Phosphorylase B and BSA; Waters Corporation).
Liquid Chromatography
NanoAcquity (Waters Corporation) configured with Trap and
Analytical columns.
Trapping Column: 5µm Symmetry C18, 180µm x 20mm.
Analytical Column: 1.85µm HSST3, 75µm x 250mm.
Solvent A: 0.1% aqueous formic acid. Solvent B: 0.1% formic acid in acetonitrile.
Trapping Phase: 5µL/min for 3min.
Analytical Phase: 300nL/min gradient from 1% to 40% B in 30 or 120min (depending on sample complexity) followed by a
column wash and re-equilibration.
Mass Spectrometry
Synapt G2-Si (Waters Corporation)
Resolution: >20k FWHM
Calibration: NaI/CsI mixture to 2000Da Wideband Enhancement Calibration:
Infusion and fragmentation of Glu[1]-Fibrinopeptide B (GFP).
IMS Data Directed Acquisition Mode: Survey 0.2s, MSMS 0.1s, Max 15 components selected for
MSMS in each survey scan. Quad window set to transmit 1Da window. Lock Mass sampled every 60s.
IMS MRM Mode: Dwell 0.1s for each analyte.
Data Processing
Wideband Enhancement Calibration: Driftscope DDA: PLGS 3.1 (Waters Corporation) and Mascot Distiller
(Matrix Science).
CONCLUSION
Ion mobility enabled Q ToF operated in
Wideband Enhancement mode improves
the duty cycle of the instrument to
approximately 89%.
The increase in signal intensity thus
afforded by this duty cycle improvement
leads to an increase in confident protein
identifications in non targeted
experiments.
Good overlap of HD-DDA protein
identifications is found when compared
to equivalent loads of ToF-DDA and
HDMSE experiments.
The use of the Wideband Enhancement
mode in ToF MRM experiments extends
the limit of detection.
Acknowledgements
The authors wish to acknowledge the useful discussions with
Dominic Helm and Bernhard Kuster from Technical University Munich. We also wish to acknowledge the assistance in
acquiring the data from Joanne Connolly and Ingvar Betner of Waters Corporation.
Figure 4. Effect of enabling Wideband Enhancement on the
intensity of ions produced during the Trap fragmentation of infused GFP, 30s of combined data. Vertical axes are linked.
Figure 6. PLGS search result from 500ng E.Coli digest. A total
of 957 proteins and 5316 peptides were identified.
Figure 5. Effect of Wideband Enhancement from the
untargeted DDA analysis of 500ng E.Coli digest. The combined TIC for each MSMS function shows significant increase for WE
ON (RED) compared to WE OFF (BLUE). Inset is an MS2 spectrum from function 16, the least intense precursor ion
selected for MS/MS at any given time.
Figure 7. Venn intersection of protein identifications from the
injections of 500ng E.Coli in three acquisition modes — HD-DDA, ToF only DDA and HDMSE.DDA data were searched
with Mascot and HDMSE with PLGS.
RESULTS
Figure 1. Schematic of the Synapt G2-Si Ion Mobility Enabled
Mass Spectrometer. Ion Mobility Separation is performed in the TriWave region of the instrument. Figure 9. Dilution series for the separation by a 30minute
gradient of MPDS Mixture1 in E. Coli background matrix. The
limit of detection was at least 5amol for the IGDYAGIK and EALDFFAR peptides, and higher for the less intense
LVNELTEFAK peptide.
OVERVIEW
ToF analyser ‘Wideband Enhancement’
mode gives sensitivity increases of 5—
10 fold.
Spectral data quality improvement in
Ion Mobility Data Directed Analysis
experiments leads to increased
protein / peptide identification rates.
Sensitivity increase extends the limit of
detection in ToF MRM experiments.
Figure 2a. Wideband Enhancement mode step 1. Ions
(represented by the red and blue lines) leave the Trap T-Wave device and enter the Ion Mobility Separator, and are separated
according to mass, shape and size.
Figure 2b. Wideband Enhancement mode step 2. The Ions
have a transit time through the Transfer T-Wave and optics towards the pusher region, whilst maintaining separation.
Figure 2c. Wideband Enhancement mode step 3. Each of the
ions are then subject to a synchronised pulse, leading to a duty cycle of approximately 89%, compared to around 15% in
the deactivated case.
Figure 3. Wideband Enhancement Calibration. M/z vs. drift
time of data acquired from partial Trap fragmentation of GFP shows distinct bands of singly and multiply charged species.
The singly charged band was selected for calibration.
1+ 2+, 3+
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300
%
0
100
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300
%
0
100684.3
480.2333.2
175.184.0
120.1
246.1
240.1337.1
382.2
627.3481.2
813.4
685.3
686.3
942.4814.4
924.4
1056.4
943.41171.5
1057.41285.5
684.3333.2 480.2
787.8 813.3
Wideband Enhancement
OFF
Wideband Enhancement
ON
Figure 8. Use of Wideband Enhancement in Targeted ToF MRM
experiments, 100amol MPDS Mixture 1 separated by a 30min gradient. Shown are the extracted product ion chromatograms
from peptides IGDYAGIK, LVNELTEFAK and EALDFFAR for WE ON (RED) and WE OFF (BLUE). Data are normalized to WE ON.
LVNELTEFAK IGDYAGIK
100amol Mix1 HDC OFF
Time28.00 29.00 30.00
%
0
100
100amol Mix1 HDC ON
Time22.00 23.00 24.00
%
0
100
100amol Mix1 HDC ON
Time30.00 31.00 32.00 33.00
%
0
100
PusherTransfer T-WAVE Ion GuideIon Mobility Separator
PusherTransfer T-WAVE Ion GuideIon Mobility Separator
Synchronised PulseSynchronised Pulse
m/z
%
m/z
%
EALDFFAR
Scatter Plot
MSMS Func
DDA 400ng EColi Fast Algorithm. No HDC. 120min. 500mDa excl. 15 cpts
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200
%
0
100
230413_003 755 (56.795) 16: TOF MSMS 705.84ES+ 5.28e31035.5
136.1
86.1
851.4
249.2
137.1
736.3
608.3
277.1 567.8447.2376.2
349.2 551.3 705.8609.3
737.4
964.5
852.4
853.4
965.4
966.4
1134.6
1036.5
1037.5
1135.5
1136.6
606
30
244
54
125
29
123
HD DDA
HDMSE
ToF DDA
606
30
244
54
125
29
123
606
30
244
54
125
29
123
HD DDA
HDMSE
ToF DDA
1.5
2
2.5
3
3.5
4
4.5
5
5.5
0.5 1 1.5 2 2.5 3 3.5 4 4.5
Log (amol on column)
Lo
g (
Resp
on
se)
EALDFFAR
IGDYAGIK
LVNELTEFAK
