a modified orbitrap tribrid mass spectrometer with real time … · 2019-08-16 · a modified...
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A Modified Orbitrap Tribrid Mass Spectrometer with Real Time Search and Advanced Spectral Processing Enhances Multiplexed Proteome Coverage and Quantification Accuracy
POSTER NOTE 65536
AuthorsAaron M. Robitaille1, Romain Huguet1, Derek J. Bailey1, Graeme McAlister1, Arne Kreutzmann2, Daniel Mourad2, Daniel Lopez-Ferrer1, Andreas Huhmer1, Vlad Zabrouskov1
1Thermo Fisher Scientific, San Jose, CA; 2ThermoFisher Scientific, Bremen, Germany
Aaron M. Robitaille 1, Romain Huguet 1, Derek J. Bailey 1, Graeme McAlister 1, Arne Kreutzmann 2, Daniel Mourad 2, Daniel Lopez-Ferrer 1, Andreas Huhmer 1, Vlad Zabrouskov 1
1 Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134. 2 Thermo Fisher Scientific Hanna-Kunath-Straße 11, 28199 Bremen, Germany
ABSTRACTIsobaric tagging strategies using Thermo Scientific™ Tandem Mass Tags™ (TMT™) are powerful tools for studying how proteins interact and function in biological systems. Up to 11 samples can be multiplexing in a single high-resolution LC/MS experiment to enable state-of-the-art quantitative analysis of peptide and protein abundance. Here we evaluate the benefits of the Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer including real time search capabilities, advanced spectral processing algorithms, and modified hardware to enhance TMT quantification accuracy and proteome coverage.
INTRODUCTIONTo assess the accuracy, precision, and sensitivity of the Orbitrap Eclipse Tribrid mass spectrometer for TMT based quantitation, we utilized the Thermo Scientific™ Pierce™ TMT11plex yeast digest standard. This standardized sample provides users with a tool to measure the accuracy, precision, and proteome depth of TMT methods across different instrumentation. Quantitative proteomics strategies using Tandem Mass Tags (TMT) allow up to 11 samples to be multiplexing in a single high resolution LC/MS experiment while enabling precise measurement of protein abundance. However, co-isolated ion interference can suppress ratio quantification and thereby mask true differences in protein abundance. Multiple methods and hardware solutions help alleviate the negative influence of interfering ions including Synchronous Precursor Selection (SPS) and a Thermo Scientific™ FAIMS Pro™ interface. However, all of these solutions come with draw backs. Furthermore, high resolution MS2 or MS3 scanning is necessary for accurate ratio determination in greater than TMT6plex experiments, which can reduce the frequency of acquisition. Here we evaluated the effects of Real Time Search on a modified Orbitrap Tribrid mass spectrometer to reduce the above stated limitations on TMT SPS MS3 quantitative experiments.
MATERIALS AND METHODSTo assess the sensitivity of the modified Orbitrap™ Tribrid™ mass spectrometer for TMT based quantitation, we utilized the Pierce TMT11plex yeast digest standard. For liquid chromatography (LC) conditions, we used an analytical gradient from 8-32% acetonitrile (vol/vol) with 0.1% (vol/vol) formic acid in 50min with a column heater set to 45°C, unless otherwise indicated. Experiments were run with a Thermo Scientific™ EASY-nLC™ 1200 HPLC system in combination with a ThermoScientific™ EASY-Spray™ C18 50cm column coupled to a Thermo Scientific™ EASY-Spray™ ion source. Samples were analyzed on a Orbitrap Eclipse Tribrid mass spectrometer. Settings for Orbitrap Tribrid Series Instrument Control Software Version 3.3 follow below (Table 1). Raw data files were processed using Thermo Scientific™ Proteome Discoverer™ 2.3 software using the SEQUEST® HT search engine with a 10ppm MS1 and 0.5 Da MS2 mass tolerance, TMT6plex (229.163 Da) set as a static modification, and a 1% false discovery rate.
CONCLUSIONSWe present a modified Orbitrap Tribrid mass spectrometer utilizing Real Time Search that improved identification rates and accuracy for TMT SPS MS3 based quantitation.
REFERENCES1. Paulo et al. A Triple Knockout (TKO) Proteomics Standard for Diagnosing Ion Interference in Isobaric
Labeling Experiments. J. Am. Soc. Mass Spectrom. 2016, 27: 16202. Erickson et al. Active Instrument Engagement Combined with a Real-Time Database Search for
Improved Performance of Sample Multiplexing Workflows. J Proteome Res. 2019 1;18(3):1299-1306. 3. Schweppe et al. Characterization and Optimization of Multiplexed Quantitative Analyses Using High-
Field Asymmetric-Waveform Ion Mobility Mass Spectrometry. Anal. Chem. 2019, 91, 6, 4010-4016
ACKNOWLEDGEMENTSWe would like to thank Ryan Bomgarden, Derek Bailey now at Google, Jimmy Eng from the University of Washington, and Steven Gygi, Devin Schweppe, and Qing Yu from the Department of Cell Biology at Harvard Medical School in Boston, USA for their collaboration and input.
TRADEMARKS/LICENSING© 2019 Thermo Fisher Scientific Inc. All rights reserved. Tandem Mass Tag and TMT are trademarks of Proteome Sciences plc. SEQUEST is a registered trademark of the University of Washington. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.
A Modified Orbitrap Tribrid Mass Spectrometer with Real Time Search and Advanced Spectral Processing Enhances Multiplexed Proteome Coverage and Quantification Accuracy
RESULTSSynchronous precursor selection (SPS) based methods provided higher accuracy compared to MS2 methods for TMT quantitation. However, depending on precursor isolation specificity and which fragments are selected for MS3 fragmentation, quantitative accuracy can still be distorted. To improve upon existing SPS methods, we implemented a Real Time Search (RTS) filter between the MS2 and MS3 scans. This feature benefits TMT SPS MS3 methods in two distinct ways. First, MS3 scans are only triggered if a peptide-spectrum match (PSM) is identified from the preceding MS2. This increased the number of peptides identified with SPS MS3. Secondly, Real Time Search identifies precursor fragments that are generated from the identified peptide on the fly, and then selects those fragment ions for MS3 quantitation. Thus, TMT SPS MS3 quantitation with Real Time Search can be improved to be 95% interference free. Next, we evaluated a new feature call TurboTMT, powered by the ΦSDM algorithm. ΦSDM is an advanced spectra processing algorithm that increases resolution within a range of the spectrum without requiring a longer transient. Applying ΦSDM specifically to the TMT reporter ions increased the resolution sufficient to baseline resolve TMT isotopologues even when using transients that produce a 30,000 or 15,000 resolving power MS2 scan. ΦSDM increased both the spectral acquisition rate for TMT11plex experiments and the number of identifications for SPS-MS3. Additionally, the modified Orbitrap Tribrid mass spectrometer has an optimized quadrupole that improves ion transmission. Thus it is possible to use narrower isolation widths to improve TMT quantitation accuracy further. Overall, the Orbitrap Eclipse Tribrid mass spectrometer includes unique features such as Real Time Search for TMT SPS MS3 based quantitation, Precursor Fit filter, and TurboTMT, which together allow for intelligent acquisition methods that improve quantitation accuracy, precision, and proteome coverage.
REAL TIME SEARCH FOR SPS MS3 TRIGGERINGFigure 4. On the fly peptide identification for TMT quantitation. Real Time Search utilizes the open source search engine Comet. We evaluated the correlation of Xcorr scores between (4A.) Real Time Search (Online) and Comet post acquisition (offline) or (4B.) Proteome Discover (PD). Secondly, we evaluated the number of fragment ions selected for a 10 notch SPS MS3 that correctly matched the post acquisition identification in RTS SPS MS3 (4C.,4E.) or SPS MS3 (4D, 4F.).
Electrodynamic Ion Funnel
High-Capacity Transfer Tube
Ion Max NG Ion Source
EASY-IC/ETD/PTCR
Ion Source
Advanced Active Ion Beam Guide
QR5 Segmented Quadrupole Mass Filter with hyperbolic surfaces
C-Trap
Ion Routing Multipole
Ultra-High-Field Orbitrap Mass Analyzer
Higher CapacityHigh Pressure
Cell
Low Pressure Cell
Modified Dual-Pressure Linear Ion Trap
Figure 1. (1A.) Schematic representation of Orbitrap Eclipse Tribrid mass spectrometer.
A MODIFIED ORBITRAP TRIBRID MASS SPECTROMETER
1A.
Figure 2. (2A.) Schematic representation of Pierce TMT11plex Yeast Digest Standard. The standard is composed of four Saccharomyces cerevisiae strains: three lines respectively lacking the non-essential proteins Met6, His4, or Ura2, and the parental strain BY4741 for reference. Modified from Schweppe et al. 2019.
A TMT11PLEX YEAST DIGEST STANDARD
126met6Δ
his4Δ
ura2Δ
127N127C128N128C129N129C130N130C131131C
BY4741
4 strains of saccharomyces cervices
11 TMT channels technical replicates N=3 or 2 (for BY4741)
Samples combined into 1 LC/MS analysis
Proteome Discoverer Software
MS1 MS3
Pierce™ TMT11plex yeast digest standard
Knoc
kout
sta
ins
Pare
ntal
C
ontro
l
2A.
MS SelectionTMT Labeled Peptides
SPS MS3 Quantitation
RTS for MS3 Triggering
MS2 Acquisition
Real Time Search
<1ms per spectrum
m/z m/z m/z
ID ID No ID
2.0x
11073
7107
1085510599
6660
9196
0
2000
4000
6000
8000
10000
12000
MS2 SPS SPS RTS
Pept
ides
Identified Quantified
RTS SPS MS3SPS MS3
+53%2207
1340
20832087
1242
1909
0
500
1000
1500
2000
2500
MS2 SPS SPS RTS
Prot
eins
Identified Quantified
RTS SPS MS3SPS MS3
+55%
9%
SPSMS3
RTSSPSMS3
Less interference
Moreinterference
Less interference
Moreinterference
6 proteinspike in
HeLa
Expected ratio
0 1 2 4 8 12 16 20 24
16 channels
Table 1. Mass spectrometer data acquisition settings.
Acquisition Settings MS2 SPS MS3 RTS SPS MS3Top Speed (sec) 2.5 2.5 2.5
RF lens 30% 30% 30%
Orbitrap MS1 resolution 120000 120000 120000
Scan range (m/z) 400-1400 400-1400 400-1400
Standardized MS1 AGC target 100% 100% 100%
MS1 max IT (mode) Auto Auto Auto
Charge state 2-5 2-5 2-5
Dynamic Exclusion (sec) 45 45 45
MS2 resolution 45000 Turbo Turbo
MS2 Scan range (m/z) First mass 110 200-1200 200-1200
MS2 Isolation Window 0.7 m/z 0.7 m/z 0.7 m/z
Standardized MS2 AGC target 500% 100% 100%
MS2 max IT (mode) Auto Auto Auto
MS2 HCD NCE% 36 36 36
SPS MS3 Resolution - 50000 60000
MS2 Scan range (m/z) - 100-500 100-500
SPS MS3 Isolation Window - 0.7 m/z 0.7 m/z
Standardized SPS MS3 AGC target - 500% 500%
SPS MS3 max IT (mode) - Auto Auto
SPS MS3 HCD NCE% - 55 55
SPS MS3 Notches - 10 10
Figure 3. Achieving improved proteome depth and accuracy for SPS MS3 quantitation (3A.) Schematic representation of TMT workflow on Orbitrap Eclipse Tribrid mass spectrometer. We evaluated how Real Time Search on the modified Orbitrap Tribrid mass spectrometer influenced TMT identification rates for (3B.) proteins and (3C.) peptides, (3D.) as well as the interference free index at the peptide level as visualized in TKOmics.com. 500ng of TMT11plex Yeast Digest Standard was measured on a 50min gradient using MS2, SPS MS3, or RTS SPS MS3 methods.
ENHANCED TMT SPS MS3 PERFORMANCE
3A.
3C.3B.
3D.
XRTS SPS MS3 Dot plot
RTS SPS MS3Histogram
SPS MS3Histogram
SPS MS3 Dot plot
Pearson’s Correlation of 0.84Pearson’s Correlation of 1.004A. 4B.
4C. 4D.
4E.4F.
REAL TIME SEARCH IMPROVES TMT QUANTITATIONFigure 6. Evaluation of TMT quantification performance. We evaluated how Real Time Search affects TMT SPS MS3 quantitation precision (6A.) and accuracy (6B.) using Pierce 6 Protein Digest Standard mixed in various ratios into the Pierce HeLa Protein Digest Standard. 1ug of peptides was analyzed on a 120min gradient.
6%
10% 11%14% 14% 13%
15%
1% 2% 3% 3% 3%2%
4%
0%
5%
10%
15%
20%
2 4 8 12 16 20 24
Coe
ffici
ent o
f Var
iatio
n (C
V)
Expected Ratio
SPS MS3 RTS MS3
N= 15 peptides per protein
RTS SPS MS3
Expected SPSMS3
RTSSPSMS3
0
5
10
15
20
25
Obs
erve
d R
atio
Quantitation
Improved Accuracy
Improved Precision6A.
6B.
REAL TIME SEARCH ENABLES CUSTOM MODIFCIATIONS Figure 5. Next generation isobaric tags increases sample multiplexing. We evaluated the potential for Real Time Search to use custom modifications using a next generation isobaric tags. (5A.) Pierce 6 Protein Digest Standard mixed in various ratios into the Pierce HeLa Protein Digest Standard and labeled. (5B). 1ug of sample was then analyzed on a 120min gradient using RTS SPS MS3 or SPS MS3.
2589 3267
1489517695
0
5000
10000
15000
20000
SPS MS3 RTS SPS MS3
Cou
nt
Proteins Peptides
5A. 5B.
PO65536-EN0519S
Aaron M. Robitaille 1, Romain Huguet 1, Derek J. Bailey 1, Graeme McAlister 1, Arne Kreutzmann 2, Daniel Mourad 2, Daniel Lopez-Ferrer 1, Andreas Huhmer 1, Vlad Zabrouskov 1
1 Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134. 2 Thermo Fisher Scientific Hanna-Kunath-Straße 11, 28199 Bremen, Germany
ABSTRACTIsobaric tagging strategies using Thermo Scientific™ Tandem Mass Tags™ (TMT™) are powerful tools for studying how proteins interact and function in biological systems. Up to 11 samples can be multiplexing in a single high-resolution LC/MS experiment to enable state-of-the-art quantitative analysis of peptide and protein abundance. Here we evaluate the benefits of the Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer including real time search capabilities, advanced spectral processing algorithms, and modified hardware to enhance TMT quantification accuracy and proteome coverage.
INTRODUCTIONTo assess the accuracy, precision, and sensitivity of the Orbitrap Eclipse Tribrid mass spectrometer for TMT based quantitation, we utilized the Thermo Scientific™ Pierce™ TMT11plex yeast digest standard. This standardized sample provides users with a tool to measure the accuracy, precision, and proteome depth of TMT methods across different instrumentation. Quantitative proteomics strategies using Tandem Mass Tags (TMT) allow up to 11 samples to be multiplexing in a single high resolution LC/MS experiment while enabling precise measurement of protein abundance. However, co-isolated ion interference can suppress ratio quantification and thereby mask true differences in protein abundance. Multiple methods and hardware solutions help alleviate the negative influence of interfering ions including Synchronous Precursor Selection (SPS) and a Thermo Scientific™ FAIMS Pro™ interface. However, all of these solutions come with draw backs. Furthermore, high resolution MS2 or MS3 scanning is necessary for accurate ratio determination in greater than TMT6plex experiments, which can reduce the frequency of acquisition. Here we evaluated the effects of Real Time Search on a modified Orbitrap Tribrid mass spectrometer to reduce the above stated limitations on TMT SPS MS3 quantitative experiments.
MATERIALS AND METHODSTo assess the sensitivity of the modified Orbitrap™ Tribrid™ mass spectrometer for TMT based quantitation, we utilized the Pierce TMT11plex yeast digest standard. For liquid chromatography (LC) conditions, we used an analytical gradient from 8-32% acetonitrile (vol/vol) with 0.1% (vol/vol) formic acid in 50min with a column heater set to 45°C, unless otherwise indicated. Experiments were run with a Thermo Scientific™ EASY-nLC™ 1200 HPLC system in combination with a ThermoScientific™ EASY-Spray™ C18 50cm column coupled to a Thermo Scientific™ EASY-Spray™ ion source. Samples were analyzed on a Orbitrap Eclipse Tribrid mass spectrometer. Settings for Orbitrap Tribrid Series Instrument Control Software Version 3.3 follow below (Table 1). Raw data files were processed using Thermo Scientific™ Proteome Discoverer™ 2.3 software using the SEQUEST® HT search engine with a 10ppm MS1 and 0.5 Da MS2 mass tolerance, TMT6plex (229.163 Da) set as a static modification, and a 1% false discovery rate.
CONCLUSIONSWe present a modified Orbitrap Tribrid mass spectrometer utilizing Real Time Search that improved identification rates and accuracy for TMT SPS MS3 based quantitation.
REFERENCES1. Paulo et al. A Triple Knockout (TKO) Proteomics Standard for Diagnosing Ion Interference in Isobaric
Labeling Experiments. J. Am. Soc. Mass Spectrom. 2016, 27: 16202. Erickson et al. Active Instrument Engagement Combined with a Real-Time Database Search for
Improved Performance of Sample Multiplexing Workflows. J Proteome Res. 2019 1;18(3):1299-1306. 3. Schweppe et al. Characterization and Optimization of Multiplexed Quantitative Analyses Using High-
Field Asymmetric-Waveform Ion Mobility Mass Spectrometry. Anal. Chem. 2019, 91, 6, 4010-4016
ACKNOWLEDGEMENTSWe would like to thank Ryan Bomgarden, Derek Bailey now at Google, Jimmy Eng from the University of Washington, and Steven Gygi, Devin Schweppe, and Qing Yu from the Department of Cell Biology at Harvard Medical School in Boston, USA for their collaboration and input.
TRADEMARKS/LICENSING© 2019 Thermo Fisher Scientific Inc. All rights reserved. Tandem Mass Tag and TMT are trademarks of Proteome Sciences plc. SEQUEST is a registered trademark of the University of Washington. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.
A Modified Orbitrap Tribrid Mass Spectrometer with Real Time Search and Advanced Spectral Processing Enhances Multiplexed Proteome Coverage and Quantification Accuracy
RESULTSSynchronous precursor selection (SPS) based methods provided higher accuracy compared to MS2 methods for TMT quantitation. However, depending on precursor isolation specificity and which fragments are selected for MS3 fragmentation, quantitative accuracy can still be distorted. To improve upon existing SPS methods, we implemented a Real Time Search (RTS) filter between the MS2 and MS3 scans. This feature benefits TMT SPS MS3 methods in two distinct ways. First, MS3 scans are only triggered if a peptide-spectrum match (PSM) is identified from the preceding MS2. This increased the number of peptides identified with SPS MS3. Secondly, Real Time Search identifies precursor fragments that are generated from the identified peptide on the fly, and then selects those fragment ions for MS3 quantitation. Thus, TMT SPS MS3 quantitation with Real Time Search can be improved to be 95% interference free. Next, we evaluated a new feature call TurboTMT, powered by the ΦSDM algorithm. ΦSDM is an advanced spectra processing algorithm that increases resolution within a range of the spectrum without requiring a longer transient. Applying ΦSDM specifically to the TMT reporter ions increased the resolution sufficient to baseline resolve TMT isotopologues even when using transients that produce a 30,000 or 15,000 resolving power MS2 scan. ΦSDM increased both the spectral acquisition rate for TMT11plex experiments and the number of identifications for SPS-MS3. Additionally, the modified Orbitrap Tribrid mass spectrometer has an optimized quadrupole that improves ion transmission. Thus it is possible to use narrower isolation widths to improve TMT quantitation accuracy further. Overall, the Orbitrap Eclipse Tribrid mass spectrometer includes unique features such as Real Time Search for TMT SPS MS3 based quantitation, Precursor Fit filter, and TurboTMT, which together allow for intelligent acquisition methods that improve quantitation accuracy, precision, and proteome coverage.
REAL TIME SEARCH FOR SPS MS3 TRIGGERINGFigure 4. On the fly peptide identification for TMT quantitation. Real Time Search utilizes the open source search engine Comet. We evaluated the correlation of Xcorr scores between (4A.) Real Time Search (Online) and Comet post acquisition (offline) or (4B.) Proteome Discover (PD). Secondly, we evaluated the number of fragment ions selected for a 10 notch SPS MS3 that correctly matched the post acquisition identification in RTS SPS MS3 (4C.,4E.) or SPS MS3 (4D, 4F.).
Electrodynamic Ion Funnel
High-Capacity Transfer Tube
Ion Max NG Ion Source
EASY-IC/ETD/PTCR
Ion Source
Advanced Active Ion Beam Guide
QR5 Segmented Quadrupole Mass Filter with hyperbolic surfaces
C-Trap
Ion Routing Multipole
Ultra-High-Field Orbitrap Mass Analyzer
Higher CapacityHigh Pressure
Cell
Low Pressure Cell
Modified Dual-Pressure Linear Ion Trap
Figure 1. (1A.) Schematic representation of Orbitrap Eclipse Tribrid mass spectrometer.
A MODIFIED ORBITRAP TRIBRID MASS SPECTROMETER
1A.
Figure 2. (2A.) Schematic representation of Pierce TMT11plex Yeast Digest Standard. The standard is composed of four Saccharomyces cerevisiae strains: three lines respectively lacking the non-essential proteins Met6, His4, or Ura2, and the parental strain BY4741 for reference. Modified from Schweppe et al. 2019.
A TMT11PLEX YEAST DIGEST STANDARD
126met6Δ
his4Δ
ura2Δ
127N127C128N128C129N129C130N130C131131C
BY4741
4 strains of saccharomyces cervices
11 TMT channels technical replicates N=3 or 2 (for BY4741)
Samples combined into 1 LC/MS analysis
Proteome Discoverer Software
MS1 MS3
Pierce™ TMT11plex yeast digest standard
Knoc
kout
sta
ins
Pare
ntal
C
ontro
l
2A.
MS SelectionTMT Labeled Peptides
SPS MS3 Quantitation
RTS for MS3 Triggering
MS2 Acquisition
Real Time Search
<1ms per spectrum
m/z m/z m/z
ID ID No ID
2.0x
11073
7107
1085510599
6660
9196
0
2000
4000
6000
8000
10000
12000
MS2 SPS SPS RTS
Pept
ides
Identified Quantified
RTS SPS MS3SPS MS3
+53%2207
1340
20832087
1242
1909
0
500
1000
1500
2000
2500
MS2 SPS SPS RTS
Prot
eins
Identified Quantified
RTS SPS MS3SPS MS3
+55%
9%
SPSMS3
RTSSPSMS3
Less interference
Moreinterference
Less interference
Moreinterference
6 proteinspike in
HeLa
Expected ratio
0 1 2 4 8 12 16 20 24
16 channels
Table 1. Mass spectrometer data acquisition settings.
Acquisition Settings MS2 SPS MS3 RTS SPS MS3Top Speed (sec) 2.5 2.5 2.5
RF lens 30% 30% 30%
Orbitrap MS1 resolution 120000 120000 120000
Scan range (m/z) 400-1400 400-1400 400-1400
Standardized MS1 AGC target 100% 100% 100%
MS1 max IT (mode) Auto Auto Auto
Charge state 2-5 2-5 2-5
Dynamic Exclusion (sec) 45 45 45
MS2 resolution 45000 Turbo Turbo
MS2 Scan range (m/z) First mass 110 200-1200 200-1200
MS2 Isolation Window 0.7 m/z 0.7 m/z 0.7 m/z
Standardized MS2 AGC target 500% 100% 100%
MS2 max IT (mode) Auto Auto Auto
MS2 HCD NCE% 36 36 36
SPS MS3 Resolution - 50000 60000
MS2 Scan range (m/z) - 100-500 100-500
SPS MS3 Isolation Window - 0.7 m/z 0.7 m/z
Standardized SPS MS3 AGC target - 500% 500%
SPS MS3 max IT (mode) - Auto Auto
SPS MS3 HCD NCE% - 55 55
SPS MS3 Notches - 10 10
Figure 3. Achieving improved proteome depth and accuracy for SPS MS3 quantitation (3A.) Schematic representation of TMT workflow on Orbitrap Eclipse Tribrid mass spectrometer. We evaluated how Real Time Search on the modified Orbitrap Tribrid mass spectrometer influenced TMT identification rates for (3B.) proteins and (3C.) peptides, (3D.) as well as the interference free index at the peptide level as visualized in TKOmics.com. 500ng of TMT11plex Yeast Digest Standard was measured on a 50min gradient using MS2, SPS MS3, or RTS SPS MS3 methods.
ENHANCED TMT SPS MS3 PERFORMANCE
3A.
3C.3B.
3D.
XRTS SPS MS3 Dot plot
RTS SPS MS3Histogram
SPS MS3Histogram
SPS MS3 Dot plot
Pearson’s Correlation of 0.84Pearson’s Correlation of 1.004A. 4B.
4C. 4D.
4E.4F.
REAL TIME SEARCH IMPROVES TMT QUANTITATIONFigure 6. Evaluation of TMT quantification performance. We evaluated how Real Time Search affects TMT SPS MS3 quantitation precision (6A.) and accuracy (6B.) using Pierce 6 Protein Digest Standard mixed in various ratios into the Pierce HeLa Protein Digest Standard. 1ug of peptides was analyzed on a 120min gradient.
6%
10% 11%14% 14% 13%
15%
1% 2% 3% 3% 3%2%
4%
0%
5%
10%
15%
20%
2 4 8 12 16 20 24
Coe
ffici
ent o
f Var
iatio
n (C
V)
Expected Ratio
SPS MS3 RTS MS3
N= 15 peptides per protein
RTS SPS MS3
Expected SPSMS3
RTSSPSMS3
0
5
10
15
20
25
Obs
erve
d R
atio
Quantitation
Improved Accuracy
Improved Precision6A.
6B.
REAL TIME SEARCH ENABLES CUSTOM MODIFCIATIONS Figure 5. Next generation isobaric tags increases sample multiplexing. We evaluated the potential for Real Time Search to use custom modifications using a next generation isobaric tags. (5A.) Pierce 6 Protein Digest Standard mixed in various ratios into the Pierce HeLa Protein Digest Standard and labeled. (5B). 1ug of sample was then analyzed on a 120min gradient using RTS SPS MS3 or SPS MS3.
2589 3267
1489517695
0
5000
10000
15000
20000
SPS MS3 RTS SPS MS3
Cou
nt
Proteins Peptides
5A. 5B.
PO65536-EN0519S
Aaron M. Robitaille 1, Romain Huguet 1, Derek J. Bailey 1, Graeme McAlister 1, Arne Kreutzmann 2, Daniel Mourad 2, Daniel Lopez-Ferrer 1, Andreas Huhmer 1, Vlad Zabrouskov 1
1 Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134. 2 Thermo Fisher Scientific Hanna-Kunath-Straße 11, 28199 Bremen, Germany
ABSTRACTIsobaric tagging strategies using Thermo Scientific™ Tandem Mass Tags™ (TMT™) are powerful tools for studying how proteins interact and function in biological systems. Up to 11 samples can be multiplexing in a single high-resolution LC/MS experiment to enable state-of-the-art quantitative analysis of peptide and protein abundance. Here we evaluate the benefits of the Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer including real time search capabilities, advanced spectral processing algorithms, and modified hardware to enhance TMT quantification accuracy and proteome coverage.
INTRODUCTIONTo assess the accuracy, precision, and sensitivity of the Orbitrap Eclipse Tribrid mass spectrometer for TMT based quantitation, we utilized the Thermo Scientific™ Pierce™ TMT11plex yeast digest standard. This standardized sample provides users with a tool to measure the accuracy, precision, and proteome depth of TMT methods across different instrumentation. Quantitative proteomics strategies using Tandem Mass Tags (TMT) allow up to 11 samples to be multiplexing in a single high resolution LC/MS experiment while enabling precise measurement of protein abundance. However, co-isolated ion interference can suppress ratio quantification and thereby mask true differences in protein abundance. Multiple methods and hardware solutions help alleviate the negative influence of interfering ions including Synchronous Precursor Selection (SPS) and a Thermo Scientific™ FAIMS Pro™ interface. However, all of these solutions come with draw backs. Furthermore, high resolution MS2 or MS3 scanning is necessary for accurate ratio determination in greater than TMT6plex experiments, which can reduce the frequency of acquisition. Here we evaluated the effects of Real Time Search on a modified Orbitrap Tribrid mass spectrometer to reduce the above stated limitations on TMT SPS MS3 quantitative experiments.
MATERIALS AND METHODSTo assess the sensitivity of the modified Orbitrap™ Tribrid™ mass spectrometer for TMT based quantitation, we utilized the Pierce TMT11plex yeast digest standard. For liquid chromatography (LC) conditions, we used an analytical gradient from 8-32% acetonitrile (vol/vol) with 0.1% (vol/vol) formic acid in 50min with a column heater set to 45°C, unless otherwise indicated. Experiments were run with a Thermo Scientific™ EASY-nLC™ 1200 HPLC system in combination with a ThermoScientific™ EASY-Spray™ C18 50cm column coupled to a Thermo Scientific™ EASY-Spray™ ion source. Samples were analyzed on a Orbitrap Eclipse Tribrid mass spectrometer. Settings for Orbitrap Tribrid Series Instrument Control Software Version 3.3 follow below (Table 1). Raw data files were processed using Thermo Scientific™ Proteome Discoverer™ 2.3 software using the SEQUEST® HT search engine with a 10ppm MS1 and 0.5 Da MS2 mass tolerance, TMT6plex (229.163 Da) set as a static modification, and a 1% false discovery rate.
CONCLUSIONSWe present a modified Orbitrap Tribrid mass spectrometer utilizing Real Time Search that improved identification rates and accuracy for TMT SPS MS3 based quantitation.
REFERENCES1. Paulo et al. A Triple Knockout (TKO) Proteomics Standard for Diagnosing Ion Interference in Isobaric
Labeling Experiments. J. Am. Soc. Mass Spectrom. 2016, 27: 16202. Erickson et al. Active Instrument Engagement Combined with a Real-Time Database Search for
Improved Performance of Sample Multiplexing Workflows. J Proteome Res. 2019 1;18(3):1299-1306. 3. Schweppe et al. Characterization and Optimization of Multiplexed Quantitative Analyses Using High-
Field Asymmetric-Waveform Ion Mobility Mass Spectrometry. Anal. Chem. 2019, 91, 6, 4010-4016
ACKNOWLEDGEMENTSWe would like to thank Ryan Bomgarden, Derek Bailey now at Google, Jimmy Eng from the University of Washington, and Steven Gygi, Devin Schweppe, and Qing Yu from the Department of Cell Biology at Harvard Medical School in Boston, USA for their collaboration and input.
TRADEMARKS/LICENSING© 2019 Thermo Fisher Scientific Inc. All rights reserved. Tandem Mass Tag and TMT are trademarks of Proteome Sciences plc. SEQUEST is a registered trademark of the University of Washington. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.
A Modified Orbitrap Tribrid Mass Spectrometer with Real Time Search and Advanced Spectral Processing Enhances Multiplexed Proteome Coverage and Quantification Accuracy
RESULTSSynchronous precursor selection (SPS) based methods provided higher accuracy compared to MS2 methods for TMT quantitation. However, depending on precursor isolation specificity and which fragments are selected for MS3 fragmentation, quantitative accuracy can still be distorted. To improve upon existing SPS methods, we implemented a Real Time Search (RTS) filter between the MS2 and MS3 scans. This feature benefits TMT SPS MS3 methods in two distinct ways. First, MS3 scans are only triggered if a peptide-spectrum match (PSM) is identified from the preceding MS2. This increased the number of peptides identified with SPS MS3. Secondly, Real Time Search identifies precursor fragments that are generated from the identified peptide on the fly, and then selects those fragment ions for MS3 quantitation. Thus, TMT SPS MS3 quantitation with Real Time Search can be improved to be 95% interference free. Next, we evaluated a new feature call TurboTMT, powered by the ΦSDM algorithm. ΦSDM is an advanced spectra processing algorithm that increases resolution within a range of the spectrum without requiring a longer transient. Applying ΦSDM specifically to the TMT reporter ions increased the resolution sufficient to baseline resolve TMT isotopologues even when using transients that produce a 30,000 or 15,000 resolving power MS2 scan. ΦSDM increased both the spectral acquisition rate for TMT11plex experiments and the number of identifications for SPS-MS3. Additionally, the modified Orbitrap Tribrid mass spectrometer has an optimized quadrupole that improves ion transmission. Thus it is possible to use narrower isolation widths to improve TMT quantitation accuracy further. Overall, the Orbitrap Eclipse Tribrid mass spectrometer includes unique features such as Real Time Search for TMT SPS MS3 based quantitation, Precursor Fit filter, and TurboTMT, which together allow for intelligent acquisition methods that improve quantitation accuracy, precision, and proteome coverage.
REAL TIME SEARCH FOR SPS MS3 TRIGGERINGFigure 4. On the fly peptide identification for TMT quantitation. Real Time Search utilizes the open source search engine Comet. We evaluated the correlation of Xcorr scores between (4A.) Real Time Search (Online) and Comet post acquisition (offline) or (4B.) Proteome Discover (PD). Secondly, we evaluated the number of fragment ions selected for a 10 notch SPS MS3 that correctly matched the post acquisition identification in RTS SPS MS3 (4C.,4E.) or SPS MS3 (4D, 4F.).
Electrodynamic Ion Funnel
High-Capacity Transfer Tube
Ion Max NG Ion Source
EASY-IC/ETD/PTCR
Ion Source
Advanced Active Ion Beam Guide
QR5 Segmented Quadrupole Mass Filter with hyperbolic surfaces
C-Trap
Ion Routing Multipole
Ultra-High-Field Orbitrap Mass Analyzer
Higher CapacityHigh Pressure
Cell
Low Pressure Cell
Modified Dual-Pressure Linear Ion Trap
Figure 1. (1A.) Schematic representation of Orbitrap Eclipse Tribrid mass spectrometer.
A MODIFIED ORBITRAP TRIBRID MASS SPECTROMETER
1A.
Figure 2. (2A.) Schematic representation of Pierce TMT11plex Yeast Digest Standard. The standard is composed of four Saccharomyces cerevisiae strains: three lines respectively lacking the non-essential proteins Met6, His4, or Ura2, and the parental strain BY4741 for reference. Modified from Schweppe et al. 2019.
A TMT11PLEX YEAST DIGEST STANDARD
126met6Δ
his4Δ
ura2Δ
127N127C128N128C129N129C130N130C131131C
BY4741
4 strains of saccharomyces cervices
11 TMT channels technical replicates N=3 or 2 (for BY4741)
Samples combined into 1 LC/MS analysis
Proteome Discoverer Software
MS1 MS3
Pierce™ TMT11plex yeast digest standard
Knoc
kout
sta
ins
Pare
ntal
C
ontro
l
2A.
MS SelectionTMT Labeled Peptides
SPS MS3 Quantitation
RTS for MS3 Triggering
MS2 Acquisition
Real Time Search
<1ms per spectrum
m/z m/z m/z
ID ID No ID
2.0x
11073
7107
1085510599
6660
9196
0
2000
4000
6000
8000
10000
12000
MS2 SPS SPS RTS
Pept
ides
Identified Quantified
RTS SPS MS3SPS MS3
+53%2207
1340
20832087
1242
1909
0
500
1000
1500
2000
2500
MS2 SPS SPS RTS
Prot
eins
Identified Quantified
RTS SPS MS3SPS MS3
+55%
9%
SPSMS3
RTSSPSMS3
Less interference
Moreinterference
Less interference
Moreinterference
6 proteinspike in
HeLa
Expected ratio
0 1 2 4 8 12 16 20 24
16 channels
Table 1. Mass spectrometer data acquisition settings.
Acquisition Settings MS2 SPS MS3 RTS SPS MS3Top Speed (sec) 2.5 2.5 2.5
RF lens 30% 30% 30%
Orbitrap MS1 resolution 120000 120000 120000
Scan range (m/z) 400-1400 400-1400 400-1400
Standardized MS1 AGC target 100% 100% 100%
MS1 max IT (mode) Auto Auto Auto
Charge state 2-5 2-5 2-5
Dynamic Exclusion (sec) 45 45 45
MS2 resolution 45000 Turbo Turbo
MS2 Scan range (m/z) First mass 110 200-1200 200-1200
MS2 Isolation Window 0.7 m/z 0.7 m/z 0.7 m/z
Standardized MS2 AGC target 500% 100% 100%
MS2 max IT (mode) Auto Auto Auto
MS2 HCD NCE% 36 36 36
SPS MS3 Resolution - 50000 60000
MS2 Scan range (m/z) - 100-500 100-500
SPS MS3 Isolation Window - 0.7 m/z 0.7 m/z
Standardized SPS MS3 AGC target - 500% 500%
SPS MS3 max IT (mode) - Auto Auto
SPS MS3 HCD NCE% - 55 55
SPS MS3 Notches - 10 10
Figure 3. Achieving improved proteome depth and accuracy for SPS MS3 quantitation (3A.) Schematic representation of TMT workflow on Orbitrap Eclipse Tribrid mass spectrometer. We evaluated how Real Time Search on the modified Orbitrap Tribrid mass spectrometer influenced TMT identification rates for (3B.) proteins and (3C.) peptides, (3D.) as well as the interference free index at the peptide level as visualized in TKOmics.com. 500ng of TMT11plex Yeast Digest Standard was measured on a 50min gradient using MS2, SPS MS3, or RTS SPS MS3 methods.
ENHANCED TMT SPS MS3 PERFORMANCE
3A.
3C.3B.
3D.
XRTS SPS MS3 Dot plot
RTS SPS MS3Histogram
SPS MS3Histogram
SPS MS3 Dot plot
Pearson’s Correlation of 0.84Pearson’s Correlation of 1.004A. 4B.
4C. 4D.
4E.4F.
REAL TIME SEARCH IMPROVES TMT QUANTITATIONFigure 6. Evaluation of TMT quantification performance. We evaluated how Real Time Search affects TMT SPS MS3 quantitation precision (6A.) and accuracy (6B.) using Pierce 6 Protein Digest Standard mixed in various ratios into the Pierce HeLa Protein Digest Standard. 1ug of peptides was analyzed on a 120min gradient.
6%
10% 11%14% 14% 13%
15%
1% 2% 3% 3% 3%2%
4%
0%
5%
10%
15%
20%
2 4 8 12 16 20 24
Coe
ffici
ent o
f Var
iatio
n (C
V)
Expected Ratio
SPS MS3 RTS MS3
N= 15 peptides per protein
RTS SPS MS3
Expected SPSMS3
RTSSPSMS3
0
5
10
15
20
25
Obs
erve
d R
atio
Quantitation
Improved Accuracy
Improved Precision6A.
6B.
REAL TIME SEARCH ENABLES CUSTOM MODIFCIATIONS Figure 5. Next generation isobaric tags increases sample multiplexing. We evaluated the potential for Real Time Search to use custom modifications using a next generation isobaric tags. (5A.) Pierce 6 Protein Digest Standard mixed in various ratios into the Pierce HeLa Protein Digest Standard and labeled. (5B). 1ug of sample was then analyzed on a 120min gradient using RTS SPS MS3 or SPS MS3.
2589 3267
1489517695
0
5000
10000
15000
20000
SPS MS3 RTS SPS MS3
Cou
nt
Proteins Peptides
5A. 5B.
PO65536-EN0519S
Aaron M. Robitaille 1, Romain Huguet 1, Derek J. Bailey 1, Graeme McAlister 1, Arne Kreutzmann 2, Daniel Mourad 2, Daniel Lopez-Ferrer 1, Andreas Huhmer 1, Vlad Zabrouskov 1
1 Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134. 2 Thermo Fisher Scientific Hanna-Kunath-Straße 11, 28199 Bremen, Germany
ABSTRACTIsobaric tagging strategies using Thermo Scientific™ Tandem Mass Tags™ (TMT™) are powerful tools for studying how proteins interact and function in biological systems. Up to 11 samples can be multiplexing in a single high-resolution LC/MS experiment to enable state-of-the-art quantitative analysis of peptide and protein abundance. Here we evaluate the benefits of the Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer including real time search capabilities, advanced spectral processing algorithms, and modified hardware to enhance TMT quantification accuracy and proteome coverage.
INTRODUCTIONTo assess the accuracy, precision, and sensitivity of the Orbitrap Eclipse Tribrid mass spectrometer for TMT based quantitation, we utilized the Thermo Scientific™ Pierce™ TMT11plex yeast digest standard. This standardized sample provides users with a tool to measure the accuracy, precision, and proteome depth of TMT methods across different instrumentation. Quantitative proteomics strategies using Tandem Mass Tags (TMT) allow up to 11 samples to be multiplexing in a single high resolution LC/MS experiment while enabling precise measurement of protein abundance. However, co-isolated ion interference can suppress ratio quantification and thereby mask true differences in protein abundance. Multiple methods and hardware solutions help alleviate the negative influence of interfering ions including Synchronous Precursor Selection (SPS) and a Thermo Scientific™ FAIMS Pro™ interface. However, all of these solutions come with draw backs. Furthermore, high resolution MS2 or MS3 scanning is necessary for accurate ratio determination in greater than TMT6plex experiments, which can reduce the frequency of acquisition. Here we evaluated the effects of Real Time Search on a modified Orbitrap Tribrid mass spectrometer to reduce the above stated limitations on TMT SPS MS3 quantitative experiments.
MATERIALS AND METHODSTo assess the sensitivity of the modified Orbitrap™ Tribrid™ mass spectrometer for TMT based quantitation, we utilized the Pierce TMT11plex yeast digest standard. For liquid chromatography (LC) conditions, we used an analytical gradient from 8-32% acetonitrile (vol/vol) with 0.1% (vol/vol) formic acid in 50min with a column heater set to 45°C, unless otherwise indicated. Experiments were run with a Thermo Scientific™ EASY-nLC™ 1200 HPLC system in combination with a ThermoScientific™ EASY-Spray™ C18 50cm column coupled to a Thermo Scientific™ EASY-Spray™ ion source. Samples were analyzed on a Orbitrap Eclipse Tribrid mass spectrometer. Settings for Orbitrap Tribrid Series Instrument Control Software Version 3.3 follow below (Table 1). Raw data files were processed using Thermo Scientific™ Proteome Discoverer™ 2.3 software using the SEQUEST® HT search engine with a 10ppm MS1 and 0.5 Da MS2 mass tolerance, TMT6plex (229.163 Da) set as a static modification, and a 1% false discovery rate.
CONCLUSIONSWe present a modified Orbitrap Tribrid mass spectrometer utilizing Real Time Search that improved identification rates and accuracy for TMT SPS MS3 based quantitation.
REFERENCES1. Paulo et al. A Triple Knockout (TKO) Proteomics Standard for Diagnosing Ion Interference in Isobaric
Labeling Experiments. J. Am. Soc. Mass Spectrom. 2016, 27: 16202. Erickson et al. Active Instrument Engagement Combined with a Real-Time Database Search for
Improved Performance of Sample Multiplexing Workflows. J Proteome Res. 2019 1;18(3):1299-1306. 3. Schweppe et al. Characterization and Optimization of Multiplexed Quantitative Analyses Using High-
Field Asymmetric-Waveform Ion Mobility Mass Spectrometry. Anal. Chem. 2019, 91, 6, 4010-4016
ACKNOWLEDGEMENTSWe would like to thank Ryan Bomgarden, Derek Bailey now at Google, Jimmy Eng from the University of Washington, and Steven Gygi, Devin Schweppe, and Qing Yu from the Department of Cell Biology at Harvard Medical School in Boston, USA for their collaboration and input.
TRADEMARKS/LICENSING© 2019 Thermo Fisher Scientific Inc. All rights reserved. Tandem Mass Tag and TMT are trademarks of Proteome Sciences plc. SEQUEST is a registered trademark of the University of Washington. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.
A Modified Orbitrap Tribrid Mass Spectrometer with Real Time Search and Advanced Spectral Processing Enhances Multiplexed Proteome Coverage and Quantification Accuracy
RESULTSSynchronous precursor selection (SPS) based methods provided higher accuracy compared to MS2 methods for TMT quantitation. However, depending on precursor isolation specificity and which fragments are selected for MS3 fragmentation, quantitative accuracy can still be distorted. To improve upon existing SPS methods, we implemented a Real Time Search (RTS) filter between the MS2 and MS3 scans. This feature benefits TMT SPS MS3 methods in two distinct ways. First, MS3 scans are only triggered if a peptide-spectrum match (PSM) is identified from the preceding MS2. This increased the number of peptides identified with SPS MS3. Secondly, Real Time Search identifies precursor fragments that are generated from the identified peptide on the fly, and then selects those fragment ions for MS3 quantitation. Thus, TMT SPS MS3 quantitation with Real Time Search can be improved to be 95% interference free. Next, we evaluated a new feature call TurboTMT, powered by the ΦSDM algorithm. ΦSDM is an advanced spectra processing algorithm that increases resolution within a range of the spectrum without requiring a longer transient. Applying ΦSDM specifically to the TMT reporter ions increased the resolution sufficient to baseline resolve TMT isotopologues even when using transients that produce a 30,000 or 15,000 resolving power MS2 scan. ΦSDM increased both the spectral acquisition rate for TMT11plex experiments and the number of identifications for SPS-MS3. Additionally, the modified Orbitrap Tribrid mass spectrometer has an optimized quadrupole that improves ion transmission. Thus it is possible to use narrower isolation widths to improve TMT quantitation accuracy further. Overall, the Orbitrap Eclipse Tribrid mass spectrometer includes unique features such as Real Time Search for TMT SPS MS3 based quantitation, Precursor Fit filter, and TurboTMT, which together allow for intelligent acquisition methods that improve quantitation accuracy, precision, and proteome coverage.
REAL TIME SEARCH FOR SPS MS3 TRIGGERINGFigure 4. On the fly peptide identification for TMT quantitation. Real Time Search utilizes the open source search engine Comet. We evaluated the correlation of Xcorr scores between (4A.) Real Time Search (Online) and Comet post acquisition (offline) or (4B.) Proteome Discover (PD). Secondly, we evaluated the number of fragment ions selected for a 10 notch SPS MS3 that correctly matched the post acquisition identification in RTS SPS MS3 (4C.,4E.) or SPS MS3 (4D, 4F.).
Electrodynamic Ion Funnel
High-Capacity Transfer Tube
Ion Max NG Ion Source
EASY-IC/ETD/PTCR
Ion Source
Advanced Active Ion Beam Guide
QR5 Segmented Quadrupole Mass Filter with hyperbolic surfaces
C-Trap
Ion Routing Multipole
Ultra-High-Field Orbitrap Mass Analyzer
Higher CapacityHigh Pressure
Cell
Low Pressure Cell
Modified Dual-Pressure Linear Ion Trap
Figure 1. (1A.) Schematic representation of Orbitrap Eclipse Tribrid mass spectrometer.
A MODIFIED ORBITRAP TRIBRID MASS SPECTROMETER
1A.
Figure 2. (2A.) Schematic representation of Pierce TMT11plex Yeast Digest Standard. The standard is composed of four Saccharomyces cerevisiae strains: three lines respectively lacking the non-essential proteins Met6, His4, or Ura2, and the parental strain BY4741 for reference. Modified from Schweppe et al. 2019.
A TMT11PLEX YEAST DIGEST STANDARD
126met6Δ
his4Δ
ura2Δ
127N127C128N128C129N129C130N130C131131C
BY4741
4 strains of saccharomyces cervices
11 TMT channels technical replicates N=3 or 2 (for BY4741)
Samples combined into 1 LC/MS analysis
Proteome Discoverer Software
MS1 MS3
Pierce™ TMT11plex yeast digest standard
Knoc
kout
sta
ins
Pare
ntal
C
ontro
l
2A.
MS SelectionTMT Labeled Peptides
SPS MS3 Quantitation
RTS for MS3 Triggering
MS2 Acquisition
Real Time Search
<1ms per spectrum
m/z m/z m/z
ID ID No ID
2.0x
11073
7107
1085510599
6660
9196
0
2000
4000
6000
8000
10000
12000
MS2 SPS SPS RTS
Pept
ides
Identified Quantified
RTS SPS MS3SPS MS3
+53%2207
1340
20832087
1242
1909
0
500
1000
1500
2000
2500
MS2 SPS SPS RTS
Prot
eins
Identified Quantified
RTS SPS MS3SPS MS3
+55%
9%
SPSMS3
RTSSPSMS3
Less interference
Moreinterference
Less interference
Moreinterference
6 proteinspike in
HeLa
Expected ratio
0 1 2 4 8 12 16 20 24
16 channels
Table 1. Mass spectrometer data acquisition settings.
Acquisition Settings MS2 SPS MS3 RTS SPS MS3Top Speed (sec) 2.5 2.5 2.5
RF lens 30% 30% 30%
Orbitrap MS1 resolution 120000 120000 120000
Scan range (m/z) 400-1400 400-1400 400-1400
Standardized MS1 AGC target 100% 100% 100%
MS1 max IT (mode) Auto Auto Auto
Charge state 2-5 2-5 2-5
Dynamic Exclusion (sec) 45 45 45
MS2 resolution 45000 Turbo Turbo
MS2 Scan range (m/z) First mass 110 200-1200 200-1200
MS2 Isolation Window 0.7 m/z 0.7 m/z 0.7 m/z
Standardized MS2 AGC target 500% 100% 100%
MS2 max IT (mode) Auto Auto Auto
MS2 HCD NCE% 36 36 36
SPS MS3 Resolution - 50000 60000
MS2 Scan range (m/z) - 100-500 100-500
SPS MS3 Isolation Window - 0.7 m/z 0.7 m/z
Standardized SPS MS3 AGC target - 500% 500%
SPS MS3 max IT (mode) - Auto Auto
SPS MS3 HCD NCE% - 55 55
SPS MS3 Notches - 10 10
Figure 3. Achieving improved proteome depth and accuracy for SPS MS3 quantitation (3A.) Schematic representation of TMT workflow on Orbitrap Eclipse Tribrid mass spectrometer. We evaluated how Real Time Search on the modified Orbitrap Tribrid mass spectrometer influenced TMT identification rates for (3B.) proteins and (3C.) peptides, (3D.) as well as the interference free index at the peptide level as visualized in TKOmics.com. 500ng of TMT11plex Yeast Digest Standard was measured on a 50min gradient using MS2, SPS MS3, or RTS SPS MS3 methods.
ENHANCED TMT SPS MS3 PERFORMANCE
3A.
3C.3B.
3D.
XRTS SPS MS3 Dot plot
RTS SPS MS3Histogram
SPS MS3Histogram
SPS MS3 Dot plot
Pearson’s Correlation of 0.84Pearson’s Correlation of 1.004A. 4B.
4C. 4D.
4E.4F.
REAL TIME SEARCH IMPROVES TMT QUANTITATIONFigure 6. Evaluation of TMT quantification performance. We evaluated how Real Time Search affects TMT SPS MS3 quantitation precision (6A.) and accuracy (6B.) using Pierce 6 Protein Digest Standard mixed in various ratios into the Pierce HeLa Protein Digest Standard. 1ug of peptides was analyzed on a 120min gradient.
6%
10% 11%14% 14% 13%
15%
1% 2% 3% 3% 3%2%
4%
0%
5%
10%
15%
20%
2 4 8 12 16 20 24
Coe
ffici
ent o
f Var
iatio
n (C
V)
Expected Ratio
SPS MS3 RTS MS3
N= 15 peptides per protein
RTS SPS MS3
Expected SPSMS3
RTSSPSMS3
0
5
10
15
20
25
Obs
erve
d R
atio
Quantitation
Improved Accuracy
Improved Precision6A.
6B.
REAL TIME SEARCH ENABLES CUSTOM MODIFCIATIONS Figure 5. Next generation isobaric tags increases sample multiplexing. We evaluated the potential for Real Time Search to use custom modifications using a next generation isobaric tags. (5A.) Pierce 6 Protein Digest Standard mixed in various ratios into the Pierce HeLa Protein Digest Standard and labeled. (5B). 1ug of sample was then analyzed on a 120min gradient using RTS SPS MS3 or SPS MS3.
2589 3267
1489517695
0
5000
10000
15000
20000
SPS MS3 RTS SPS MS3
Cou
nt
Proteins Peptides
5A. 5B.
PO65536-EN0519S
Aaron M. Robitaille 1, Romain Huguet 1, Derek J. Bailey 1, Graeme McAlister 1, Arne Kreutzmann 2, Daniel Mourad 2, Daniel Lopez-Ferrer 1, Andreas Huhmer 1, Vlad Zabrouskov 1
1 Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134. 2 Thermo Fisher Scientific Hanna-Kunath-Straße 11, 28199 Bremen, Germany
ABSTRACTIsobaric tagging strategies using Thermo Scientific™ Tandem Mass Tags™ (TMT™) are powerful tools for studying how proteins interact and function in biological systems. Up to 11 samples can be multiplexing in a single high-resolution LC/MS experiment to enable state-of-the-art quantitative analysis of peptide and protein abundance. Here we evaluate the benefits of the Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer including real time search capabilities, advanced spectral processing algorithms, and modified hardware to enhance TMT quantification accuracy and proteome coverage.
INTRODUCTIONTo assess the accuracy, precision, and sensitivity of the Orbitrap Eclipse Tribrid mass spectrometer for TMT based quantitation, we utilized the Thermo Scientific™ Pierce™ TMT11plex yeast digest standard. This standardized sample provides users with a tool to measure the accuracy, precision, and proteome depth of TMT methods across different instrumentation. Quantitative proteomics strategies using Tandem Mass Tags (TMT) allow up to 11 samples to be multiplexing in a single high resolution LC/MS experiment while enabling precise measurement of protein abundance. However, co-isolated ion interference can suppress ratio quantification and thereby mask true differences in protein abundance. Multiple methods and hardware solutions help alleviate the negative influence of interfering ions including Synchronous Precursor Selection (SPS) and a Thermo Scientific™ FAIMS Pro™ interface. However, all of these solutions come with draw backs. Furthermore, high resolution MS2 or MS3 scanning is necessary for accurate ratio determination in greater than TMT6plex experiments, which can reduce the frequency of acquisition. Here we evaluated the effects of Real Time Search on a modified Orbitrap Tribrid mass spectrometer to reduce the above stated limitations on TMT SPS MS3 quantitative experiments.
MATERIALS AND METHODSTo assess the sensitivity of the modified Orbitrap™ Tribrid™ mass spectrometer for TMT based quantitation, we utilized the Pierce TMT11plex yeast digest standard. For liquid chromatography (LC) conditions, we used an analytical gradient from 8-32% acetonitrile (vol/vol) with 0.1% (vol/vol) formic acid in 50min with a column heater set to 45°C, unless otherwise indicated. Experiments were run with a Thermo Scientific™ EASY-nLC™ 1200 HPLC system in combination with a ThermoScientific™ EASY-Spray™ C18 50cm column coupled to a Thermo Scientific™ EASY-Spray™ ion source. Samples were analyzed on a Orbitrap Eclipse Tribrid mass spectrometer. Settings for Orbitrap Tribrid Series Instrument Control Software Version 3.3 follow below (Table 1). Raw data files were processed using Thermo Scientific™ Proteome Discoverer™ 2.3 software using the SEQUEST® HT search engine with a 10ppm MS1 and 0.5 Da MS2 mass tolerance, TMT6plex (229.163 Da) set as a static modification, and a 1% false discovery rate.
CONCLUSIONSWe present a modified Orbitrap Tribrid mass spectrometer utilizing Real Time Search that improved identification rates and accuracy for TMT SPS MS3 based quantitation.
REFERENCES1. Paulo et al. A Triple Knockout (TKO) Proteomics Standard for Diagnosing Ion Interference in Isobaric
Labeling Experiments. J. Am. Soc. Mass Spectrom. 2016, 27: 16202. Erickson et al. Active Instrument Engagement Combined with a Real-Time Database Search for
Improved Performance of Sample Multiplexing Workflows. J Proteome Res. 2019 1;18(3):1299-1306. 3. Schweppe et al. Characterization and Optimization of Multiplexed Quantitative Analyses Using High-
Field Asymmetric-Waveform Ion Mobility Mass Spectrometry. Anal. Chem. 2019, 91, 6, 4010-4016
ACKNOWLEDGEMENTSWe would like to thank Ryan Bomgarden, Derek Bailey now at Google, Jimmy Eng from the University of Washington, and Steven Gygi, Devin Schweppe, and Qing Yu from the Department of Cell Biology at Harvard Medical School in Boston, USA for their collaboration and input.
TRADEMARKS/LICENSING© 2019 Thermo Fisher Scientific Inc. All rights reserved. Tandem Mass Tag and TMT are trademarks of Proteome Sciences plc. SEQUEST is a registered trademark of the University of Washington. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.
A Modified Orbitrap Tribrid Mass Spectrometer with Real Time Search and Advanced Spectral Processing Enhances Multiplexed Proteome Coverage and Quantification Accuracy
RESULTSSynchronous precursor selection (SPS) based methods provided higher accuracy compared to MS2 methods for TMT quantitation. However, depending on precursor isolation specificity and which fragments are selected for MS3 fragmentation, quantitative accuracy can still be distorted. To improve upon existing SPS methods, we implemented a Real Time Search (RTS) filter between the MS2 and MS3 scans. This feature benefits TMT SPS MS3 methods in two distinct ways. First, MS3 scans are only triggered if a peptide-spectrum match (PSM) is identified from the preceding MS2. This increased the number of peptides identified with SPS MS3. Secondly, Real Time Search identifies precursor fragments that are generated from the identified peptide on the fly, and then selects those fragment ions for MS3 quantitation. Thus, TMT SPS MS3 quantitation with Real Time Search can be improved to be 95% interference free. Next, we evaluated a new feature call TurboTMT, powered by the ΦSDM algorithm. ΦSDM is an advanced spectra processing algorithm that increases resolution within a range of the spectrum without requiring a longer transient. Applying ΦSDM specifically to the TMT reporter ions increased the resolution sufficient to baseline resolve TMT isotopologues even when using transients that produce a 30,000 or 15,000 resolving power MS2 scan. ΦSDM increased both the spectral acquisition rate for TMT11plex experiments and the number of identifications for SPS-MS3. Additionally, the modified Orbitrap Tribrid mass spectrometer has an optimized quadrupole that improves ion transmission. Thus it is possible to use narrower isolation widths to improve TMT quantitation accuracy further. Overall, the Orbitrap Eclipse Tribrid mass spectrometer includes unique features such as Real Time Search for TMT SPS MS3 based quantitation, Precursor Fit filter, and TurboTMT, which together allow for intelligent acquisition methods that improve quantitation accuracy, precision, and proteome coverage.
REAL TIME SEARCH FOR SPS MS3 TRIGGERINGFigure 4. On the fly peptide identification for TMT quantitation. Real Time Search utilizes the open source search engine Comet. We evaluated the correlation of Xcorr scores between (4A.) Real Time Search (Online) and Comet post acquisition (offline) or (4B.) Proteome Discover (PD). Secondly, we evaluated the number of fragment ions selected for a 10 notch SPS MS3 that correctly matched the post acquisition identification in RTS SPS MS3 (4C.,4E.) or SPS MS3 (4D, 4F.).
Electrodynamic Ion Funnel
High-Capacity Transfer Tube
Ion Max NG Ion Source
EASY-IC/ETD/PTCR
Ion Source
Advanced Active Ion Beam Guide
QR5 Segmented Quadrupole Mass Filter with hyperbolic surfaces
C-Trap
Ion Routing Multipole
Ultra-High-Field Orbitrap Mass Analyzer
Higher CapacityHigh Pressure
Cell
Low Pressure Cell
Modified Dual-Pressure Linear Ion Trap
Figure 1. (1A.) Schematic representation of Orbitrap Eclipse Tribrid mass spectrometer.
A MODIFIED ORBITRAP TRIBRID MASS SPECTROMETER
1A.
Figure 2. (2A.) Schematic representation of Pierce TMT11plex Yeast Digest Standard. The standard is composed of four Saccharomyces cerevisiae strains: three lines respectively lacking the non-essential proteins Met6, His4, or Ura2, and the parental strain BY4741 for reference. Modified from Schweppe et al. 2019.
A TMT11PLEX YEAST DIGEST STANDARD
126met6Δ
his4Δ
ura2Δ
127N127C128N128C129N129C130N130C131131C
BY4741
4 strains of saccharomyces cervices
11 TMT channels technical replicates N=3 or 2 (for BY4741)
Samples combined into 1 LC/MS analysis
Proteome Discoverer Software
MS1 MS3
Pierce™ TMT11plex yeast digest standard
Knoc
kout
sta
ins
Pare
ntal
C
ontro
l
2A.
MS SelectionTMT Labeled Peptides
SPS MS3 Quantitation
RTS for MS3 Triggering
MS2 Acquisition
Real Time Search
<1ms per spectrum
m/z m/z m/z
ID ID No ID
2.0x
11073
7107
1085510599
6660
9196
0
2000
4000
6000
8000
10000
12000
MS2 SPS SPS RTS
Pept
ides
Identified Quantified
RTS SPS MS3SPS MS3
+53%2207
1340
20832087
1242
1909
0
500
1000
1500
2000
2500
MS2 SPS SPS RTS
Prot
eins
Identified Quantified
RTS SPS MS3SPS MS3
+55%
9%
SPSMS3
RTSSPSMS3
Less interference
Moreinterference
Less interference
Moreinterference
6 proteinspike in
HeLa
Expected ratio
0 1 2 4 8 12 16 20 24
16 channels
Table 1. Mass spectrometer data acquisition settings.
Acquisition Settings MS2 SPS MS3 RTS SPS MS3Top Speed (sec) 2.5 2.5 2.5
RF lens 30% 30% 30%
Orbitrap MS1 resolution 120000 120000 120000
Scan range (m/z) 400-1400 400-1400 400-1400
Standardized MS1 AGC target 100% 100% 100%
MS1 max IT (mode) Auto Auto Auto
Charge state 2-5 2-5 2-5
Dynamic Exclusion (sec) 45 45 45
MS2 resolution 45000 Turbo Turbo
MS2 Scan range (m/z) First mass 110 200-1200 200-1200
MS2 Isolation Window 0.7 m/z 0.7 m/z 0.7 m/z
Standardized MS2 AGC target 500% 100% 100%
MS2 max IT (mode) Auto Auto Auto
MS2 HCD NCE% 36 36 36
SPS MS3 Resolution - 50000 60000
MS2 Scan range (m/z) - 100-500 100-500
SPS MS3 Isolation Window - 0.7 m/z 0.7 m/z
Standardized SPS MS3 AGC target - 500% 500%
SPS MS3 max IT (mode) - Auto Auto
SPS MS3 HCD NCE% - 55 55
SPS MS3 Notches - 10 10
Figure 3. Achieving improved proteome depth and accuracy for SPS MS3 quantitation (3A.) Schematic representation of TMT workflow on Orbitrap Eclipse Tribrid mass spectrometer. We evaluated how Real Time Search on the modified Orbitrap Tribrid mass spectrometer influenced TMT identification rates for (3B.) proteins and (3C.) peptides, (3D.) as well as the interference free index at the peptide level as visualized in TKOmics.com. 500ng of TMT11plex Yeast Digest Standard was measured on a 50min gradient using MS2, SPS MS3, or RTS SPS MS3 methods.
ENHANCED TMT SPS MS3 PERFORMANCE
3A.
3C.3B.
3D.
XRTS SPS MS3 Dot plot
RTS SPS MS3Histogram
SPS MS3Histogram
SPS MS3 Dot plot
Pearson’s Correlation of 0.84Pearson’s Correlation of 1.004A. 4B.
4C. 4D.
4E.4F.
REAL TIME SEARCH IMPROVES TMT QUANTITATIONFigure 6. Evaluation of TMT quantification performance. We evaluated how Real Time Search affects TMT SPS MS3 quantitation precision (6A.) and accuracy (6B.) using Pierce 6 Protein Digest Standard mixed in various ratios into the Pierce HeLa Protein Digest Standard. 1ug of peptides was analyzed on a 120min gradient.
6%
10% 11%14% 14% 13%
15%
1% 2% 3% 3% 3%2%
4%
0%
5%
10%
15%
20%
2 4 8 12 16 20 24
Coe
ffici
ent o
f Var
iatio
n (C
V)
Expected Ratio
SPS MS3 RTS MS3
N= 15 peptides per protein
RTS SPS MS3
Expected SPSMS3
RTSSPSMS3
0
5
10
15
20
25
Obs
erve
d R
atio
Quantitation
Improved Accuracy
Improved Precision6A.
6B.
REAL TIME SEARCH ENABLES CUSTOM MODIFCIATIONS Figure 5. Next generation isobaric tags increases sample multiplexing. We evaluated the potential for Real Time Search to use custom modifications using a next generation isobaric tags. (5A.) Pierce 6 Protein Digest Standard mixed in various ratios into the Pierce HeLa Protein Digest Standard and labeled. (5B). 1ug of sample was then analyzed on a 120min gradient using RTS SPS MS3 or SPS MS3.
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SPS MS3 RTS SPS MS3
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5A. 5B.
PO65536-EN0519S
Find out more at thermofisher.com/OrbitrapEclipse
© 2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representatives for details. PN65536-EN 0819M
Aaron M. Robitaille 1, Romain Huguet 1, Derek J. Bailey 1, Graeme McAlister 1, Arne Kreutzmann 2, Daniel Mourad 2, Daniel Lopez-Ferrer 1, Andreas Huhmer 1, Vlad Zabrouskov 1
1 Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134. 2 Thermo Fisher Scientific Hanna-Kunath-Straße 11, 28199 Bremen, Germany
ABSTRACTIsobaric tagging strategies using Thermo Scientific™ Tandem Mass Tags™ (TMT™) are powerful tools for studying how proteins interact and function in biological systems. Up to 11 samples can be multiplexing in a single high-resolution LC/MS experiment to enable state-of-the-art quantitative analysis of peptide and protein abundance. Here we evaluate the benefits of the Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer including real time search capabilities, advanced spectral processing algorithms, and modified hardware to enhance TMT quantification accuracy and proteome coverage.
INTRODUCTIONTo assess the accuracy, precision, and sensitivity of the Orbitrap Eclipse Tribrid mass spectrometer for TMT based quantitation, we utilized the Thermo Scientific™ Pierce™ TMT11plex yeast digest standard. This standardized sample provides users with a tool to measure the accuracy, precision, and proteome depth of TMT methods across different instrumentation. Quantitative proteomics strategies using Tandem Mass Tags (TMT) allow up to 11 samples to be multiplexing in a single high resolution LC/MS experiment while enabling precise measurement of protein abundance. However, co-isolated ion interference can suppress ratio quantification and thereby mask true differences in protein abundance. Multiple methods and hardware solutions help alleviate the negative influence of interfering ions including Synchronous Precursor Selection (SPS) and a Thermo Scientific™ FAIMS Pro™ interface. However, all of these solutions come with draw backs. Furthermore, high resolution MS2 or MS3 scanning is necessary for accurate ratio determination in greater than TMT6plex experiments, which can reduce the frequency of acquisition. Here we evaluated the effects of Real Time Search on a modified Orbitrap Tribrid mass spectrometer to reduce the above stated limitations on TMT SPS MS3 quantitative experiments.
MATERIALS AND METHODSTo assess the sensitivity of the modified Orbitrap™ Tribrid™ mass spectrometer for TMT based quantitation, we utilized the Pierce TMT11plex yeast digest standard. For liquid chromatography (LC) conditions, we used an analytical gradient from 8-32% acetonitrile (vol/vol) with 0.1% (vol/vol) formic acid in 50min with a column heater set to 45°C, unless otherwise indicated. Experiments were run with a Thermo Scientific™ EASY-nLC™ 1200 HPLC system in combination with a ThermoScientific™ EASY-Spray™ C18 50cm column coupled to a Thermo Scientific™ EASY-Spray™ ion source. Samples were analyzed on a Orbitrap Eclipse Tribrid mass spectrometer. Settings for Orbitrap Tribrid Series Instrument Control Software Version 3.3 follow below (Table 1). Raw data files were processed using Thermo Scientific™ Proteome Discoverer™ 2.3 software using the SEQUEST® HT search engine with a 10ppm MS1 and 0.5 Da MS2 mass tolerance, TMT6plex (229.163 Da) set as a static modification, and a 1% false discovery rate.
CONCLUSIONSWe present a modified Orbitrap Tribrid mass spectrometer utilizing Real Time Search that improved identification rates and accuracy for TMT SPS MS3 based quantitation.
REFERENCES1. Paulo et al. A Triple Knockout (TKO) Proteomics Standard for Diagnosing Ion Interference in Isobaric
Labeling Experiments. J. Am. Soc. Mass Spectrom. 2016, 27: 16202. Erickson et al. Active Instrument Engagement Combined with a Real-Time Database Search for
Improved Performance of Sample Multiplexing Workflows. J Proteome Res. 2019 1;18(3):1299-1306. 3. Schweppe et al. Characterization and Optimization of Multiplexed Quantitative Analyses Using High-
Field Asymmetric-Waveform Ion Mobility Mass Spectrometry. Anal. Chem. 2019, 91, 6, 4010-4016
ACKNOWLEDGEMENTSWe would like to thank Ryan Bomgarden, Derek Bailey now at Google, Jimmy Eng from the University of Washington, and Steven Gygi, Devin Schweppe, and Qing Yu from the Department of Cell Biology at Harvard Medical School in Boston, USA for their collaboration and input.
TRADEMARKS/LICENSING© 2019 Thermo Fisher Scientific Inc. All rights reserved. Tandem Mass Tag and TMT are trademarks of Proteome Sciences plc. SEQUEST is a registered trademark of the University of Washington. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.
A Modified Orbitrap Tribrid Mass Spectrometer with Real Time Search and Advanced Spectral Processing Enhances Multiplexed Proteome Coverage and Quantification Accuracy
RESULTSSynchronous precursor selection (SPS) based methods provided higher accuracy compared to MS2 methods for TMT quantitation. However, depending on precursor isolation specificity and which fragments are selected for MS3 fragmentation, quantitative accuracy can still be distorted. To improve upon existing SPS methods, we implemented a Real Time Search (RTS) filter between the MS2 and MS3 scans. This feature benefits TMT SPS MS3 methods in two distinct ways. First, MS3 scans are only triggered if a peptide-spectrum match (PSM) is identified from the preceding MS2. This increased the number of peptides identified with SPS MS3. Secondly, Real Time Search identifies precursor fragments that are generated from the identified peptide on the fly, and then selects those fragment ions for MS3 quantitation. Thus, TMT SPS MS3 quantitation with Real Time Search can be improved to be 95% interference free. Next, we evaluated a new feature call TurboTMT, powered by the ΦSDM algorithm. ΦSDM is an advanced spectra processing algorithm that increases resolution within a range of the spectrum without requiring a longer transient. Applying ΦSDM specifically to the TMT reporter ions increased the resolution sufficient to baseline resolve TMT isotopologues even when using transients that produce a 30,000 or 15,000 resolving power MS2 scan. ΦSDM increased both the spectral acquisition rate for TMT11plex experiments and the number of identifications for SPS-MS3. Additionally, the modified Orbitrap Tribrid mass spectrometer has an optimized quadrupole that improves ion transmission. Thus it is possible to use narrower isolation widths to improve TMT quantitation accuracy further. Overall, the Orbitrap Eclipse Tribrid mass spectrometer includes unique features such as Real Time Search for TMT SPS MS3 based quantitation, Precursor Fit filter, and TurboTMT, which together allow for intelligent acquisition methods that improve quantitation accuracy, precision, and proteome coverage.
REAL TIME SEARCH FOR SPS MS3 TRIGGERINGFigure 4. On the fly peptide identification for TMT quantitation. Real Time Search utilizes the open source search engine Comet. We evaluated the correlation of Xcorr scores between (4A.) Real Time Search (Online) and Comet post acquisition (offline) or (4B.) Proteome Discover (PD). Secondly, we evaluated the number of fragment ions selected for a 10 notch SPS MS3 that correctly matched the post acquisition identification in RTS SPS MS3 (4C.,4E.) or SPS MS3 (4D, 4F.).
Electrodynamic Ion Funnel
High-Capacity Transfer Tube
Ion Max NG Ion Source
EASY-IC/ETD/PTCR
Ion Source
Advanced Active Ion Beam Guide
QR5 Segmented Quadrupole Mass Filter with hyperbolic surfaces
C-Trap
Ion Routing Multipole
Ultra-High-Field Orbitrap Mass Analyzer
Higher CapacityHigh Pressure
Cell
Low Pressure Cell
Modified Dual-Pressure Linear Ion Trap
Figure 1. (1A.) Schematic representation of Orbitrap Eclipse Tribrid mass spectrometer.
A MODIFIED ORBITRAP TRIBRID MASS SPECTROMETER
1A.
Figure 2. (2A.) Schematic representation of Pierce TMT11plex Yeast Digest Standard. The standard is composed of four Saccharomyces cerevisiae strains: three lines respectively lacking the non-essential proteins Met6, His4, or Ura2, and the parental strain BY4741 for reference. Modified from Schweppe et al. 2019.
A TMT11PLEX YEAST DIGEST STANDARD
126met6Δ
his4Δ
ura2Δ
127N127C128N128C129N129C130N130C131131C
BY4741
4 strains of saccharomyces cervices
11 TMT channels technical replicates N=3 or 2 (for BY4741)
Samples combined into 1 LC/MS analysis
Proteome Discoverer Software
MS1 MS3
Pierce™ TMT11plex yeast digest standard
Knoc
kout
sta
ins
Pare
ntal
C
ontro
l
2A.
MS SelectionTMT Labeled Peptides
SPS MS3 Quantitation
RTS for MS3 Triggering
MS2 Acquisition
Real Time Search
<1ms per spectrum
m/z m/z m/z
ID ID No ID
2.0x
11073
7107
1085510599
6660
9196
0
2000
4000
6000
8000
10000
12000
MS2 SPS SPS RTS
Pept
ides
Identified Quantified
RTS SPS MS3SPS MS3
+53%2207
1340
20832087
1242
1909
0
500
1000
1500
2000
2500
MS2 SPS SPS RTS
Prot
eins
Identified Quantified
RTS SPS MS3SPS MS3
+55%
9%
SPSMS3
RTSSPSMS3
Less interference
Moreinterference
Less interference
Moreinterference
6 proteinspike in
HeLa
Expected ratio
0 1 2 4 8 12 16 20 24
16 channels
Table 1. Mass spectrometer data acquisition settings.
Acquisition Settings MS2 SPS MS3 RTS SPS MS3Top Speed (sec) 2.5 2.5 2.5
RF lens 30% 30% 30%
Orbitrap MS1 resolution 120000 120000 120000
Scan range (m/z) 400-1400 400-1400 400-1400
Standardized MS1 AGC target 100% 100% 100%
MS1 max IT (mode) Auto Auto Auto
Charge state 2-5 2-5 2-5
Dynamic Exclusion (sec) 45 45 45
MS2 resolution 45000 Turbo Turbo
MS2 Scan range (m/z) First mass 110 200-1200 200-1200
MS2 Isolation Window 0.7 m/z 0.7 m/z 0.7 m/z
Standardized MS2 AGC target 500% 100% 100%
MS2 max IT (mode) Auto Auto Auto
MS2 HCD NCE% 36 36 36
SPS MS3 Resolution - 50000 60000
MS2 Scan range (m/z) - 100-500 100-500
SPS MS3 Isolation Window - 0.7 m/z 0.7 m/z
Standardized SPS MS3 AGC target - 500% 500%
SPS MS3 max IT (mode) - Auto Auto
SPS MS3 HCD NCE% - 55 55
SPS MS3 Notches - 10 10
Figure 3. Achieving improved proteome depth and accuracy for SPS MS3 quantitation (3A.) Schematic representation of TMT workflow on Orbitrap Eclipse Tribrid mass spectrometer. We evaluated how Real Time Search on the modified Orbitrap Tribrid mass spectrometer influenced TMT identification rates for (3B.) proteins and (3C.) peptides, (3D.) as well as the interference free index at the peptide level as visualized in TKOmics.com. 500ng of TMT11plex Yeast Digest Standard was measured on a 50min gradient using MS2, SPS MS3, or RTS SPS MS3 methods.
ENHANCED TMT SPS MS3 PERFORMANCE
3A.
3C.3B.
3D.
XRTS SPS MS3 Dot plot
RTS SPS MS3Histogram
SPS MS3Histogram
SPS MS3 Dot plot
Pearson’s Correlation of 0.84Pearson’s Correlation of 1.004A. 4B.
4C. 4D.
4E.4F.
REAL TIME SEARCH IMPROVES TMT QUANTITATIONFigure 6. Evaluation of TMT quantification performance. We evaluated how Real Time Search affects TMT SPS MS3 quantitation precision (6A.) and accuracy (6B.) using Pierce 6 Protein Digest Standard mixed in various ratios into the Pierce HeLa Protein Digest Standard. 1ug of peptides was analyzed on a 120min gradient.
6%
10% 11%14% 14% 13%
15%
1% 2% 3% 3% 3%2%
4%
0%
5%
10%
15%
20%
2 4 8 12 16 20 24
Coe
ffici
ent o
f Var
iatio
n (C
V)
Expected Ratio
SPS MS3 RTS MS3
N= 15 peptides per protein
RTS SPS MS3
Expected SPSMS3
RTSSPSMS3
0
5
10
15
20
25
Obs
erve
d R
atio
Quantitation
Improved Accuracy
Improved Precision6A.
6B.
REAL TIME SEARCH ENABLES CUSTOM MODIFCIATIONS Figure 5. Next generation isobaric tags increases sample multiplexing. We evaluated the potential for Real Time Search to use custom modifications using a next generation isobaric tags. (5A.) Pierce 6 Protein Digest Standard mixed in various ratios into the Pierce HeLa Protein Digest Standard and labeled. (5B). 1ug of sample was then analyzed on a 120min gradient using RTS SPS MS3 or SPS MS3.
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5000
10000
15000
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SPS MS3 RTS SPS MS3
Cou
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5A. 5B.
PO65536-EN0519S