Second-Generation Electron Transfer Dissociation (ETD) on the Thermo Scientifi c Orbitrap Fusion Mass Spectrometer with Improved Functionality, Increased Speed, and Improved Robustness of Data

Christopher Mullen,1 Lee Earley,1 Jean-Jacques Dunyach,1 John E.P. Syka,1 Philip D. Compton,2 Dina L. Bai,3 Jefferey Shabanowitz,3 and Donald F. Hunt3 1Thermo Fisher Scientifi c, San Jose, CA; 2Kelleher Lab, Northwestern University, Evanston, IL; 3Department of Chemistry, University of Virginia, Charlottesville, VA Second-Generation Electron Transfer Dissociation (ETD) on the Thermo Scientific Orbitrap Fusion Mass Spectrometer with Improved Functionality, Increased Speed, and Improved Robustness of Data Christopher Mullen1, Lee Earley1, Jean-Jacques Dunyach1, John E.P. Syka1, Philip D. Compton2, Dina L. Bai3, Jefferey Shabanowitz3, and Donald F. Hunt3 1Thermo Fisher Scientific, San Jose, CA; 2Kelleher Lab, Northwestern University, Evanston, IL; 3Department of Chemistry, University of Virginia, Charlottesville, VA

TABLE 1. Calculated charge state dependent reaction times based on a Verification of the Calibration Unique ETD Capabilities Overview Results saturated reaction rate coefficient of 58.2 sec–1 for charge state 3+, based on 5% The calibrated ETD reaction conditions are verified by using an infusion of ubiquitin The location of the ion-routing multipole within the Orbitrap Fusion mass spectrometer of the precursor remaining after reaction. Purpose: Improve ETD robustness, functionality, and speed on the Thermo Calibrating the reaction kinetics ensures that the ETD fragmentation efficiency is from bovine erythrocytes. Spectra of the 12+ charge state at 714.7 m/z were obtained allows for a parallel ITMS2 acquisition mode, enabling ITMS2 CID and HCD spectral Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer optimized and that the maximum duty cycle for ETD can be accomplished. as a function of the ETD reaction time at an FT resolution of 120K and averaged for acquisition rates up to 20 Hz. ITMS2 ETD spectral acquisition rates are slightly reduced Charge State Reaction Time (msec) Methods: Orbitrap Fusion mass spectrometer with the Thermo Scientific Calibrating the reaction kinetics is a multi-step process in which the decay of the 100 micro scans. The spectra were then searched using ProSightPC™. Figure 6 due to the additional time requirement of the ion-ion reaction, but rates up to 12 Hz are Easy-ETD™ source angiotensin I (433 m/z) precursor is monitored as a function of reaction time at a 2 116 shows two representative ubiquitin spectra, obtained at 3.25 msec and 8 msec of attainable. In addition, the ability to perform the ion-ion reaction and m/z analysis in number of reagent targets (Figure 2). From the slope of the individual decay curves, 3 52 reaction time, respectively. The calibrated reaction conditions predict an optimal parallel with the precursor injection means that the spectral acquisition rate at a Results: Demonstrated increased ETD functionality and usability by using a reaction time of 3.25 msec for the 12+ precursor, and while the spectrum visually looks particular reaction time can be maintained for a significantly longer precursor injection the reaction rate coefficient is extracted, and plotted as a function of the reagent target 4 29 combination of hardware and software improvements under reacted, it yields the most total c and z fragments from the ProSightPC v3.0 time than in the absence of parallelization (Figure 9). at which it was acquired (Figure 3). The data are then fitted to find the target at which 5 19 the reaction rate coefficient saturates, and combinations of this target with the reaction search (Figure 7). In addition, we demonstrate the ability to obtain nearly complete 6 13 rate coefficient are used to calculate the optimal charge state dependent reaction times ubiquitin sequence coverage on a LC timescale using a combination of ProSightPC Introduction 7 9.5 searching and manual interpretation of the spectra. The results presented in Figure 8 (Table 1). The reaction rate coefficient as a function of the precursor charge state FIGURE 9. Ion-trap ETD MSMS cycle rate dependence on reaction and precursor The Orbitrap Fusion platform incorporates a second-generation Easy-ETD reagent ion were achieved by averaging 20 FT micro scans, corresponding to a total acquisition squared has been demonstrated to be linear by J. L. Stephenson Jr. and S. A. 8 7.2 injection times using parallel acquisition. The reagent injection time was fixed at source. The Easy-ETD source improvements include a bright and stable glow- 3 time of 6.7 seconds. McLuckey , which is verified in Figure 4, and used to calculate the optimal reaction 9 5.7 5 msec, corresponding to a reagent anion population of 2e5 for all experiments. discharge-based ETD reagent ion source located between the S-Lens and the Active time per charge state, based on a desired amount of reaction completeness. We find 10 4.6 Beam Guide, and a higher frequency RF axial trapping field (trap end-lens voltage) to 95% consumption of the initial precursor intensity to yield high quality ETD spectra. FIGURE 6. ETD spectra obtained on the 12+ charge state of ubiquitin at improve ion confinement during ETD (Figure 1). The Orbitrap Fusion mass Figure 5 shows the relationship between the reaction time and the amount of precursor 11 3.8 714.7 m/z at two different reaction times. A) 3.25 msec, the calibrated reaction 100 spectrometer design enables previously unavailable parallel/pipelined scan modes to remaining after reaction for the angiotensin I (433 m/z) precursor. 12 3.2 time. B) 8 msec.

minimize overall scan cycle times. Further, calibration of the ETD reaction kinetics Ubiquitin_3pt25msec_2e5_100uscans #1 RT: 0.00 AV: 1 NL: 7.96E6 T: FTMS + p ESI Full ms2 [email protected] [150.00-2000.00] 90 714.6434 ensures the shortest possible reaction times while maximizing product ion yields and 100 A) Frequency (Hz) FIGURE 2. Angiotensen I (433 m/z) precursor decay curves as a function of the 90 spectral reproducibility. Collectively, these developments constitute a new-generation 779.6105 80 reagent anion target under pseudo 1st order reaction conditions. The reaction FIGURE 4. The maximum rate coefficient vs. the charge state squared of the 80 ETD platform on the Orbitrap Fusion mass spectrometer. 70 1 progress is monitored for up to four half-lives, and the slope of the individual precursor is linear , allowing extrapolation of the optimal reaction times 60 70 5. 000 50 691.5779 818.7508 6. 000 decays is equal to the negative of the rate coefficient. obtained for a single charge state to all charge states 40 857.5713 Relative Abundance Relative 30 640.3763 277.1327 60 Methods 390.2168 537.2855 898.4890 7. 000 20 978.5804 243.6364 433.2516 1136.6495 1.2 10 1023.5648 1159.3170 328.2096 502.8156 1347.2285 1,2 167.9136 1264.7439 1580.9261 8. 000 Reagent anions from a glow discharge source (previously described) are introduced 0 50 0.0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 into the ion optics path ahead of the quadrupole mass filter where they are m/z 1.1 m/z 9. 000 ubiquitin_8msec_2e5_100uscans #1 RT: 0.00 AV: 1 NL: 3.24E6 T: FTMS + p ESI Full ms2 [email protected] [150.00-2000.00] 40 selected, accumulated in the ion-routing multipole, and then transferred into the high 277.1326 10 .00 1.0 100

pressure trap (HPT) of the dual-pressure linear ion trap for the ETD reaction. -0.5 Ubiquitin 90 B) 30 11 .00 0.9 Angiotensen 80 390.2166 640.3759 Increasing the frequency of the RF axial confinement field during ETD from ½ to 2 537.2852 70 1136.6490 12 .00 times the quadrupole field frequency avoids parametric resonance excitation and 0.8 60 20 898.4885 961.0632 -1.0 50 664.3729 (msec) Time Reaction ETD 13 .00 ejection of low m/z (typically 120–130 Th) ions. ETD products may be directly 602.3616 717.9185 40 433.2514 ) 243.6363 Relative Abundance Relative

) 0.7 790.4649 -1 30 10 0 1079.6276 1347.2284 transferred to the low pressure trap (LPT) or to the Orbitrap Fusion mass spectrometer 1023.5646 20 1159.3162 328.2094 2 167.9123 1282.7065 502.8152 for m/z analysis. ITMS ETD scan rates of up to 12 Hz are attainable using a parallel -1.5 Targ et: 0.1e 5 0.6 10 1414.2650 1518.3324 1580.9260 1705.9368 1921.1191 Targ et: 0.2e 5 0 acquisition mode. 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 ln(A/A (msec 10 20 30 40 50 60 70 80 90 100 Targ et: 0.4e 5 0.5 m/z

Targ et: 0.6e 5 m ax FIGURE 1. Schematic of the Orbitrap Fusion tribrid mass spectrometer showing -2.0 Targ et: 0.7e 5 k 0.4 Precursor Injection Time (msec) Targ et: 0.8e 5 FIGURE 7. ProSightPC search results for the 12+ charge state of ubiquitin (714.7 the location of the Easy-ETD reagent ion source within the overall ion optics Targ et: 1.0e 5 0.3 m/z) as a function of the ETD reaction time, demonstrating that the calibrated path. The exploded view shows how the reagent ion source is incorporated into -2.5 Targ et: 2.0e 5 Targ et: 4.0e 5 0.2 kinetics chooses appropriate reaction times up to at least charge state 12. the S-Lens/Q00 region. Targ et: 9.9e 5 Dual-pressure linear ion trap 0.1 Fragments ions using either CID -3.0 Conclusion or optional ETD and provides Ion-routing multipole fast, sensitive mass analysis 0 10 20 30 40 50 . A second-generation glow-discharge-based Easy-ETD source has been . High-pressure cell produces 0 20 40 60 80 100 120 140 160 180 110 highly efficient storage of ions developed for the Orbitrap Fusion MS that incorporates significant hardware and . Voltages can be adjusted to Reaction Time (msec) 2 fragment ions at higher energies Charge State Square (z ) c software advancements. 100 z . Calibration of the reaction kinetics removes the guesswork from ETD, and leads to Ultra-high-field Orbitrap mass analyzer FIGURE 3. The reaction rate coefficient versus reagent anion target, showing total conditions that optimizes the ETD reaction and scan cycle time. . Compact Orbitrap mass spectrometer FIGURE 5. Plot of the reaction time required to reduce the reactant precursor by the fit to the data and the optimal reagent anion target leading to at least 90% 90 operating at ultra-high voltage a fixed amount, calculated as a function of the reaction rate coefficient. We find . . Produces 500K resolution every of the k observed. The calibrated reaction conditions are demonstrated to provide optimal conditions 1.2 seconds max that 3 to 6% remaining precursor is optimal for most compounds. for ETD identifications and sequence coverage. . Ultra-fast operation produces 80 15 spectra/sec with 15K resolution . 2 0.07 Parallel acquisition provides ITMS ETD scan rate cycle times of up to 12 Hz. 100 50 C-Trap 0.06 Rate Coefficient References Active beam guide (ABG) Ions are focused and -1 . Prevents neutral species from injected into the Orbitrap 40 sec Number of Fragments 1. Earley et al., 61st ASMS Conference on Mass Spectrometry and Allied Topics, entering Q1 mass spectrometer 80 -1 . Axial fields improves 0.05 60 sec 40 Minneapolis, MN, June 9–13, 2013; Poster Th101 – Implementation of a operational robustness Quadrupole mass filter High Selectivity and -1 Multipurpose Glow Discharge Ion Source for the Introduction of Reagent/ excellent transmission 80 sec

) 0.04 60 Calibrant Ions Into a Hybrid Mass Spectrometer, poster number: 101, Thursday,

-1 k 30 Halls B&C. Easy-ETD ion source Fit to Data 0 1 2 3 4 5 6 7 8 9 . Discharge-based source 0.03 2. Earley et al., Presented at the 58th ASMS Conference on Mass Spectrometry and . Robust design with an extremely Best Target 40 ETD Reaction Time (msec) stable source of ions

k (msec Allied Topics, Salt Lake City, Utah, May 23–27, 2010; Poster T600. S-Lens . High sensitivity 0.02 FIGURE 8. Ubiquitin sequence coverage resulting from averaging 20 FT micro 3. Stephenson, J. L., Jr. and McLuckey, S.A. J. Am. Chem. Soc. 1996, 118, . Robust ion optics 20 scans for a total acquisition time of 6.7 seconds. Fragments denoted by the red 7390–7397.

Precursor Remaining (%) Remaining Precursor (c ion), blue (z ion), and green (y ion) asterisks were found using manual 0.01 interpretation of the data. 0 ProSightPC is a trademark of Proteinaceous Inc. All other trademarks are the property of Thermo Fisher 0.00 Scientific and its subsidiaries. 0 1x10 5 2x105 3x105 4x105 5x105 0 20 40 60 80 100 120 This information is not intended to encourage use of these products in any manners that might infringe the Reagent Target Reaction Time (msec) intellectual property rights of others.

2 Second-Generation Electron Transfer Dissociation (ETD) on the Thermo Scienti c Orbitrap Fusion Mass Spectrometer with Improved Functionality, Increased Speed, and Improved Robustness of Data Second-Generation Electron Transfer Dissociation (ETD) on the Thermo Scientific Orbitrap Fusion Mass Spectrometer with Improved Functionality, Increased Speed, and Improved Robustness of Data Christopher Mullen1, Lee Earley1, Jean-Jacques Dunyach1, John E.P. Syka1, Philip D. Compton2, Dina L. Bai3, Jefferey Shabanowitz3, and Donald F. Hunt3 1Thermo Fisher Scientific, San Jose, CA; 2Kelleher Lab, Northwestern University, Evanston, IL; 3Department of Chemistry, University of Virginia, Charlottesville, VA

TABLE 1. Calculated charge state dependent reaction times based on a Verification of the Calibration Unique ETD Capabilities Overview Results saturated reaction rate coefficient of 58.2 sec–1 for charge state 3+, based on 5% The calibrated ETD reaction conditions are verified by using an infusion of ubiquitin The location of the ion-routing multipole within the Orbitrap Fusion mass spectrometer of the precursor remaining after reaction. Purpose: Improve ETD robustness, functionality, and speed on the Thermo Calibrating the reaction kinetics ensures that the ETD fragmentation efficiency is from bovine erythrocytes. Spectra of the 12+ charge state at 714.7 m/z were obtained allows for a parallel ITMS2 acquisition mode, enabling ITMS2 CID and HCD spectral Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer optimized and that the maximum duty cycle for ETD can be accomplished. as a function of the ETD reaction time at an FT resolution of 120K and averaged for acquisition rates up to 20 Hz. ITMS2 ETD spectral acquisition rates are slightly reduced Charge State Reaction Time (msec) Methods: Orbitrap Fusion mass spectrometer with the Thermo Scientific Calibrating the reaction kinetics is a multi-step process in which the decay of the 100 micro scans. The spectra were then searched using ProSightPC™. Figure 6 due to the additional time requirement of the ion-ion reaction, but rates up to 12 Hz are Easy-ETD™ source angiotensin I (433 m/z) precursor is monitored as a function of reaction time at a 2 116 shows two representative ubiquitin spectra, obtained at 3.25 msec and 8 msec of attainable. In addition, the ability to perform the ion-ion reaction and m/z analysis in number of reagent targets (Figure 2). From the slope of the individual decay curves, 3 52 reaction time, respectively. The calibrated reaction conditions predict an optimal parallel with the precursor injection means that the spectral acquisition rate at a Results: Demonstrated increased ETD functionality and usability by using a reaction time of 3.25 msec for the 12+ precursor, and while the spectrum visually looks particular reaction time can be maintained for a significantly longer precursor injection the reaction rate coefficient is extracted, and plotted as a function of the reagent target 4 29 combination of hardware and software improvements under reacted, it yields the most total c and z fragments from the ProSightPC v3.0 time than in the absence of parallelization (Figure 9). at which it was acquired (Figure 3). The data are then fitted to find the target at which 5 19 the reaction rate coefficient saturates, and combinations of this target with the reaction search (Figure 7). In addition, we demonstrate the ability to obtain nearly complete 6 13 rate coefficient are used to calculate the optimal charge state dependent reaction times ubiquitin sequence coverage on a LC timescale using a combination of ProSightPC Introduction 7 9.5 searching and manual interpretation of the spectra. The results presented in Figure 8 (Table 1). The reaction rate coefficient as a function of the precursor charge state FIGURE 9. Ion-trap ETD MSMS cycle rate dependence on reaction and precursor The Orbitrap Fusion platform incorporates a second-generation Easy-ETD reagent ion were achieved by averaging 20 FT micro scans, corresponding to a total acquisition squared has been demonstrated to be linear by J. L. Stephenson Jr. and S. A. 8 7.2 injection times using parallel acquisition. The reagent injection time was fixed at source. The Easy-ETD source improvements include a bright and stable glow- 3 time of 6.7 seconds. McLuckey , which is verified in Figure 4, and used to calculate the optimal reaction 9 5.7 5 msec, corresponding to a reagent anion population of 2e5 for all experiments. discharge-based ETD reagent ion source located between the S-Lens and the Active time per charge state, based on a desired amount of reaction completeness. We find 10 4.6 Beam Guide, and a higher frequency RF axial trapping field (trap end-lens voltage) to 95% consumption of the initial precursor intensity to yield high quality ETD spectra. FIGURE 6. ETD spectra obtained on the 12+ charge state of ubiquitin at improve ion confinement during ETD (Figure 1). The Orbitrap Fusion mass Figure 5 shows the relationship between the reaction time and the amount of precursor 11 3.8 714.7 m/z at two different reaction times. A) 3.25 msec, the calibrated reaction 100 spectrometer design enables previously unavailable parallel/pipelined scan modes to remaining after reaction for the angiotensin I (433 m/z) precursor. 12 3.2 time. B) 8 msec. minimize overall scan cycle times. Further, calibration of the ETD reaction kinetics Ubiquitin_3pt25msec_2e5_100uscans #1 RT: 0.00 AV: 1 NL: 7.96E6 T: FTMS + p ESI Full ms2 [email protected] [150.00-2000.00] 90 714.6434 ensures the shortest possible reaction times while maximizing product ion yields and 100 A) Frequency (Hz) FIGURE 2. Angiotensen I (433 m/z) precursor decay curves as a function of the 90 spectral reproducibility. Collectively, these developments constitute a new-generation 779.6105 80 reagent anion target under pseudo 1st order reaction conditions. The reaction FIGURE 4. The maximum rate coefficient vs. the charge state squared of the 80 ETD platform on the Orbitrap Fusion mass spectrometer. 70 1 progress is monitored for up to four half-lives, and the slope of the individual precursor is linear , allowing extrapolation of the optimal reaction times 60 70 5. 000 50 691.5779 818.7508 6. 000 decays is equal to the negative of the rate coefficient. obtained for a single charge state to all charge states 40 857.5713 Relative Abundance Relative 30 640.3763 277.1327 60 Methods 390.2168 537.2855 898.4890 7. 000 20 978.5804 243.6364 433.2516 1136.6495 1.2 10 1023.5648 1159.3170 328.2096 502.8156 1347.2285 1,2 167.9136 1264.7439 1580.9261 8. 000 Reagent anions from a glow discharge source (previously described) are introduced 0 50 0.0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 into the ion optics path ahead of the quadrupole mass filter where they are m/z 1.1 m/z 9. 000 ubiquitin_8msec_2e5_100uscans #1 RT: 0.00 AV: 1 NL: 3.24E6 T: FTMS + p ESI Full ms2 [email protected] [150.00-2000.00] 40 selected, accumulated in the ion-routing multipole, and then transferred into the high 277.1326 10 .00 1.0 100 pressure trap (HPT) of the dual-pressure linear ion trap for the ETD reaction. -0.5 Ubiquitin 90 B) 30 11 .00 0.9 Angiotensen 80 390.2166 640.3759 Increasing the frequency of the RF axial confinement field during ETD from ½ to 2 537.2852 70 1136.6490 12 .00 times the quadrupole field frequency avoids parametric resonance excitation and 0.8 60 20 898.4885 961.0632 -1.0 50 664.3729 (msec) Time Reaction ETD 13 .00 ejection of low m/z (typically 120–130 Th) ions. ETD products may be directly 602.3616 717.9185 40 433.2514 ) 243.6363 Relative Abundance Relative

) 0.7 790.4649 -1 30 10 0 1079.6276 1347.2284 transferred to the low pressure trap (LPT) or to the Orbitrap Fusion mass spectrometer 1023.5646 20 1159.3162 328.2094 2 167.9123 1282.7065 502.8152 for m/z analysis. ITMS ETD scan rates of up to 12 Hz are attainable using a parallel -1.5 Targ et: 0.1e 5 0.6 10 1414.2650 1518.3324 1580.9260 1705.9368 1921.1191 Targ et: 0.2e 5 0 acquisition mode. 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 ln(A/A (msec 10 20 30 40 50 60 70 80 90 100 Targ et: 0.4e 5 0.5 m/z

Targ et: 0.6e 5 m ax FIGURE 1. Schematic of the Orbitrap Fusion tribrid mass spectrometer showing -2.0 Targ et: 0.7e 5 k 0.4 Precursor Injection Time (msec) Targ et: 0.8e 5 FIGURE 7. ProSightPC search results for the 12+ charge state of ubiquitin (714.7 the location of the Easy-ETD reagent ion source within the overall ion optics Targ et: 1.0e 5 0.3 m/z) as a function of the ETD reaction time, demonstrating that the calibrated path. The exploded view shows how the reagent ion source is incorporated into -2.5 Targ et: 2.0e 5 Targ et: 4.0e 5 0.2 kinetics chooses appropriate reaction times up to at least charge state 12. the S-Lens/Q00 region. Targ et: 9.9e 5 Dual-pressure linear ion trap 0.1 Fragments ions using either CID -3.0 Conclusion or optional ETD and provides Ion-routing multipole fast, sensitive mass analysis 0 10 20 30 40 50 . A second-generation glow-discharge-based Easy-ETD source has been . High-pressure cell produces 0 20 40 60 80 100 120 140 160 180 110 highly efficient storage of ions developed for the Orbitrap Fusion MS that incorporates significant hardware and . Voltages can be adjusted to Reaction Time (msec) 2 fragment ions at higher energies Charge State Square (z ) c software advancements. 100 z . Calibration of the reaction kinetics removes the guesswork from ETD, and leads to Ultra-high-field Orbitrap mass analyzer FIGURE 3. The reaction rate coefficient versus reagent anion target, showing total conditions that optimizes the ETD reaction and scan cycle time. . Compact Orbitrap mass spectrometer FIGURE 5. Plot of the reaction time required to reduce the reactant precursor by the fit to the data and the optimal reagent anion target leading to at least 90% 90 operating at ultra-high voltage a fixed amount, calculated as a function of the reaction rate coefficient. We find . . Produces 500K resolution every of the k observed. The calibrated reaction conditions are demonstrated to provide optimal conditions 1.2 seconds max that 3 to 6% remaining precursor is optimal for most compounds. for ETD identifications and sequence coverage. . Ultra-fast operation produces 80 15 spectra/sec with 15K resolution . 2 0.07 Parallel acquisition provides ITMS ETD scan rate cycle times of up to 12 Hz. 100 50 C-Trap 0.06 Rate Coefficient References Active beam guide (ABG) Ions are focused and -1 . Prevents neutral species from injected into the Orbitrap 40 sec Number of Fragments 1. Earley et al., 61st ASMS Conference on Mass Spectrometry and Allied Topics, entering Q1 mass spectrometer 80 -1 . Axial fields improves 0.05 60 sec 40 Minneapolis, MN, June 9–13, 2013; Poster Th101 – Implementation of a operational robustness Quadrupole mass filter High Selectivity and -1 Multipurpose Glow Discharge Ion Source for the Introduction of Reagent/ excellent transmission 80 sec

) 0.04 60 Calibrant Ions Into a Hybrid Mass Spectrometer, poster number: 101, Thursday,

-1 k 30 Halls B&C. Easy-ETD ion source Fit to Data 0 1 2 3 4 5 6 7 8 9 . Discharge-based source 0.03 2. Earley et al., Presented at the 58th ASMS Conference on Mass Spectrometry and . Robust design with an extremely Best Target 40 ETD Reaction Time (msec) stable source of ions

k (msec Allied Topics, Salt Lake City, Utah, May 23–27, 2010; Poster T600. S-Lens . High sensitivity 0.02 FIGURE 8. Ubiquitin sequence coverage resulting from averaging 20 FT micro 3. Stephenson, J. L., Jr. and McLuckey, S.A. J. Am. Chem. Soc. 1996, 118, . Robust ion optics 20 scans for a total acquisition time of 6.7 seconds. Fragments denoted by the red 7390–7397.

Precursor Remaining (%) Remaining Precursor (c ion), blue (z ion), and green (y ion) asterisks were found using manual 0.01 interpretation of the data. 0 ProSightPC is a trademark of Proteinaceous Inc. All other trademarks are the property of Thermo Fisher 0.00 Scientific and its subsidiaries. 0 1x10 5 2x105 3x105 4x105 5x105 0 20 40 60 80 100 120 This information is not intended to encourage use of these products in any manners that might infringe the Reagent Target Reaction Time (msec) intellectual property rights of others.

Thermo Scienti c Poster Note • PN ASMS13_T019_CMullen_E 07/13S 3 Second-Generation Electron Transfer Dissociation (ETD) on the Thermo Scientific Orbitrap Fusion Mass Spectrometer with Improved Functionality, Increased Speed, and Improved Robustness of Data Christopher Mullen1, Lee Earley1, Jean-Jacques Dunyach1, John E.P. Syka1, Philip D. Compton2, Dina L. Bai3, Jefferey Shabanowitz3, and Donald F. Hunt3 1Thermo Fisher Scientific, San Jose, CA; 2Kelleher Lab, Northwestern University, Evanston, IL; 3Department of Chemistry, University of Virginia, Charlottesville, VA

TABLE 1. Calculated charge state dependent reaction times based on a Verification of the Calibration Unique ETD Capabilities Overview Results saturated reaction rate coefficient of 58.2 sec–1 for charge state 3+, based on 5% The calibrated ETD reaction conditions are verified by using an infusion of ubiquitin The location of the ion-routing multipole within the Orbitrap Fusion mass spectrometer of the precursor remaining after reaction. Purpose: Improve ETD robustness, functionality, and speed on the Thermo Calibrating the reaction kinetics ensures that the ETD fragmentation efficiency is from bovine erythrocytes. Spectra of the 12+ charge state at 714.7 m/z were obtained allows for a parallel ITMS2 acquisition mode, enabling ITMS2 CID and HCD spectral Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer optimized and that the maximum duty cycle for ETD can be accomplished. as a function of the ETD reaction time at an FT resolution of 120K and averaged for acquisition rates up to 20 Hz. ITMS2 ETD spectral acquisition rates are slightly reduced Charge State Reaction Time (msec) Methods: Orbitrap Fusion mass spectrometer with the Thermo Scientific Calibrating the reaction kinetics is a multi-step process in which the decay of the 100 micro scans. The spectra were then searched using ProSightPC™. Figure 6 due to the additional time requirement of the ion-ion reaction, but rates up to 12 Hz are Easy-ETD™ source angiotensin I (433 m/z) precursor is monitored as a function of reaction time at a 2 116 shows two representative ubiquitin spectra, obtained at 3.25 msec and 8 msec of attainable. In addition, the ability to perform the ion-ion reaction and m/z analysis in number of reagent targets (Figure 2). From the slope of the individual decay curves, 3 52 reaction time, respectively. The calibrated reaction conditions predict an optimal parallel with the precursor injection means that the spectral acquisition rate at a Results: Demonstrated increased ETD functionality and usability by using a reaction time of 3.25 msec for the 12+ precursor, and while the spectrum visually looks particular reaction time can be maintained for a significantly longer precursor injection the reaction rate coefficient is extracted, and plotted as a function of the reagent target 4 29 combination of hardware and software improvements under reacted, it yields the most total c and z fragments from the ProSightPC v3.0 time than in the absence of parallelization (Figure 9). at which it was acquired (Figure 3). The data are then fitted to find the target at which 5 19 the reaction rate coefficient saturates, and combinations of this target with the reaction search (Figure 7). In addition, we demonstrate the ability to obtain nearly complete 6 13 rate coefficient are used to calculate the optimal charge state dependent reaction times ubiquitin sequence coverage on a LC timescale using a combination of ProSightPC Introduction 7 9.5 searching and manual interpretation of the spectra. The results presented in Figure 8 (Table 1). The reaction rate coefficient as a function of the precursor charge state FIGURE 9. Ion-trap ETD MSMS cycle rate dependence on reaction and precursor The Orbitrap Fusion platform incorporates a second-generation Easy-ETD reagent ion were achieved by averaging 20 FT micro scans, corresponding to a total acquisition squared has been demonstrated to be linear by J. L. Stephenson Jr. and S. A. 8 7.2 injection times using parallel acquisition. The reagent injection time was fixed at source. The Easy-ETD source improvements include a bright and stable glow- 3 time of 6.7 seconds. McLuckey , which is verified in Figure 4, and used to calculate the optimal reaction 9 5.7 5 msec, corresponding to a reagent anion population of 2e5 for all experiments. discharge-based ETD reagent ion source located between the S-Lens and the Active time per charge state, based on a desired amount of reaction completeness. We find 10 4.6 Beam Guide, and a higher frequency RF axial trapping field (trap end-lens voltage) to 95% consumption of the initial precursor intensity to yield high quality ETD spectra. FIGURE 6. ETD spectra obtained on the 12+ charge state of ubiquitin at improve ion confinement during ETD (Figure 1). The Orbitrap Fusion mass Figure 5 shows the relationship between the reaction time and the amount of precursor 11 3.8 714.7 m/z at two different reaction times. A) 3.25 msec, the calibrated reaction 100 spectrometer design enables previously unavailable parallel/pipelined scan modes to remaining after reaction for the angiotensin I (433 m/z) precursor. 12 3.2 time. B) 8 msec. minimize overall scan cycle times. Further, calibration of the ETD reaction kinetics Ubiquitin_3pt25msec_2e5_100uscans #1 RT: 0.00 AV: 1 NL: 7.96E6 T: FTMS + p ESI Full ms2 [email protected] [150.00-2000.00] 90 714.6434 ensures the shortest possible reaction times while maximizing product ion yields and 100 A) Frequency (Hz) FIGURE 2. Angiotensen I (433 m/z) precursor decay curves as a function of the 90 spectral reproducibility. Collectively, these developments constitute a new-generation 779.6105 80 reagent anion target under pseudo 1st order reaction conditions. The reaction FIGURE 4. The maximum rate coefficient vs. the charge state squared of the 80 ETD platform on the Orbitrap Fusion mass spectrometer. 70 1 progress is monitored for up to four half-lives, and the slope of the individual precursor is linear , allowing extrapolation of the optimal reaction times 60 70 5. 000 50 691.5779 818.7508 6. 000 decays is equal to the negative of the rate coefficient. obtained for a single charge state to all charge states 40 857.5713 Relative Abundance Relative 30 640.3763 277.1327 60 Methods 390.2168 537.2855 898.4890 7. 000 20 978.5804 243.6364 433.2516 1136.6495 1.2 10 1023.5648 1159.3170 328.2096 502.8156 1347.2285 1,2 167.9136 1264.7439 1580.9261 8. 000 Reagent anions from a glow discharge source (previously described) are introduced 0 50 0.0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 into the ion optics path ahead of the quadrupole mass filter where they are m/z 1.1 m/z 9. 000 ubiquitin_8msec_2e5_100uscans #1 RT: 0.00 AV: 1 NL: 3.24E6 T: FTMS + p ESI Full ms2 [email protected] [150.00-2000.00] 40 selected, accumulated in the ion-routing multipole, and then transferred into the high 277.1326 10 .00 1.0 100 pressure trap (HPT) of the dual-pressure linear ion trap for the ETD reaction. -0.5 Ubiquitin 90 B) 30 11 .00 0.9 Angiotensen 80 390.2166 640.3759 Increasing the frequency of the RF axial confinement field during ETD from ½ to 2 537.2852 70 1136.6490 12 .00 times the quadrupole field frequency avoids parametric resonance excitation and 0.8 60 20 898.4885 961.0632 -1.0 50 664.3729 (msec) Time Reaction ETD 13 .00 ejection of low m/z (typically 120–130 Th) ions. ETD products may be directly 602.3616 717.9185 40 433.2514 ) 243.6363 Relative Abundance Relative

) 0.7 790.4649 -1 30 10 0 1079.6276 1347.2284 transferred to the low pressure trap (LPT) or to the Orbitrap Fusion mass spectrometer 1023.5646 20 1159.3162 328.2094 2 167.9123 1282.7065 502.8152 for m/z analysis. ITMS ETD scan rates of up to 12 Hz are attainable using a parallel -1.5 Targ et: 0.1e 5 0.6 10 1414.2650 1518.3324 1580.9260 1705.9368 1921.1191 Targ et: 0.2e 5 0 acquisition mode. 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 ln(A/A (msec 10 20 30 40 50 60 70 80 90 100 Targ et: 0.4e 5 0.5 m/z

Targ et: 0.6e 5 m ax FIGURE 1. Schematic of the Orbitrap Fusion tribrid mass spectrometer showing -2.0 Targ et: 0.7e 5 k 0.4 Precursor Injection Time (msec) Targ et: 0.8e 5 FIGURE 7. ProSightPC search results for the 12+ charge state of ubiquitin (714.7 the location of the Easy-ETD reagent ion source within the overall ion optics Targ et: 1.0e 5 0.3 m/z) as a function of the ETD reaction time, demonstrating that the calibrated path. The exploded view shows how the reagent ion source is incorporated into -2.5 Targ et: 2.0e 5 Targ et: 4.0e 5 0.2 kinetics chooses appropriate reaction times up to at least charge state 12. the S-Lens/Q00 region. Targ et: 9.9e 5 Dual-pressure linear ion trap 0.1 Fragments ions using either CID -3.0 Conclusion or optional ETD and provides Ion-routing multipole fast, sensitive mass analysis 0 10 20 30 40 50 . A second-generation glow-discharge-based Easy-ETD source has been . High-pressure cell produces 0 20 40 60 80 100 120 140 160 180 110 highly efficient storage of ions developed for the Orbitrap Fusion MS that incorporates significant hardware and . Voltages can be adjusted to Reaction Time (msec) 2 fragment ions at higher energies Charge State Square (z ) c software advancements. 100 z . Calibration of the reaction kinetics removes the guesswork from ETD, and leads to Ultra-high-field Orbitrap mass analyzer FIGURE 3. The reaction rate coefficient versus reagent anion target, showing total conditions that optimizes the ETD reaction and scan cycle time. . Compact Orbitrap mass spectrometer FIGURE 5. Plot of the reaction time required to reduce the reactant precursor by the fit to the data and the optimal reagent anion target leading to at least 90% 90 operating at ultra-high voltage a fixed amount, calculated as a function of the reaction rate coefficient. We find . . Produces 500K resolution every of the k observed. The calibrated reaction conditions are demonstrated to provide optimal conditions 1.2 seconds max that 3 to 6% remaining precursor is optimal for most compounds. for ETD identifications and sequence coverage. . Ultra-fast operation produces 80 15 spectra/sec with 15K resolution . 2 0.07 Parallel acquisition provides ITMS ETD scan rate cycle times of up to 12 Hz. 100 50 C-Trap 0.06 Rate Coefficient References Active beam guide (ABG) Ions are focused and -1 . Prevents neutral species from injected into the Orbitrap 40 sec Number of Fragments 1. Earley et al., 61st ASMS Conference on Mass Spectrometry and Allied Topics, entering Q1 mass spectrometer 80 -1 . Axial fields improves 0.05 60 sec 40 Minneapolis, MN, June 9–13, 2013; Poster Th101 – Implementation of a operational robustness Quadrupole mass filter High Selectivity and -1 Multipurpose Glow Discharge Ion Source for the Introduction of Reagent/ excellent transmission 80 sec

) 0.04 60 Calibrant Ions Into a Hybrid Mass Spectrometer, poster number: 101, Thursday,

-1 k 30 Halls B&C. Easy-ETD ion source Fit to Data 0 1 2 3 4 5 6 7 8 9 . Discharge-based source 0.03 2. Earley et al., Presented at the 58th ASMS Conference on Mass Spectrometry and . Robust design with an extremely Best Target 40 ETD Reaction Time (msec) stable source of ions k (msec Allied Topics, Salt Lake City, Utah, May 23–27, 2010; Poster T600. S-Lens . High sensitivity 0.02 FIGURE 8. Ubiquitin sequence coverage resulting from averaging 20 FT micro 3. Stephenson, J. L., Jr. and McLuckey, S.A. J. Am. Chem. Soc. 1996, 118, . Robust ion optics 20 scans for a total acquisition time of 6.7 seconds. Fragments denoted by the red 7390–7397.

Precursor Remaining (%) Remaining Precursor (c ion), blue (z ion), and green (y ion) asterisks were found using manual 0.01 interpretation of the data. 0 ProSightPC is a trademark of Proteinaceous Inc. All other trademarks are the property of Thermo Fisher 0.00 Scientific and its subsidiaries. 0 1x10 5 2x105 3x105 4x105 5x105 0 20 40 60 80 100 120 This information is not intended to encourage use of these products in any manners that might infringe the Reagent Target Reaction Time (msec) intellectual property rights of others.

4 Second-Generation Electron Transfer Dissociation (ETD) on the Thermo Scienti c Orbitrap Fusion Mass Spectrometer with Improved Functionality, Increased Speed, and Improved Robustness of Data Second-Generation Electron Transfer Dissociation (ETD) on the Thermo Scientific Orbitrap Fusion Mass Spectrometer with Improved Functionality, Increased Speed, and Improved Robustness of Data Christopher Mullen1, Lee Earley1, Jean-Jacques Dunyach1, John E.P. Syka1, Philip D. Compton2, Dina L. Bai3, Jefferey Shabanowitz3, and Donald F. Hunt3 1Thermo Fisher Scientific, San Jose, CA; 2Kelleher Lab, Northwestern University, Evanston, IL; 3Department of Chemistry, University of Virginia, Charlottesville, VA

TABLE 1. Calculated charge state dependent reaction times based on a Verification of the Calibration Unique ETD Capabilities Overview Results saturated reaction rate coefficient of 58.2 sec–1 for charge state 3+, based on 5% The calibrated ETD reaction conditions are verified by using an infusion of ubiquitin The location of the ion-routing multipole within the Orbitrap Fusion mass spectrometer of the precursor remaining after reaction. Purpose: Improve ETD robustness, functionality, and speed on the Thermo Calibrating the reaction kinetics ensures that the ETD fragmentation efficiency is from bovine erythrocytes. Spectra of the 12+ charge state at 714.7 m/z were obtained allows for a parallel ITMS2 acquisition mode, enabling ITMS2 CID and HCD spectral Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer optimized and that the maximum duty cycle for ETD can be accomplished. as a function of the ETD reaction time at an FT resolution of 120K and averaged for acquisition rates up to 20 Hz. ITMS2 ETD spectral acquisition rates are slightly reduced Charge State Reaction Time (msec) Methods: Orbitrap Fusion mass spectrometer with the Thermo Scientific Calibrating the reaction kinetics is a multi-step process in which the decay of the 100 micro scans. The spectra were then searched using ProSightPC™. Figure 6 due to the additional time requirement of the ion-ion reaction, but rates up to 12 Hz are Easy-ETD™ source angiotensin I (433 m/z) precursor is monitored as a function of reaction time at a 2 116 shows two representative ubiquitin spectra, obtained at 3.25 msec and 8 msec of attainable. In addition, the ability to perform the ion-ion reaction and m/z analysis in number of reagent targets (Figure 2). From the slope of the individual decay curves, 3 52 reaction time, respectively. The calibrated reaction conditions predict an optimal parallel with the precursor injection means that the spectral acquisition rate at a Results: Demonstrated increased ETD functionality and usability by using a reaction time of 3.25 msec for the 12+ precursor, and while the spectrum visually looks particular reaction time can be maintained for a significantly longer precursor injection the reaction rate coefficient is extracted, and plotted as a function of the reagent target 4 29 combination of hardware and software improvements under reacted, it yields the most total c and z fragments from the ProSightPC v3.0 time than in the absence of parallelization (Figure 9). at which it was acquired (Figure 3). The data are then fitted to find the target at which 5 19 the reaction rate coefficient saturates, and combinations of this target with the reaction search (Figure 7). In addition, we demonstrate the ability to obtain nearly complete 6 13 rate coefficient are used to calculate the optimal charge state dependent reaction times ubiquitin sequence coverage on a LC timescale using a combination of ProSightPC Introduction 7 9.5 searching and manual interpretation of the spectra. The results presented in Figure 8 (Table 1). The reaction rate coefficient as a function of the precursor charge state FIGURE 9. Ion-trap ETD MSMS cycle rate dependence on reaction and precursor The Orbitrap Fusion platform incorporates a second-generation Easy-ETD reagent ion were achieved by averaging 20 FT micro scans, corresponding to a total acquisition squared has been demonstrated to be linear by J. L. Stephenson Jr. and S. A. 8 7.2 injection times using parallel acquisition. The reagent injection time was fixed at source. The Easy-ETD source improvements include a bright and stable glow- 3 time of 6.7 seconds. McLuckey , which is verified in Figure 4, and used to calculate the optimal reaction 9 5.7 5 msec, corresponding to a reagent anion population of 2e5 for all experiments. discharge-based ETD reagent ion source located between the S-Lens and the Active time per charge state, based on a desired amount of reaction completeness. We find 10 4.6 Beam Guide, and a higher frequency RF axial trapping field (trap end-lens voltage) to 95% consumption of the initial precursor intensity to yield high quality ETD spectra. FIGURE 6. ETD spectra obtained on the 12+ charge state of ubiquitin at improve ion confinement during ETD (Figure 1). The Orbitrap Fusion mass Figure 5 shows the relationship between the reaction time and the amount of precursor 11 3.8 714.7 m/z at two different reaction times. A) 3.25 msec, the calibrated reaction 100 spectrometer design enables previously unavailable parallel/pipelined scan modes to remaining after reaction for the angiotensin I (433 m/z) precursor. 12 3.2 time. B) 8 msec. minimize overall scan cycle times. Further, calibration of the ETD reaction kinetics Ubiquitin_3pt25msec_2e5_100uscans #1 RT: 0.00 AV: 1 NL: 7.96E6 T: FTMS + p ESI Full ms2 [email protected] [150.00-2000.00] 90 714.6434 ensures the shortest possible reaction times while maximizing product ion yields and 100 A) Frequency (Hz) FIGURE 2. Angiotensen I (433 m/z) precursor decay curves as a function of the 90 spectral reproducibility. Collectively, these developments constitute a new-generation 779.6105 80 reagent anion target under pseudo 1st order reaction conditions. The reaction FIGURE 4. The maximum rate coefficient vs. the charge state squared of the 80 ETD platform on the Orbitrap Fusion mass spectrometer. 70 1 progress is monitored for up to four half-lives, and the slope of the individual precursor is linear , allowing extrapolation of the optimal reaction times 60 70 5. 000 50 691.5779 818.7508 6. 000 decays is equal to the negative of the rate coefficient. obtained for a single charge state to all charge states 40 857.5713 Relative Abundance Relative 30 640.3763 277.1327 60 Methods 390.2168 537.2855 898.4890 7. 000 20 978.5804 243.6364 433.2516 1136.6495 1.2 10 1023.5648 1159.3170 328.2096 502.8156 1347.2285 1,2 167.9136 1264.7439 1580.9261 8. 000 Reagent anions from a glow discharge source (previously described) are introduced 0 50 0.0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 into the ion optics path ahead of the quadrupole mass filter where they are m/z 1.1 m/z 9. 000 ubiquitin_8msec_2e5_100uscans #1 RT: 0.00 AV: 1 NL: 3.24E6 T: FTMS + p ESI Full ms2 [email protected] [150.00-2000.00] 40 selected, accumulated in the ion-routing multipole, and then transferred into the high 277.1326 10 .00 1.0 100 pressure trap (HPT) of the dual-pressure linear ion trap for the ETD reaction. -0.5 Ubiquitin 90 B) 30 11 .00 0.9 Angiotensen 80 390.2166 640.3759 Increasing the frequency of the RF axial confinement field during ETD from ½ to 2 537.2852 70 1136.6490 12 .00 times the quadrupole field frequency avoids parametric resonance excitation and 0.8 60 20 898.4885 961.0632 -1.0 50 664.3729 (msec) Time Reaction ETD 13 .00 ejection of low m/z (typically 120–130 Th) ions. ETD products may be directly 602.3616 717.9185 40 433.2514 ) 243.6363 Relative Abundance Relative

) 0.7 790.4649 -1 30 10 0 1079.6276 1347.2284 transferred to the low pressure trap (LPT) or to the Orbitrap Fusion mass spectrometer 1023.5646 20 1159.3162 328.2094 2 167.9123 1282.7065 502.8152 for m/z analysis. ITMS ETD scan rates of up to 12 Hz are attainable using a parallel -1.5 Targ et: 0.1e 5 0.6 10 1414.2650 1518.3324 1580.9260 1705.9368 1921.1191 Targ et: 0.2e 5 0 acquisition mode. 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 ln(A/A (msec 10 20 30 40 50 60 70 80 90 100 Targ et: 0.4e 5 0.5 m/z

Targ et: 0.6e 5 m ax FIGURE 1. Schematic of the Orbitrap Fusion tribrid mass spectrometer showing -2.0 Targ et: 0.7e 5 k 0.4 Precursor Injection Time (msec) Targ et: 0.8e 5 FIGURE 7. ProSightPC search results for the 12+ charge state of ubiquitin (714.7 the location of the Easy-ETD reagent ion source within the overall ion optics Targ et: 1.0e 5 0.3 m/z) as a function of the ETD reaction time, demonstrating that the calibrated path. The exploded view shows how the reagent ion source is incorporated into -2.5 Targ et: 2.0e 5 Targ et: 4.0e 5 0.2 kinetics chooses appropriate reaction times up to at least charge state 12. the S-Lens/Q00 region. Targ et: 9.9e 5 Dual-pressure linear ion trap 0.1 Fragments ions using either CID -3.0 Conclusion or optional ETD and provides Ion-routing multipole fast, sensitive mass analysis 0 10 20 30 40 50 . A second-generation glow-discharge-based Easy-ETD source has been . High-pressure cell produces 0 20 40 60 80 100 120 140 160 180 110 highly efficient storage of ions developed for the Orbitrap Fusion MS that incorporates significant hardware and . Voltages can be adjusted to Reaction Time (msec) 2 fragment ions at higher energies Charge State Square (z ) c software advancements. 100 z . Calibration of the reaction kinetics removes the guesswork from ETD, and leads to Ultra-high-field Orbitrap mass analyzer FIGURE 3. The reaction rate coefficient versus reagent anion target, showing total conditions that optimizes the ETD reaction and scan cycle time. . Compact Orbitrap mass spectrometer FIGURE 5. Plot of the reaction time required to reduce the reactant precursor by the fit to the data and the optimal reagent anion target leading to at least 90% 90 operating at ultra-high voltage a fixed amount, calculated as a function of the reaction rate coefficient. We find . . Produces 500K resolution every of the k observed. The calibrated reaction conditions are demonstrated to provide optimal conditions 1.2 seconds max that 3 to 6% remaining precursor is optimal for most compounds. for ETD identifications and sequence coverage. . Ultra-fast operation produces 80 15 spectra/sec with 15K resolution . 2 0.07 Parallel acquisition provides ITMS ETD scan rate cycle times of up to 12 Hz. 100 50 C-Trap 0.06 Rate Coefficient References Active beam guide (ABG) Ions are focused and -1 . Prevents neutral species from injected into the Orbitrap 40 sec Number of Fragments 1. Earley et al., 61st ASMS Conference on Mass Spectrometry and Allied Topics, entering Q1 mass spectrometer 80 -1 . Axial fields improves 0.05 60 sec 40 Minneapolis, MN, June 9–13, 2013; Poster Th101 – Implementation of a operational robustness Quadrupole mass filter High Selectivity and -1 Multipurpose Glow Discharge Ion Source for the Introduction of Reagent/ excellent transmission 80 sec

) 0.04 60 Calibrant Ions Into a Hybrid Mass Spectrometer, poster number: 101, Thursday,

-1 k 30 Halls B&C. Easy-ETD ion source Fit to Data 0 1 2 3 4 5 6 7 8 9 . Discharge-based source 0.03 2. Earley et al., Presented at the 58th ASMS Conference on Mass Spectrometry and . Robust design with an extremely Best Target 40 ETD Reaction Time (msec) stable source of ions k (msec Allied Topics, Salt Lake City, Utah, May 23–27, 2010; Poster T600. S-Lens . High sensitivity 0.02 FIGURE 8. Ubiquitin sequence coverage resulting from averaging 20 FT micro 3. Stephenson, J. L., Jr. and McLuckey, S.A. J. Am. Chem. Soc. 1996, 118, . Robust ion optics 20 scans for a total acquisition time of 6.7 seconds. Fragments denoted by the red 7390–7397.

Precursor Remaining (%) Remaining Precursor (c ion), blue (z ion), and green (y ion) asterisks were found using manual 0.01 interpretation of the data. 0 ProSightPC is a trademark of Proteinaceous Inc. All other trademarks are the property of Thermo Fisher 0.00 Scientific and its subsidiaries. 0 1x10 5 2x105 3x105 4x105 5x105 0 20 40 60 80 100 120 This information is not intended to encourage use of these products in any manners that might infringe the Reagent Target Reaction Time (msec) intellectual property rights of others.

Thermo Scienti c Poster Note • PN ASMS13_T019_CMullen_E 07/13S 5 Second-Generation Electron Transfer Dissociation (ETD) on the Thermo Scientific Orbitrap Fusion Mass Spectrometer with Improved Functionality, Increased Speed, and Improved Robustness of Data Christopher Mullen1, Lee Earley1, Jean-Jacques Dunyach1, John E.P. Syka1, Philip D. Compton2, Dina L. Bai3, Jefferey Shabanowitz3, and Donald F. Hunt3 1Thermo Fisher Scientific, San Jose, CA; 2Kelleher Lab, Northwestern University, Evanston, IL; 3Department of Chemistry, University of Virginia, Charlottesville, VA

TABLE 1. Calculated charge state dependent reaction times based on a Verification of the Calibration Unique ETD Capabilities Overview Results saturated reaction rate coefficient of 58.2 sec–1 for charge state 3+, based on 5% The calibrated ETD reaction conditions are verified by using an infusion of ubiquitin The location of the ion-routing multipole within the Orbitrap Fusion mass spectrometer of the precursor remaining after reaction. Purpose: Improve ETD robustness, functionality, and speed on the Thermo Calibrating the reaction kinetics ensures that the ETD fragmentation efficiency is from bovine erythrocytes. Spectra of the 12+ charge state at 714.7 m/z were obtained allows for a parallel ITMS2 acquisition mode, enabling ITMS2 CID and HCD spectral Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer optimized and that the maximum duty cycle for ETD can be accomplished. as a function of the ETD reaction time at an FT resolution of 120K and averaged for acquisition rates up to 20 Hz. ITMS2 ETD spectral acquisition rates are slightly reduced Charge State Reaction Time (msec) Methods: Orbitrap Fusion mass spectrometer with the Thermo Scientific Calibrating the reaction kinetics is a multi-step process in which the decay of the 100 micro scans. The spectra were then searched using ProSightPC™. Figure 6 due to the additional time requirement of the ion-ion reaction, but rates up to 12 Hz are Easy-ETD™ source angiotensin I (433 m/z) precursor is monitored as a function of reaction time at a 2 116 shows two representative ubiquitin spectra, obtained at 3.25 msec and 8 msec of attainable. In addition, the ability to perform the ion-ion reaction and m/z analysis in number of reagent targets (Figure 2). From the slope of the individual decay curves, 3 52 reaction time, respectively. The calibrated reaction conditions predict an optimal parallel with the precursor injection means that the spectral acquisition rate at a Results: Demonstrated increased ETD functionality and usability by using a reaction time of 3.25 msec for the 12+ precursor, and while the spectrum visually looks particular reaction time can be maintained for a significantly longer precursor injection the reaction rate coefficient is extracted, and plotted as a function of the reagent target 4 29 combination of hardware and software improvements under reacted, it yields the most total c and z fragments from the ProSightPC v3.0 time than in the absence of parallelization (Figure 9). at which it was acquired (Figure 3). The data are then fitted to find the target at which 5 19 the reaction rate coefficient saturates, and combinations of this target with the reaction search (Figure 7). In addition, we demonstrate the ability to obtain nearly complete 6 13 rate coefficient are used to calculate the optimal charge state dependent reaction times ubiquitin sequence coverage on a LC timescale using a combination of ProSightPC Introduction 7 9.5 searching and manual interpretation of the spectra. The results presented in Figure 8 (Table 1). The reaction rate coefficient as a function of the precursor charge state FIGURE 9. Ion-trap ETD MSMS cycle rate dependence on reaction and precursor The Orbitrap Fusion platform incorporates a second-generation Easy-ETD reagent ion were achieved by averaging 20 FT micro scans, corresponding to a total acquisition squared has been demonstrated to be linear by J. L. Stephenson Jr. and S. A. 8 7.2 injection times using parallel acquisition. The reagent injection time was fixed at source. The Easy-ETD source improvements include a bright and stable glow- 3 time of 6.7 seconds. McLuckey , which is verified in Figure 4, and used to calculate the optimal reaction 9 5.7 5 msec, corresponding to a reagent anion population of 2e5 for all experiments. discharge-based ETD reagent ion source located between the S-Lens and the Active time per charge state, based on a desired amount of reaction completeness. We find 10 4.6 Beam Guide, and a higher frequency RF axial trapping field (trap end-lens voltage) to 95% consumption of the initial precursor intensity to yield high quality ETD spectra. FIGURE 6. ETD spectra obtained on the 12+ charge state of ubiquitin at improve ion confinement during ETD (Figure 1). The Orbitrap Fusion mass Figure 5 shows the relationship between the reaction time and the amount of precursor 11 3.8 714.7 m/z at two different reaction times. A) 3.25 msec, the calibrated reaction 100 spectrometer design enables previously unavailable parallel/pipelined scan modes to remaining after reaction for the angiotensin I (433 m/z) precursor. 12 3.2 time. B) 8 msec. minimize overall scan cycle times. Further, calibration of the ETD reaction kinetics Ubiquitin_3pt25msec_2e5_100uscans #1 RT: 0.00 AV: 1 NL: 7.96E6 T: FTMS + p ESI Full ms2 [email protected] [150.00-2000.00] 90 714.6434 ensures the shortest possible reaction times while maximizing product ion yields and 100 A) Frequency (Hz) FIGURE 2. Angiotensen I (433 m/z) precursor decay curves as a function of the 90 spectral reproducibility. Collectively, these developments constitute a new-generation 779.6105 80 reagent anion target under pseudo 1st order reaction conditions. The reaction FIGURE 4. The maximum rate coefficient vs. the charge state squared of the 80 ETD platform on the Orbitrap Fusion mass spectrometer. 70 1 progress is monitored for up to four half-lives, and the slope of the individual precursor is linear , allowing extrapolation of the optimal reaction times 60 70 5. 000 50 691.5779 818.7508 6. 000 decays is equal to the negative of the rate coefficient. obtained for a single charge state to all charge states 40 857.5713 Relative Abundance Relative 30 640.3763 277.1327 60 Methods 390.2168 537.2855 898.4890 7. 000 20 978.5804 243.6364 433.2516 1136.6495 1.2 10 1023.5648 1159.3170 328.2096 502.8156 1347.2285 1,2 167.9136 1264.7439 1580.9261 8. 000 Reagent anions from a glow discharge source (previously described) are introduced 0 50 0.0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 into the ion optics path ahead of the quadrupole mass filter where they are m/z 1.1 m/z 9. 000 ubiquitin_8msec_2e5_100uscans #1 RT: 0.00 AV: 1 NL: 3.24E6 T: FTMS + p ESI Full ms2 [email protected] [150.00-2000.00] 40 selected, accumulated in the ion-routing multipole, and then transferred into the high 277.1326 10 .00 1.0 100 pressure trap (HPT) of the dual-pressure linear ion trap for the ETD reaction. -0.5 Ubiquitin 90 B) 30 11 .00 0.9 Angiotensen 80 390.2166 640.3759 Increasing the frequency of the RF axial confinement field during ETD from ½ to 2 537.2852 70 1136.6490 12 .00 times the quadrupole field frequency avoids parametric resonance excitation and 0.8 60 20 898.4885 961.0632 -1.0 50 664.3729 (msec) Time Reaction ETD 13 .00 ejection of low m/z (typically 120–130 Th) ions. ETD products may be directly 602.3616 717.9185 40 433.2514 ) 243.6363 Relative Abundance Relative

) 0.7 790.4649 -1 30 10 0 1079.6276 1347.2284 transferred to the low pressure trap (LPT) or to the Orbitrap Fusion mass spectrometer 1023.5646 20 1159.3162 328.2094 2 167.9123 1282.7065 502.8152 for m/z analysis. ITMS ETD scan rates of up to 12 Hz are attainable using a parallel -1.5 Targ et: 0.1e 5 0.6 10 1414.2650 1518.3324 1580.9260 1705.9368 1921.1191 Targ et: 0.2e 5 0 acquisition mode. 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 ln(A/A (msec 10 20 30 40 50 60 70 80 90 100 Targ et: 0.4e 5 0.5 m/z

Targ et: 0.6e 5 m ax FIGURE 1. Schematic of the Orbitrap Fusion tribrid mass spectrometer showing -2.0 Targ et: 0.7e 5 k 0.4 Precursor Injection Time (msec) Targ et: 0.8e 5 FIGURE 7. ProSightPC search results for the 12+ charge state of ubiquitin (714.7 the location of the Easy-ETD reagent ion source within the overall ion optics Targ et: 1.0e 5 0.3 m/z) as a function of the ETD reaction time, demonstrating that the calibrated path. The exploded view shows how the reagent ion source is incorporated into -2.5 Targ et: 2.0e 5 Targ et: 4.0e 5 0.2 kinetics chooses appropriate reaction times up to at least charge state 12. the S-Lens/Q00 region. Targ et: 9.9e 5 Dual-pressure linear ion trap 0.1 Fragments ions using either CID -3.0 Conclusion or optional ETD and provides Ion-routing multipole fast, sensitive mass analysis 0 10 20 30 40 50 . A second-generation glow-discharge-based Easy-ETD source has been . High-pressure cell produces 0 20 40 60 80 100 120 140 160 180 110 highly efficient storage of ions developed for the Orbitrap Fusion MS that incorporates significant hardware and . Voltages can be adjusted to Reaction Time (msec) 2 fragment ions at higher energies Charge State Square (z ) c software advancements. 100 z . Calibration of the reaction kinetics removes the guesswork from ETD, and leads to Ultra-high-field Orbitrap mass analyzer FIGURE 3. The reaction rate coefficient versus reagent anion target, showing total conditions that optimizes the ETD reaction and scan cycle time. . Compact Orbitrap mass spectrometer FIGURE 5. Plot of the reaction time required to reduce the reactant precursor by the fit to the data and the optimal reagent anion target leading to at least 90% 90 operating at ultra-high voltage a fixed amount, calculated as a function of the reaction rate coefficient. We find . . Produces 500K resolution every of the k observed. The calibrated reaction conditions are demonstrated to provide optimal conditions 1.2 seconds max that 3 to 6% remaining precursor is optimal for most compounds. for ETD identifications and sequence coverage. . Ultra-fast operation produces 80 15 spectra/sec with 15K resolution . 2 0.07 Parallel acquisition provides ITMS ETD scan rate cycle times of up to 12 Hz. 100 50 C-Trap 0.06 Rate Coefficient References Active beam guide (ABG) Ions are focused and -1 . Prevents neutral species from injected into the Orbitrap 40 sec Number of Fragments 1. Earley et al., 61st ASMS Conference on Mass Spectrometry and Allied Topics, entering Q1 mass spectrometer 80 -1 . Axial fields improves 0.05 60 sec 40 Minneapolis, MN, June 9–13, 2013; Poster Th101 – Implementation of a operational robustness Quadrupole mass filter High Selectivity and -1 Multipurpose Glow Discharge Ion Source for the Introduction of Reagent/ excellent transmission 80 sec

) 0.04 60 Calibrant Ions Into a Hybrid Mass Spectrometer, poster number: 101, Thursday,

-1 k 30 Halls B&C. Easy-ETD ion source Fit to Data 0 1 2 3 4 5 6 7 8 9 . Discharge-based source 0.03 2. Earley et al., Presented at the 58th ASMS Conference on Mass Spectrometry and . Robust design with an extremely Best Target 40 ETD Reaction Time (msec) stable source of ions k (msec Allied Topics, Salt Lake City, Utah, May 23–27, 2010; Poster T600. S-Lens . High sensitivity 0.02 FIGURE 8. Ubiquitin sequence coverage resulting from averaging 20 FT micro 3. Stephenson, J. L., Jr. and McLuckey, S.A. J. Am. Chem. Soc. 1996, 118, . Robust ion optics 20 scans for a total acquisition time of 6.7 seconds. Fragments denoted by the red 7390–7397.

Precursor Remaining (%) Remaining Precursor (c ion), blue (z ion), and green (y ion) asterisks were found using manual 0.01 interpretation of the data. 0 ProSightPC is a trademark of Proteinaceous Inc. All other trademarks are the property of Thermo Fisher 0.00 Scientific and its subsidiaries. 0 1x10 5 2x105 3x105 4x105 5x105 0 20 40 60 80 100 120 This information is not intended to encourage use of these products in any manners that might infringe the Reagent Target Reaction Time (msec) intellectual property rights of others.

6 Second-Generation Electron Transfer Dissociation (ETD) on the Thermo Scienti c Orbitrap Fusion Mass Spectrometer with Improved Functionality, Increased Speed, and Improved Robustness of Data www.thermoscientific.com ©2013 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. Thermo Fisher Scientific, ProSightPC is a trademark of Proteinaceous Inc. All other trademarks are the property of Thermo Fisher Scientific, Inc. and its San Jose, CA USA subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult is ISO 9001:2008 Certified. your local sales representative for details.

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