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1 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 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. Hunt 3 1 Thermo Fisher Scientific, San Jose, CA; 2 Kelleher Lab, Northwestern University, Evanston, IL; 3 Department of Chemistry, University of Virginia, Charlottesville, VA

2 Overview Purpose: Improve ETD robustness, functionality, and speed on the Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer Methods: Orbitrap Fusion mass spectrometer with the Thermo Scientific EASY-ETD ion source Results: Demonstrated increased ETD functionality and usability by using a combination of hardware and software improvements Introduction The Orbitrap Fusion platform incorporates a second-generation EASY-ETD reagent ion source. The EASY-ETD source improvements include a bright and stable glow-dischargebased ETD reagent ion source located between the S-Lens and the Active Beam Guide, and a higher frequency RF axial trapping field (trap end-lens voltage) to improve ion confinement during ETD (Figure 1). The Orbitrap Fusion mass spectrometer design enables previously unavailable parallel/pipelined scan modes to minimize overall scan cycle times. Further, calibration of the ETD reaction kinetics ensures the shortest possible reaction times while maximizing product ion yields and spectral reproducibility. Collectively, these developments constitute a new-generation ETD platform on the Orbitrap Fusion mass spectrometer. Methods Reagent anions from a glow discharge source (previously described) 1,2 are introduced into the ion optics path ahead of the quadrupole mass filter where they are m/z selected, accumulated in the ion-routing multipole, and then transferred into the high pressure trap (HPT) of the dual-pressure linear ion trap for the ETD reaction. Increasing the frequency of the RF axial confinement field during ETD from ½ to 2 times the quadrupole field frequency avoids parametric resonance excitation and ejection of low m/z (typically 1 1 Th) ions. ETD products may be directly transferred to the low pressure trap (LPT) or to the Orbitrap Fusion mass spectrometer for m/z analysis. ITMS 2 ETD scan rates of up to 12 Hz are attainable using a parallel acquisition mode. 2 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

3 FIGURE 1. Schematic of the Orbitrap Fusion tribrid mass spectrometer showing the location of the EASY-ETD reagent ion source within the overall ion optics path. The exploded view shows how the reagent ion source is incorporated into the S-Lens/Q region. Ion-routing multipole High-pressure cell produces highly efficient storage of ions Voltages can be adjusted to fragment ions at higher energies Dual-pressure linear ion trap Fragments ions using either CID or optional ETD and provides fast, sensitive mass analysis Ultra-high-field Orbitrap mass analyzer Compact Orbitrap mass spectrometer operating at ultra-high voltage Produces K resolution every 1.2 seconds Ultra-fast operation produces 15 spectra/sec with 15K resolution Active beam guide (ABG) Prevents neutral species from entering Q1 Axial fields improves operational robustness Quadrupole mass filter High Selectivity and excellent transmission C-Trap Ions are focused and injected into the Orbitrap mass spectrometer EASY-ETD ion source Discharge-based source Robust design with an extremely stable source of ions S-Lens High sensitivity Robust ion optics Results Calibrating the reaction kinetics ensures that the ETD fragmentation efficiency is optimized and that the maximum duty cycle for ETD can be accomplished. Calibrating the reaction kinetics is a multi-step process in which the decay of the angiotensin I (433 m/z) precursor is monitored as a function of reaction time at a number of reagent targets (Figure 2). From the slope of the individual decay curves, the reaction rate coefficient is extracted, and plotted as a function of the reagent target at which it was acquired (Figure 3). The data are then fitted to find the target at which the reaction rate coefficient saturates, and combinations of this target with the reaction rate coefficient are used to calculate the optimal charge state dependent reaction times (Table 1). The reaction rate coefficient as a function of the precursor charge state squared has been demonstrated to be linear by J. L. Stephenson Jr. and S. A. McLuckey 3, which is verified in Figure 4, and used to calculate the optimal reaction time per charge state, based on a desired amount of reaction completeness. We find 95% consumption of the initial precursor intensity to yield high quality ETD spectra. Figure 5 shows the relationship between the reaction time and the amount of precursor remaining after reaction for the angiotensin I (433 m/z) precursor. Thermo Scientifi c Poster Note PN HUPO13_POS-3-68_CMullen_E 9/13S 3

4 Results Calibrating the reaction kinetics ensures that the ETD fragmentation efficiency is optimized and that the maximum duty cycle for ETD can be accomplished. Calibrating the reaction kinetics is a multi-step process in which the decay of the angiotensin I (433 m/z) precursor is monitored as a function of reaction time at a number of reagent targets (Figure 2). From the slope of the individual decay curves, the reaction rate coefficient is extracted, and plotted as a function of the reagent target at which it was acquired (Figure 3). The data are then fitted to find the target at which the reaction rate coefficient saturates, and combinations of this target with the reaction rate coefficient are used to calculate the optimal charge state dependent reaction times (Table 1). The reaction rate coefficient as a function of the precursor charge state squared has been demonstrated to be linear by J. L. Stephenson Jr. and S. A. McLuckey 3, which is verified in Figure 4, and used to calculate the optimal reaction time per charge state, based on a desired amount of reaction completeness. We find 95% consumption of the initial precursor intensity to yield high quality ETD spectra. Figure 5 shows the relationship between the reaction time and the amount of precursor remaining after reaction for the angiotensin I (433 m/z) precursor. Precursor Remaining (%) React FIGURE 2. Angiotensen I (433 m/z) precursor decay curves as a function of the reagent anion target under pseudo 1 st order reaction conditions. The reaction progress is monitored for up to four half-lives, and the slope of the individual decays is equal to the negative of the rate coefficient.. FIGURE 6. ETD spectra o of ubiquitin at m/z A) 3.25 msec, the calibra -.5 Ubiquitin_3pt25msec_2e5_uscans #1 RT:. AV: 1 NL: 7.96E6 T: FTMS + p ESI Full ms2 [1.-.] ln(a/a ) Target:.1e5 Target:.2e5 Target:.4e5 Target:.6e5 Target:.7e5 Target:.8e5 Target: 1.e5 Target: 2.e5 Target: 4.e5 Target: 9.9e5-3. Relative Abundance Relative Abundance ubiquitin_8msec_2e5_uscans #1 RT:. AV: 1 NL: 3.24E6 T: FTMS + p ESI Full ms2 [1.-.] Reaction Time (msec) 4 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

5 FIGURE 9. Ion-trap ETD MSM reaction and precursor injec acquisition. The reagent inje msec, corresponding to a rea for all experiments. FIGURE 3. The reaction rate coefficient versus reagent anion target, showing the fit to the data and the optimal reagent anion target leading to at least % of the k max observed k (msec -1 ).4.3 k Fit to Data Best Target x 5 2x 5 3x 5 4x 5 5x 5 Reagent Target TABLE 1. Calculated charge state dependent reaction times based on a saturated reaction rate coefficient of 58.2 sec 1 for charge state 3+, based on 5% of the precursor remaining after reaction. Charge State Reaction Time (msec) FIGURE 8. Ubiquitin sequenc averaging FT micro scans 6.7 seconds. Fragments den (z ion), and green (y ion) aste manual interpretation of the Unique ETD Capabilities The location of the ion-routing Fusion mass spectrometer allo acquisition mode, enabling ITM acquisition rates up to Hz. I rates are slightly reduced due t requirement of the ion-ion reac attainable. In addition, the abili and m/z analysis in parallel wit that the spectral acquisition rat can be maintained for a signific time than in the absence of par Thermo Scientifi c Poster Note PN HUPO13_POS-3-68_CMullen_E 9/13S ime (msec) 5

6 FIGURE 4. The maximum rate coefficient vs. the charge state squared of the precursor is linear 1, allowing extrapolation of the optimal reaction times obtained for a single charge state to all charge states k max (msec -1 ) Ubiquitin Angiotensen Charge State Square (z 2 ) FIGURE 5. Plot of the reaction time required to reduce the reactant precursor by a fixed amount, calculated as a function of the reaction rate coefficient. We find that 3 to 6% remaining precursor is optimal for most compounds. acquisition rates up to H rates are slightly reduced d requirement of the ion-ion attainable. In addition, the and m/z analysis in paralle that the spectral acquisitio can be maintained for a sig time than in the absence o FIGURE 9. Ion-trap ETD M reaction and precursor in acquisition. The reagent msec, corresponding to for all experiments. ETD Reaction Time (msec) Precursor Remaining (%) Rate Coefficient sec -1 sec -1 sec -1 1 Reaction Time (msec) Precursor Inje Conclusion A second-generation ETD source has bee Fusion MS that incor software advanceme Calibration of the rea guesswork from ETD optimizes the ETD re The calibrated reacti provide optimal cond sequence coverage. Parallel acquisition p cycle times of up to 1 References 1.Earley et al., 61 st ASM Spectrometry and All June 9 13, 13; Po Multipurpose Glow D Introduction of Reage Mass Spectrometer, Halls B&C. 2. Earley et al., Present 6 Second-Generation Electron Transfer Dissociation (ETD) on the Thermo Scientifi c Orbitrap Fusion Mass Spectrometer with Improved Functionality, on Increased Mass Spectromet Speed, and Improved Robustness of Data City, Utah, May 23 2

7 Verification of the Calibration The calibrated ETD reaction conditions are verified by using an infusion of ubiquitin from bovine erythrocytes. Spectra of the 12+ charge state at m/z were obtained as a function of the ETD reaction time at an FT resolution of 1K and averaged for micro scans. The spectra were then searched using ProSightPC. Figure 6 shows two representative ubiquitin spectra, obtained at 3.25 msec and 8 msec of reaction time, respectively. The calibrated reaction conditions predict an optimal reaction time of 3.25 msec for the 12+ precursor, and while the spectrum visually looks under reacted, it yields the most total c and z fragments from the ProSightPC v3. search (Figure 7). In addition, we demonstrate the ability to obtain nearly complete ubiquitin sequence coverage on a LC timescale using a combination of ProSightPC searching and manual interpretation of the spectra. The results presented in Figure 8 were achieved by averaging FT micro scans, corresponding to a total acquisition time of 6.7 seconds. FIGURE 6. ETD spectra obtained on the 12+ charge state of ubiquitin at m/z at two different reaction times. A) 3.25 msec, the calibrated reaction time. B) 8 msec. optimizes the ETD react The calibrated reaction c provide optimal condition sequence coverage. Parallel acquisition provi cycle times of up to 12 H References 1.Earley et al., 61 st ASMS Spectrometry and Allied June 9 13, 13; Poster Multipurpose Glow Disch Introduction of Reagent/ Mass Spectrometer, pos Halls B&C. 2.Earley et al., Presented on Mass Spectrometry a City, Utah, May 23 27, 2 3.Stephenson, J. L., Jr. an Chem. Soc. 1996, 118, Ubiquitin_3pt25msec_2e5_uscans #1 RT:. AV: 1 NL: 7.96E6 T: FTMS + p ESI Full ms2 [1.-.] A) ProSightPC is a trademark of Proteinaceous Inc. All oth and its subsidiaries. This information is not intended to encourage use of the intellectual property rights of others. Relative Abundance m/z ubiquitin_8msec_2e5_uscans #1 RT:. AV: 1 NL: 3.24E6 T: FTMS + p ESI Full ms2 [1.-.] B) Relative Abundance m/z Thermo Scientifi c Poster Note PN HUPO13_POS-3-68_CMullen_E 9/13S 7

8 FIGURE 7. ProSightPC search results for the 12+ charge state of ubiquitin (714.7 m/z) as a function of the ETD reaction time, demonstrating that the calibrated kinetics chooses appropriate reaction times up to at least charge state 12 Number of Fragments 1 c z total ETD Reaction Time (msec) FIGURE 8. Ubiquitin sequence coverage resulting from averaging FT micro scans for a total acquisition time of 6.7 seconds. Fragments denoted by the red (c ion), blue (z ion), and green (y ion) asterisks were found using manual interpretation of the data. Unique ETD Capabilities The location of the ion-routing multipole within the Orbitrap Fusion mass spectrometer allows for a parallel ITMS 2 acquisition mode, enabling ITMS 2 CID and HCD spectral acquisition rates up to Hz. ITMS 2 ETD spectral acquisition rates are slightly reduced due to the additional time requirement of the ion-ion reaction, but rates up to 12 Hz are attainable. In addition, the ability to perform the ion-ion reaction and m/z analysis in parallel with the precursor injection means that the spectral acquisition rate at a particular reaction time can be maintained for a significantly longer precursor injection time than in the absence of parallelization (Figure 9). FIGURE 9. Ion-trap ETD MSMS cycle rate dependence on reaction and precursor injection times using parallel acquisition. The reagent injection time was fixed at 5 msec, corresponding to a reagent anion population of 2e5 for all experiments. ime (msec) Frequency (Hz) 6. 8 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 7. 5.

9 time than in the absence of parallelization (Figure 9). FIGURE 9. Ion-trap ETD MSMS cycle rate dependence on reaction and precursor injection times using parallel acquisition. The reagent injection time was fixed at 5 msec, corresponding to a reagent anion population of 2e5 for all experiments. ETD Reaction Time (msec) Frequency (Hz) Precursor Injection Time (msec) Conclusion A second-generation glow-discharge-based EASY- ETD source has been developed for the Orbitrap Fusion MS that incorporates significant hardware and software advancements. Calibration of the reaction kinetics removes the guesswork from ETD, and leads to conditions that optimizes the ETD reaction and scan cycle time. The calibrated reaction conditions are demonstrated to provide optimal conditions for ETD identifications and sequence coverage. Parallel acquisition provides ITMS 2 ETD scan rate cycle times of up to 12 Hz. References 1.Earley et al., 61 st ASMS Conference on Mass Spectrometry and Allied Topics, Minneapolis, MN, June 9 13, 13; Poster Th1 Implementation of a Multipurpose Glow Discharge Ion Source for the Introduction of Reagent/Calibrant Ions Into a Hybrid Mass Spectrometer, poster number: 1, Thursday, Halls B&C. 2.Earley et al., Presented at the 58 th ASMS Conference on Mass Spectrometry and Allied Topics, Salt Lake City, Utah, May 23 27, ; Poster T. 3.Stephenson, J. L., Jr. and McLuckey, S.A. J. Am. Chem. Soc. 1996, 118, A) ProSightPC is a trademark of Proteinaceous Inc. 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 manners that might infringe the intellectual property rights of others. Thermo Scientifi c Poster Note PN HUPO13_POS-3-68_CMullen_E 9/13S 9

10 sec -1 sec -1 sec charge state action times.. B) 8 msec. A) Conclusion A second-generation glow-discharge-based EASY- ETD source has been developed for the Orbitrap Fusion MS that incorporates significant hardware and software advancements. Calibration of the reaction kinetics removes the guesswork from ETD, and leads to conditions that optimizes the ETD reaction and scan cycle time. The calibrated reaction conditions are demonstrated to provide optimal conditions for ETD identifications and sequence coverage. Parallel acquisition provides ITMS 2 ETD scan rate cycle times of up to 12 Hz. References Precursor Injection Time (msec) 1.Earley et al., 61 st ASMS Conference on Mass Spectrometry and Allied Topics, Minneapolis, MN, June 9 13, 13; Poster Th1 Implementation of a Multipurpose Glow Discharge Ion Source for the Introduction of Reagent/Calibrant Ions Into a Hybrid Mass Spectrometer, poster number: 1, Thursday, Halls B&C. 2.Earley et al., Presented at the 58 th ASMS Conference on Mass Spectrometry and Allied Topics, Salt Lake City, Utah, May 23 27, ; Poster T. 3.Stephenson, J. L., Jr. and McLuckey, S.A. J. Am. Chem. Soc. 1996, 118, ProSightPC is a trademark of Proteinaceous Inc. 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 manners that might infringe the intellectual property rights of others B) Thermo Fisher Scientifi c Inc. All rights reserved. ISO is a trademark of the International Standards Organization. ProSightPC is a trademark of Proteinaceous Inc. All other trademarks are the property of Thermo Fisher Scientifi c, Inc. and its subsidiaries. Specifi cations, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Thermo Fisher Scientifi c, San Jose, CA USA is ISO 1:8 Certifi ed. Africa-Other Australia Austria Belgium Canada China Denmark Europe-Other Finland France Germany India Italy Japan Latin America Middle East Netherlands New Zealand Norway Russia/CIS South Africa Spain Sweden Switzerland UK USA HUPO13_POS-3-68_CMullen_E 9/13S

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