Improved Electron Transfer Dissociation (ETD) Duty Cycle and Spectral Signal to Noise Ratio in a Dual Cell Linear Ion Trap

Christopher Mullen,1 Lee Earley,1 Jean-Jacques Dunyach,1 John E.P. Syka,1 Jeffrey Shabanowitz,2 A. Michelle English,2 Donald F. Hunt2 1Thermo Fisher Scientific, San Jose, CA; 2Department of Chemistry, University of Virginia, Charlottesville, VA Improved Electron Transfer Dissociation (ETD) Duty Cycle and Spectral Signal to Noise Ratio in a Dual Cell Linear Ion Trap

Christopher Mullen1, Lee Earley1, Jean-Jacques Dunyach1, John E.P. Syka1, Jeffrey Shabanowitz2, A. Michelle English2, Donald F. Hunt3 1Thermo Fisher Scientific, San Jose, CA; 2Department of Chemistry, University of Virginia, Charlottesville, VA

Unfortunately, certain combinations of axial well depth and RF pseudopotential can FIGURE 6. Comparison of the Angiotensin I ETD c and z ion fragment TIC for an The geometry of the Orbitrap Fusion Tribrid MS in conjunction with it’s ability to run Overview Results lead to ion excitation as the ions spend more time at greater radii. The increased up to 8-fills ETD approach versus a single fill. The multiple fill data was taken at scans in a parallel/pipelined fashion affords a significant decrease in scan acquisition kinetic energy in these situations causes fragmentation which generates significant three individual precursor targets per fill corresponding to 1e4 (blue squares), time per spectrum for the multiple fills approach (Figure 8) as the ETD fills are run in Purpose: Improve ETD Signal to Noise (S/N) ratio and scan duty cycle. Motivation for Employing Multiple Fills ETD 2 amounts of b and y series ions , and contributes to overall lower signal to noise (S/N) 5e4 (red circles), and 1e5 (green triangles) charges/fill. parallel with OT m/z analysis. Methods: Thermo Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer with the Performing the ETD reaction in the HPT of the Orbitrap Fusion Tribrid MS requires the ratio ETD spectra. We demonstrate that the precursor fragmentation is occurring Thermo Scientific™ EASY-ETD™ ion source. cation precursor population to be sequestered to the back section of the HPT, so that during the sequester events by monitoring the fragment TIC in the absence of ion-ion the reagent anion species can be injected into the trap prior to the charge sign 5 Results: Developed a methodology incorporating multiple fills of ETD products into a reaction time, Figure 4. 1.50x10 independent trapping (CSIT) and ETD reaction events. As a result, the maximum FIGURE 8. Scan acquisition time per spectrum for the ETD multiple fills storage cell followed by a single m/z analysis leading to improved spectral S/N ratio MultFills 1e4 Target/Fill number of cation precursor charges that can be employed per ETD reaction is a experiment and the uScans approach taken at a variety of Orbitrap transient and acquisition speed. MultFills 5e4 Target/Fill durations. function of the space charge capacity of the section employed for sequestration. FIGURE 4. a) Angiotensin I b and y ion formation observed at high precursor 5 1.25x10 MultFills 1e5 Target/Fill Figure 2 shows the potentials employed during the cation sequestration/reagent initial targets resulting from harsh sequestration conditions. b) Fragment yield Single Fill Introduction injection events and the relative locations of the cationic and anionic species. versus precursor target demonstrating the onset of b and y ion formation. 1.2 12 5 Electron transfer dissociation (ETD) has been demonstrated to be a useful tool for the AngioVsTarget_ETD_50msec #548 RT: 2.58 AV: 1 NL: 6.18E5 1.00x10 FIGURE 2. Cation sequestration and reagent injection conditions prior to the T: FTMS + p ESI Full ms2 [email protected] [150.0000-1500.0000] 32 msec analysis of polypeptide compounds including peptides with labile PTM’s, peptides with x2 ETD reaction in the high pressure cell of the dual cell linear trap 432.90 64 msec 100 many basic sites, and large proteins. One drawback of the approach is that it leads to a 128 msec reduction of both scan duty cycle and total MS2 ion signal, compared to conventional 95 4 1.0 10 Accumulation of Precursor Cations and Reagent Anions TIC ETD Fragement 7.50x10 256 msec 90 resonant CID, due to necessity to perform the ion-ion reaction and the fact that the a) 3+ 512 msec 85 130620 CaP4 Area vs. Precursor Target reaction consumes charge. This research explores the possibility to circumvent these Reagent = 2e5. Rxn Time = 70 msec 1024 msec 80 Precursor = Angio3+ 433.23 6 limitations by performing multiple ETD reactions per m/z analysis in a 2D linear ion trap 10 Analyzer = Orbitrap 4 75 0.8 8 on a three-analyzer hybrid instrument based upon a mass resolving quadrupole, b) 5.00x10 70 Orbitrap (OT), collision cell, and dual linear ion trap (Q-OT-LT) architecture. 5 65 10

60 2.50x104 0.6 6 Methods 55 104 50

HPT LPT I [M+3H] Angiotensin 513.28 45 The multi-fill per m/z analysis scan mode in the dual cell linear ion trap is accomplished Area 3

Relative Abundance 10 by using the high pressure trap (HPT) as a reaction vessel, and utilizing the low 40 0.00 0.4 4 Previously m/z Isolated Precursor Cations Held In Back Section 5 5 5 5 6 pressure trap (LPT) as an accumulation and m/z analysis cell. In this fashion, the 35 tic 0.0

2 2.0x10 4.0x10 6.0x10 8.0x10 1.0x10

multiple fills ETD scan cycle becomes a loop over n (the number of fills requested) 30 10 precursor

c and z - - -

ETD reaction, transfer and storage cycles, followed by a single m/z analysis in either +10 V - 25 b and y Precursor Target

20 1 0.2 2 the low pressure trap of the dual cell or, after the appropriate transfer, in the OT mass 10 (sec) Spectrum per Scan Time

- 3 4 5 6 analyzer. All experiments were performed on an Orbitrap Fusion Tribrid MS. 15 10 10 10 10 583.31 10 Precursor Target 0 V + + 619.37 784.44 514.77 647.37 1028.59 It is apparent (Figure 6) that the working range using the multiple fills approach is 5 785.45 500.77 569.31 592.30 649.37 434.72 756.45 786.45 1000.60 1030.60 0.0 0 0 much greater than that for a single fill, while preserving soft sequestration conditions. 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 0 2 4 6 8 10 0 2 4 6 8 10 m/z The fit to the data (Figure 7) represents the theoretical gains that could be realized if The charge capacity of the back section of the HPT is determined by the axial potential the S/N ratio increases linearly with the ion population and as a square root function well and radial pseudopotential, and can be measured by a number of methods. We ETD Fills uScans for scan averaging. FIGURE 1. Schematic of the Orbitrap Fusion Tribrid mass spectrometer choose to examine the product yield from the ETD reaction of Angiotensin I as a showing the location of the Easy-ETD reagent ion source within the overall ion function of the starting precursor ion population. Fragment TIC plotted as a function of ETD Multiple Fills Proof of Concept optics path. The exploded view shows how the reagent ion source is the axial potential in the back section of the high pressure trap during the ion The multiple fills ETD approach has been previously demonstrated3,4 and is incorporated into the S-Lens/Q00 region. sequester event (Figure 3) is characterized by a linear region where ETD products advantageous as it has the capability to provide higher S/N ratio ETD spectra, while increase with the initial precursor population, followed by a transition to a plateau providing facile ETD sequestration conditions. A flow diagram detailing the approach is FIGURE 7. Comparison of the S/N ratio of ETD c and z fragments from tetrapeptide MRFA [M+2H]2+ after reaction for 100 msec versus the number of Ion-routing multipole region where additional precursor does not yield additional product species. The presented in Figure 5. Figures 6, 7, and 8 compare and contrast the multiple fills Conclusions HCD Cell location of the plateau is dictated by the balance of space charge forces with that of approach to the scan averaging approach in terms of spectral S/N ratio scan injection events. For the case of multiple ETD fills this represents the number of 1 . Single fill ETD limited by space charge capacity of the back section of the ion the effective RF focusing forces, and has been well modeled in the literature. acquisition speed. precursor fills/ETD cycles before m/z analysis; uScans represents the number of scans averaged. trap in the Orbitrap Fusion Tribrid MS. Dual-pressure . linear ion trap FIGURE 5. Flow diagram describing the ETD multiple fills approach when the 4.0 The sequestration conditions during ETD need careful consideration in order to FIGURE 3. Angiotensin I ETD product ion yield vs. the starting precursor ETD reaction is conducted in the HPT and product accumulation is conducted minimize trap-like collision induced dissociation. population for a single ETD reaction. ETD ion source Orbitrap Mass in the LPT. Mass analysis can be conducted in either analyzer. . A multiple fills methodology has been presented based on ETD reaction in the Analyzer 3.5 Quadrupole 1.0x105 high pressure trap followed by accumulation of ETD products in the low pressure Mass Filter -0.5 Volts trap. -1 Volts 3.0 . The geometry of the Orbitrap Fusion Tribrid MS in conjunction with the ability to HPT LPT 4 -2 Volts 8.0x10 run both analyzers in parallel allows for acquisition of high S/N ratio spectra in a much shorter time than can be done with scan averaging alone. 2.5 6.0x104 References 2.0 ETD Fragement TIC ETD Fragement 1. Tolmachev, A. V., Udseth, H. R., Smith, R.D., IJMS 222 (2003) pg 155-174. 3+ MRFA_c2 4.0x104 MRFA_c3 2. Sannes-Lowery, K., Griffey, R.H., Kruppa, G.H., Speir, J.P., Hofstadler, S.A., 1.5 MRFA_z3 Rapid Comm in Mass Spec 12 (1998) pg 1957-1961. MRFA 3. Rose, C.M. et al., J Am Soc Mass Spectrom 24 (2013) pg 816-827. 4 2.0x10 S/N(uscans) / S/N(multFills) n/sqrt(n) 1.0 4. Earley, L. et al., Anal Chem 85:17 (2013) pg 8385-8390.

Angiotensin I [M+3H] Angiotensin 0.5 0.0 0.0 2.0x105 4.0x105 6.0x105 8.0x105 1.0x106 1 2 3 4 5 6 7 8 9

Precursor Target number of injection events All 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.

PO64107-EN 0614S

2 Improved Electron Transfer Dissociation (ETD) Duty Cycle and Spectral Signal to Noise Ratio in a Dual Cell Linear Ion Trap Improved Electron Transfer Dissociation (ETD) Duty Cycle and Spectral Signal to Noise Ratio in a Dual Cell Linear Ion Trap

Christopher Mullen1, Lee Earley1, Jean-Jacques Dunyach1, John E.P. Syka1, Jeffrey Shabanowitz2, A. Michelle English2, Donald F. Hunt3 1Thermo Fisher Scientific, San Jose, CA; 2Department of Chemistry, University of Virginia, Charlottesville, VA

Unfortunately, certain combinations of axial well depth and RF pseudopotential can FIGURE 6. Comparison of the Angiotensin I ETD c and z ion fragment TIC for an The geometry of the Orbitrap Fusion Tribrid MS in conjunction with it’s ability to run Overview Results lead to ion excitation as the ions spend more time at greater radii. The increased up to 8-fills ETD approach versus a single fill. The multiple fill data was taken at scans in a parallel/pipelined fashion affords a significant decrease in scan acquisition kinetic energy in these situations causes fragmentation which generates significant three individual precursor targets per fill corresponding to 1e4 (blue squares), time per spectrum for the multiple fills approach (Figure 8) as the ETD fills are run in Purpose: Improve ETD Signal to Noise (S/N) ratio and scan duty cycle. Motivation for Employing Multiple Fills ETD 2 amounts of b and y series ions , and contributes to overall lower signal to noise (S/N) 5e4 (red circles), and 1e5 (green triangles) charges/fill. parallel with OT m/z analysis. Methods: Thermo Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer with the Performing the ETD reaction in the HPT of the Orbitrap Fusion Tribrid MS requires the ratio ETD spectra. We demonstrate that the precursor fragmentation is occurring Thermo Scientific™ EASY-ETD™ ion source. cation precursor population to be sequestered to the back section of the HPT, so that during the sequester events by monitoring the fragment TIC in the absence of ion-ion the reagent anion species can be injected into the trap prior to the charge sign 5 Results: Developed a methodology incorporating multiple fills of ETD products into a reaction time, Figure 4. 1.50x10 independent trapping (CSIT) and ETD reaction events. As a result, the maximum FIGURE 8. Scan acquisition time per spectrum for the ETD multiple fills storage cell followed by a single m/z analysis leading to improved spectral S/N ratio MultFills 1e4 Target/Fill number of cation precursor charges that can be employed per ETD reaction is a experiment and the uScans approach taken at a variety of Orbitrap transient and acquisition speed. MultFills 5e4 Target/Fill durations. function of the space charge capacity of the section employed for sequestration. FIGURE 4. a) Angiotensin I b and y ion formation observed at high precursor 5 1.25x10 MultFills 1e5 Target/Fill Figure 2 shows the potentials employed during the cation sequestration/reagent initial targets resulting from harsh sequestration conditions. b) Fragment yield Single Fill Introduction injection events and the relative locations of the cationic and anionic species. versus precursor target demonstrating the onset of b and y ion formation. 1.2 12 5 Electron transfer dissociation (ETD) has been demonstrated to be a useful tool for the AngioVsTarget_ETD_50msec #548 RT: 2.58 AV: 1 NL: 6.18E5 1.00x10 FIGURE 2. Cation sequestration and reagent injection conditions prior to the T: FTMS + p ESI Full ms2 [email protected] [150.0000-1500.0000] 32 msec analysis of polypeptide compounds including peptides with labile PTM’s, peptides with x2 ETD reaction in the high pressure cell of the dual cell linear trap 432.90 64 msec 100 many basic sites, and large proteins. One drawback of the approach is that it leads to a 128 msec reduction of both scan duty cycle and total MS2 ion signal, compared to conventional 95 4 1.0 10 Accumulation of Precursor Cations and Reagent Anions TIC ETD Fragement 7.50x10 256 msec 90 resonant CID, due to necessity to perform the ion-ion reaction and the fact that the a) 3+ 512 msec 85 130620 CaP4 Area vs. Precursor Target reaction consumes charge. This research explores the possibility to circumvent these Reagent = 2e5. Rxn Time = 70 msec 1024 msec 80 Precursor = Angio3+ 433.23 6 limitations by performing multiple ETD reactions per m/z analysis in a 2D linear ion trap 10 Analyzer = Orbitrap 4 75 0.8 8 on a three-analyzer hybrid instrument based upon a mass resolving quadrupole, b) 5.00x10 70 Orbitrap (OT), collision cell, and dual linear ion trap (Q-OT-LT) architecture. 5 65 10

60 2.50x104 0.6 6 Methods 55 104 50

HPT LPT I [M+3H] Angiotensin 513.28 45 The multi-fill per m/z analysis scan mode in the dual cell linear ion trap is accomplished Area 3

Relative Abundance 10 by using the high pressure trap (HPT) as a reaction vessel, and utilizing the low 40 0.00 0.4 4 Previously m/z Isolated Precursor Cations Held In Back Section 5 5 5 5 6 pressure trap (LPT) as an accumulation and m/z analysis cell. In this fashion, the 35 tic 0.0

2 2.0x10 4.0x10 6.0x10 8.0x10 1.0x10

multiple fills ETD scan cycle becomes a loop over n (the number of fills requested) 30 10 precursor

c and z - - -

ETD reaction, transfer and storage cycles, followed by a single m/z analysis in either +10 V - 25 b and y Precursor Target

20 1 0.2 2 the low pressure trap of the dual cell or, after the appropriate transfer, in the OT mass 10 (sec) Spectrum per Scan Time

- 3 4 5 6 analyzer. All experiments were performed on an Orbitrap Fusion Tribrid MS. 15 10 10 10 10 583.31 10 Precursor Target 0 V + + 619.37 784.44 514.77 647.37 1028.59 It is apparent (Figure 6) that the working range using the multiple fills approach is 5 785.45 500.77 569.31 592.30 649.37 434.72 756.45 786.45 1000.60 1030.60 0.0 0 0 much greater than that for a single fill, while preserving soft sequestration conditions. 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 0 2 4 6 8 10 0 2 4 6 8 10 m/z The fit to the data (Figure 7) represents the theoretical gains that could be realized if The charge capacity of the back section of the HPT is determined by the axial potential the S/N ratio increases linearly with the ion population and as a square root function well and radial pseudopotential, and can be measured by a number of methods. We ETD Fills uScans for scan averaging. FIGURE 1. Schematic of the Orbitrap Fusion Tribrid mass spectrometer choose to examine the product yield from the ETD reaction of Angiotensin I as a showing the location of the Easy-ETD reagent ion source within the overall ion function of the starting precursor ion population. Fragment TIC plotted as a function of ETD Multiple Fills Proof of Concept optics path. The exploded view shows how the reagent ion source is the axial potential in the back section of the high pressure trap during the ion The multiple fills ETD approach has been previously demonstrated3,4 and is incorporated into the S-Lens/Q00 region. sequester event (Figure 3) is characterized by a linear region where ETD products advantageous as it has the capability to provide higher S/N ratio ETD spectra, while increase with the initial precursor population, followed by a transition to a plateau providing facile ETD sequestration conditions. A flow diagram detailing the approach is FIGURE 7. Comparison of the S/N ratio of ETD c and z fragments from tetrapeptide MRFA [M+2H]2+ after reaction for 100 msec versus the number of Ion-routing multipole region where additional precursor does not yield additional product species. The presented in Figure 5. Figures 6, 7, and 8 compare and contrast the multiple fills Conclusions HCD Cell location of the plateau is dictated by the balance of space charge forces with that of approach to the scan averaging approach in terms of spectral S/N ratio scan injection events. For the case of multiple ETD fills this represents the number of 1 . Single fill ETD limited by space charge capacity of the back section of the ion the effective RF focusing forces, and has been well modeled in the literature. acquisition speed. precursor fills/ETD cycles before m/z analysis; uScans represents the number of scans averaged. trap in the Orbitrap Fusion Tribrid MS. Dual-pressure . linear ion trap FIGURE 5. Flow diagram describing the ETD multiple fills approach when the 4.0 The sequestration conditions during ETD need careful consideration in order to FIGURE 3. Angiotensin I ETD product ion yield vs. the starting precursor ETD reaction is conducted in the HPT and product accumulation is conducted minimize trap-like collision induced dissociation. population for a single ETD reaction. ETD ion source Orbitrap Mass in the LPT. Mass analysis can be conducted in either analyzer. . A multiple fills methodology has been presented based on ETD reaction in the Analyzer 3.5 Quadrupole 1.0x105 high pressure trap followed by accumulation of ETD products in the low pressure Mass Filter -0.5 Volts trap. -1 Volts 3.0 . The geometry of the Orbitrap Fusion Tribrid MS in conjunction with the ability to HPT LPT 4 -2 Volts 8.0x10 run both analyzers in parallel allows for acquisition of high S/N ratio spectra in a much shorter time than can be done with scan averaging alone. 2.5 6.0x104 References 2.0 ETD Fragement TIC ETD Fragement 1. Tolmachev, A. V., Udseth, H. R., Smith, R.D., IJMS 222 (2003) pg 155-174. 3+ MRFA_c2 4.0x104 MRFA_c3 2. Sannes-Lowery, K., Griffey, R.H., Kruppa, G.H., Speir, J.P., Hofstadler, S.A., 1.5 MRFA_z3 Rapid Comm in Mass Spec 12 (1998) pg 1957-1961. MRFA 3. Rose, C.M. et al., J Am Soc Mass Spectrom 24 (2013) pg 816-827. 4 2.0x10 S/N(uscans) / S/N(multFills) n/sqrt(n) 1.0 4. Earley, L. et al., Anal Chem 85:17 (2013) pg 8385-8390.

Angiotensin I [M+3H] Angiotensin 0.5 0.0 0.0 2.0x105 4.0x105 6.0x105 8.0x105 1.0x106 1 2 3 4 5 6 7 8 9

Precursor Target number of injection events All 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.

PO64107-EN 0614S

Thermo Scientific Poster Note• PN-64107-ASMS-EN-0614S 3 Improved Electron Transfer Dissociation (ETD) Duty Cycle and Spectral Signal to Noise Ratio in a Dual Cell Linear Ion Trap

Christopher Mullen1, Lee Earley1, Jean-Jacques Dunyach1, John E.P. Syka1, Jeffrey Shabanowitz2, A. Michelle English2, Donald F. Hunt3 1Thermo Fisher Scientific, San Jose, CA; 2Department of Chemistry, University of Virginia, Charlottesville, VA

Unfortunately, certain combinations of axial well depth and RF pseudopotential can FIGURE 6. Comparison of the Angiotensin I ETD c and z ion fragment TIC for an The geometry of the Orbitrap Fusion Tribrid MS in conjunction with it’s ability to run Overview Results lead to ion excitation as the ions spend more time at greater radii. The increased up to 8-fills ETD approach versus a single fill. The multiple fill data was taken at scans in a parallel/pipelined fashion affords a significant decrease in scan acquisition kinetic energy in these situations causes fragmentation which generates significant three individual precursor targets per fill corresponding to 1e4 (blue squares), time per spectrum for the multiple fills approach (Figure 8) as the ETD fills are run in Purpose: Improve ETD Signal to Noise (S/N) ratio and scan duty cycle. Motivation for Employing Multiple Fills ETD 2 amounts of b and y series ions , and contributes to overall lower signal to noise (S/N) 5e4 (red circles), and 1e5 (green triangles) charges/fill. parallel with OT m/z analysis. Methods: Thermo Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer with the Performing the ETD reaction in the HPT of the Orbitrap Fusion Tribrid MS requires the ratio ETD spectra. We demonstrate that the precursor fragmentation is occurring Thermo Scientific™ EASY-ETD™ ion source. cation precursor population to be sequestered to the back section of the HPT, so that during the sequester events by monitoring the fragment TIC in the absence of ion-ion the reagent anion species can be injected into the trap prior to the charge sign 5 Results: Developed a methodology incorporating multiple fills of ETD products into a reaction time, Figure 4. 1.50x10 independent trapping (CSIT) and ETD reaction events. As a result, the maximum FIGURE 8. Scan acquisition time per spectrum for the ETD multiple fills storage cell followed by a single m/z analysis leading to improved spectral S/N ratio MultFills 1e4 Target/Fill number of cation precursor charges that can be employed per ETD reaction is a experiment and the uScans approach taken at a variety of Orbitrap transient and acquisition speed. MultFills 5e4 Target/Fill durations. function of the space charge capacity of the section employed for sequestration. FIGURE 4. a) Angiotensin I b and y ion formation observed at high precursor 5 1.25x10 MultFills 1e5 Target/Fill Figure 2 shows the potentials employed during the cation sequestration/reagent initial targets resulting from harsh sequestration conditions. b) Fragment yield Single Fill Introduction injection events and the relative locations of the cationic and anionic species. versus precursor target demonstrating the onset of b and y ion formation. 1.2 12 5 Electron transfer dissociation (ETD) has been demonstrated to be a useful tool for the AngioVsTarget_ETD_50msec #548 RT: 2.58 AV: 1 NL: 6.18E5 1.00x10 FIGURE 2. Cation sequestration and reagent injection conditions prior to the T: FTMS + p ESI Full ms2 [email protected] [150.0000-1500.0000] 32 msec analysis of polypeptide compounds including peptides with labile PTM’s, peptides with x2 ETD reaction in the high pressure cell of the dual cell linear trap 432.90 64 msec 100 many basic sites, and large proteins. One drawback of the approach is that it leads to a 128 msec reduction of both scan duty cycle and total MS2 ion signal, compared to conventional 95 4 1.0 10 Accumulation of Precursor Cations and Reagent Anions TIC ETD Fragement 7.50x10 256 msec 90 resonant CID, due to necessity to perform the ion-ion reaction and the fact that the a) 3+ 512 msec 85 130620 CaP4 Area vs. Precursor Target reaction consumes charge. This research explores the possibility to circumvent these Reagent = 2e5. Rxn Time = 70 msec 1024 msec 80 Precursor = Angio3+ 433.23 6 limitations by performing multiple ETD reactions per m/z analysis in a 2D linear ion trap 10 Analyzer = Orbitrap 4 75 0.8 8 on a three-analyzer hybrid instrument based upon a mass resolving quadrupole, b) 5.00x10 70 Orbitrap (OT), collision cell, and dual linear ion trap (Q-OT-LT) architecture. 5 65 10

60 2.50x104 0.6 6 Methods 55 104 50

HPT LPT I [M+3H] Angiotensin 513.28 45 The multi-fill per m/z analysis scan mode in the dual cell linear ion trap is accomplished Area 3

Relative Abundance 10 by using the high pressure trap (HPT) as a reaction vessel, and utilizing the low 40 0.00 0.4 4 Previously m/z Isolated Precursor Cations Held In Back Section 5 5 5 5 6 pressure trap (LPT) as an accumulation and m/z analysis cell. In this fashion, the 35 tic 0.0

2 2.0x10 4.0x10 6.0x10 8.0x10 1.0x10

multiple fills ETD scan cycle becomes a loop over n (the number of fills requested) 30 10 precursor

c and z - - -

ETD reaction, transfer and storage cycles, followed by a single m/z analysis in either +10 V - 25 b and y Precursor Target

20 1 0.2 2 the low pressure trap of the dual cell or, after the appropriate transfer, in the OT mass 10 (sec) Spectrum per Scan Time

- 3 4 5 6 analyzer. All experiments were performed on an Orbitrap Fusion Tribrid MS. 15 10 10 10 10 583.31 10 Precursor Target 0 V + + 619.37 784.44 514.77 647.37 1028.59 It is apparent (Figure 6) that the working range using the multiple fills approach is 5 785.45 500.77 569.31 592.30 649.37 434.72 756.45 786.45 1000.60 1030.60 0.0 0 0 much greater than that for a single fill, while preserving soft sequestration conditions. 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 0 2 4 6 8 10 0 2 4 6 8 10 m/z The fit to the data (Figure 7) represents the theoretical gains that could be realized if The charge capacity of the back section of the HPT is determined by the axial potential the S/N ratio increases linearly with the ion population and as a square root function well and radial pseudopotential, and can be measured by a number of methods. We ETD Fills uScans for scan averaging. FIGURE 1. Schematic of the Orbitrap Fusion Tribrid mass spectrometer choose to examine the product yield from the ETD reaction of Angiotensin I as a showing the location of the Easy-ETD reagent ion source within the overall ion function of the starting precursor ion population. Fragment TIC plotted as a function of ETD Multiple Fills Proof of Concept optics path. The exploded view shows how the reagent ion source is the axial potential in the back section of the high pressure trap during the ion The multiple fills ETD approach has been previously demonstrated3,4 and is incorporated into the S-Lens/Q00 region. sequester event (Figure 3) is characterized by a linear region where ETD products advantageous as it has the capability to provide higher S/N ratio ETD spectra, while increase with the initial precursor population, followed by a transition to a plateau providing facile ETD sequestration conditions. A flow diagram detailing the approach is FIGURE 7. Comparison of the S/N ratio of ETD c and z fragments from tetrapeptide MRFA [M+2H]2+ after reaction for 100 msec versus the number of Ion-routing multipole region where additional precursor does not yield additional product species. The presented in Figure 5. Figures 6, 7, and 8 compare and contrast the multiple fills Conclusions HCD Cell location of the plateau is dictated by the balance of space charge forces with that of approach to the scan averaging approach in terms of spectral S/N ratio scan injection events. For the case of multiple ETD fills this represents the number of 1 . Single fill ETD limited by space charge capacity of the back section of the ion the effective RF focusing forces, and has been well modeled in the literature. acquisition speed. precursor fills/ETD cycles before m/z analysis; uScans represents the number of scans averaged. trap in the Orbitrap Fusion Tribrid MS. Dual-pressure . linear ion trap FIGURE 5. Flow diagram describing the ETD multiple fills approach when the 4.0 The sequestration conditions during ETD need careful consideration in order to FIGURE 3. Angiotensin I ETD product ion yield vs. the starting precursor ETD reaction is conducted in the HPT and product accumulation is conducted minimize trap-like collision induced dissociation. population for a single ETD reaction. ETD ion source Orbitrap Mass in the LPT. Mass analysis can be conducted in either analyzer. . A multiple fills methodology has been presented based on ETD reaction in the Analyzer 3.5 Quadrupole 1.0x105 high pressure trap followed by accumulation of ETD products in the low pressure Mass Filter -0.5 Volts trap. -1 Volts 3.0 . The geometry of the Orbitrap Fusion Tribrid MS in conjunction with the ability to HPT LPT 4 -2 Volts 8.0x10 run both analyzers in parallel allows for acquisition of high S/N ratio spectra in a much shorter time than can be done with scan averaging alone. 2.5 6.0x104 References 2.0 ETD Fragement TIC ETD Fragement 1. Tolmachev, A. V., Udseth, H. R., Smith, R.D., IJMS 222 (2003) pg 155-174. 3+ MRFA_c2 4.0x104 MRFA_c3 2. Sannes-Lowery, K., Griffey, R.H., Kruppa, G.H., Speir, J.P., Hofstadler, S.A., 1.5 MRFA_z3 Rapid Comm in Mass Spec 12 (1998) pg 1957-1961. MRFA 3. Rose, C.M. et al., J Am Soc Mass Spectrom 24 (2013) pg 816-827. 4 2.0x10 S/N(uscans) / S/N(multFills) n/sqrt(n) 1.0 4. Earley, L. et al., Anal Chem 85:17 (2013) pg 8385-8390.

Angiotensin I [M+3H] Angiotensin 0.5 0.0 0.0 2.0x105 4.0x105 6.0x105 8.0x105 1.0x106 1 2 3 4 5 6 7 8 9

Precursor Target number of injection events All 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.

PO64107-EN 0614S

4 Improved Electron Transfer Dissociation (ETD) Duty Cycle and Spectral Signal to Noise Ratio in a Dual Cell Linear Ion Trap Improved Electron Transfer Dissociation (ETD) Duty Cycle and Spectral Signal to Noise Ratio in a Dual Cell Linear Ion Trap

Christopher Mullen1, Lee Earley1, Jean-Jacques Dunyach1, John E.P. Syka1, Jeffrey Shabanowitz2, A. Michelle English2, Donald F. Hunt3 1Thermo Fisher Scientific, San Jose, CA; 2Department of Chemistry, University of Virginia, Charlottesville, VA

Unfortunately, certain combinations of axial well depth and RF pseudopotential can FIGURE 6. Comparison of the Angiotensin I ETD c and z ion fragment TIC for an The geometry of the Orbitrap Fusion Tribrid MS in conjunction with it’s ability to run Overview Results lead to ion excitation as the ions spend more time at greater radii. The increased up to 8-fills ETD approach versus a single fill. The multiple fill data was taken at scans in a parallel/pipelined fashion affords a significant decrease in scan acquisition kinetic energy in these situations causes fragmentation which generates significant three individual precursor targets per fill corresponding to 1e4 (blue squares), time per spectrum for the multiple fills approach (Figure 8) as the ETD fills are run in Purpose: Improve ETD Signal to Noise (S/N) ratio and scan duty cycle. Motivation for Employing Multiple Fills ETD 2 amounts of b and y series ions , and contributes to overall lower signal to noise (S/N) 5e4 (red circles), and 1e5 (green triangles) charges/fill. parallel with OT m/z analysis. Methods: Thermo Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer with the Performing the ETD reaction in the HPT of the Orbitrap Fusion Tribrid MS requires the ratio ETD spectra. We demonstrate that the precursor fragmentation is occurring Thermo Scientific™ EASY-ETD™ ion source. cation precursor population to be sequestered to the back section of the HPT, so that during the sequester events by monitoring the fragment TIC in the absence of ion-ion the reagent anion species can be injected into the trap prior to the charge sign 5 Results: Developed a methodology incorporating multiple fills of ETD products into a reaction time, Figure 4. 1.50x10 independent trapping (CSIT) and ETD reaction events. As a result, the maximum FIGURE 8. Scan acquisition time per spectrum for the ETD multiple fills storage cell followed by a single m/z analysis leading to improved spectral S/N ratio MultFills 1e4 Target/Fill number of cation precursor charges that can be employed per ETD reaction is a experiment and the uScans approach taken at a variety of Orbitrap transient and acquisition speed. MultFills 5e4 Target/Fill durations. function of the space charge capacity of the section employed for sequestration. FIGURE 4. a) Angiotensin I b and y ion formation observed at high precursor 5 1.25x10 MultFills 1e5 Target/Fill Figure 2 shows the potentials employed during the cation sequestration/reagent initial targets resulting from harsh sequestration conditions. b) Fragment yield Single Fill Introduction injection events and the relative locations of the cationic and anionic species. versus precursor target demonstrating the onset of b and y ion formation. 1.2 12 5 Electron transfer dissociation (ETD) has been demonstrated to be a useful tool for the AngioVsTarget_ETD_50msec #548 RT: 2.58 AV: 1 NL: 6.18E5 1.00x10 FIGURE 2. Cation sequestration and reagent injection conditions prior to the T: FTMS + p ESI Full ms2 [email protected] [150.0000-1500.0000] 32 msec analysis of polypeptide compounds including peptides with labile PTM’s, peptides with x2 ETD reaction in the high pressure cell of the dual cell linear trap 432.90 64 msec 100 many basic sites, and large proteins. One drawback of the approach is that it leads to a 128 msec reduction of both scan duty cycle and total MS2 ion signal, compared to conventional 95 4 1.0 10 Accumulation of Precursor Cations and Reagent Anions TIC ETD Fragement 7.50x10 256 msec 90 resonant CID, due to necessity to perform the ion-ion reaction and the fact that the a) 3+ 512 msec 85 130620 CaP4 Area vs. Precursor Target reaction consumes charge. This research explores the possibility to circumvent these Reagent = 2e5. Rxn Time = 70 msec 1024 msec 80 Precursor = Angio3+ 433.23 6 limitations by performing multiple ETD reactions per m/z analysis in a 2D linear ion trap 10 Analyzer = Orbitrap 4 75 0.8 8 on a three-analyzer hybrid instrument based upon a mass resolving quadrupole, b) 5.00x10 70 Orbitrap (OT), collision cell, and dual linear ion trap (Q-OT-LT) architecture. 5 65 10

60 2.50x104 0.6 6 Methods 55 104 50

HPT LPT I [M+3H] Angiotensin 513.28 45 The multi-fill per m/z analysis scan mode in the dual cell linear ion trap is accomplished Area 3

Relative Abundance 10 by using the high pressure trap (HPT) as a reaction vessel, and utilizing the low 40 0.00 0.4 4 Previously m/z Isolated Precursor Cations Held In Back Section 5 5 5 5 6 pressure trap (LPT) as an accumulation and m/z analysis cell. In this fashion, the 35 tic 0.0

2 2.0x10 4.0x10 6.0x10 8.0x10 1.0x10

multiple fills ETD scan cycle becomes a loop over n (the number of fills requested) 30 10 precursor

c and z - - -

ETD reaction, transfer and storage cycles, followed by a single m/z analysis in either +10 V - 25 b and y Precursor Target

20 1 0.2 2 the low pressure trap of the dual cell or, after the appropriate transfer, in the OT mass 10 (sec) Spectrum per Scan Time

- 3 4 5 6 analyzer. All experiments were performed on an Orbitrap Fusion Tribrid MS. 15 10 10 10 10 583.31 10 Precursor Target 0 V + + 619.37 784.44 514.77 647.37 1028.59 It is apparent (Figure 6) that the working range using the multiple fills approach is 5 785.45 500.77 569.31 592.30 649.37 434.72 756.45 786.45 1000.60 1030.60 0.0 0 0 much greater than that for a single fill, while preserving soft sequestration conditions. 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 0 2 4 6 8 10 0 2 4 6 8 10 m/z The fit to the data (Figure 7) represents the theoretical gains that could be realized if The charge capacity of the back section of the HPT is determined by the axial potential the S/N ratio increases linearly with the ion population and as a square root function well and radial pseudopotential, and can be measured by a number of methods. We ETD Fills uScans for scan averaging. FIGURE 1. Schematic of the Orbitrap Fusion Tribrid mass spectrometer choose to examine the product yield from the ETD reaction of Angiotensin I as a showing the location of the Easy-ETD reagent ion source within the overall ion function of the starting precursor ion population. Fragment TIC plotted as a function of ETD Multiple Fills Proof of Concept optics path. The exploded view shows how the reagent ion source is the axial potential in the back section of the high pressure trap during the ion The multiple fills ETD approach has been previously demonstrated3,4 and is incorporated into the S-Lens/Q00 region. sequester event (Figure 3) is characterized by a linear region where ETD products advantageous as it has the capability to provide higher S/N ratio ETD spectra, while increase with the initial precursor population, followed by a transition to a plateau providing facile ETD sequestration conditions. A flow diagram detailing the approach is FIGURE 7. Comparison of the S/N ratio of ETD c and z fragments from tetrapeptide MRFA [M+2H]2+ after reaction for 100 msec versus the number of Ion-routing multipole region where additional precursor does not yield additional product species. The presented in Figure 5. Figures 6, 7, and 8 compare and contrast the multiple fills Conclusions HCD Cell location of the plateau is dictated by the balance of space charge forces with that of approach to the scan averaging approach in terms of spectral S/N ratio scan injection events. For the case of multiple ETD fills this represents the number of 1 . Single fill ETD limited by space charge capacity of the back section of the ion the effective RF focusing forces, and has been well modeled in the literature. acquisition speed. precursor fills/ETD cycles before m/z analysis; uScans represents the number of scans averaged. trap in the Orbitrap Fusion Tribrid MS. Dual-pressure . linear ion trap FIGURE 5. Flow diagram describing the ETD multiple fills approach when the 4.0 The sequestration conditions during ETD need careful consideration in order to FIGURE 3. Angiotensin I ETD product ion yield vs. the starting precursor ETD reaction is conducted in the HPT and product accumulation is conducted minimize trap-like collision induced dissociation. population for a single ETD reaction. ETD ion source Orbitrap Mass in the LPT. Mass analysis can be conducted in either analyzer. . A multiple fills methodology has been presented based on ETD reaction in the Analyzer 3.5 Quadrupole 1.0x105 high pressure trap followed by accumulation of ETD products in the low pressure Mass Filter -0.5 Volts trap. -1 Volts 3.0 . The geometry of the Orbitrap Fusion Tribrid MS in conjunction with the ability to HPT LPT 4 -2 Volts 8.0x10 run both analyzers in parallel allows for acquisition of high S/N ratio spectra in a much shorter time than can be done with scan averaging alone. 2.5 6.0x104 References 2.0 ETD Fragement TIC ETD Fragement 1. Tolmachev, A. V., Udseth, H. R., Smith, R.D., IJMS 222 (2003) pg 155-174. 3+ MRFA_c2 4.0x104 MRFA_c3 2. Sannes-Lowery, K., Griffey, R.H., Kruppa, G.H., Speir, J.P., Hofstadler, S.A., 1.5 MRFA_z3 Rapid Comm in Mass Spec 12 (1998) pg 1957-1961. MRFA 3. Rose, C.M. et al., J Am Soc Mass Spectrom 24 (2013) pg 816-827. 4 2.0x10 S/N(uscans) / S/N(multFills) n/sqrt(n) 1.0 4. Earley, L. et al., Anal Chem 85:17 (2013) pg 8385-8390.

Angiotensin I [M+3H] Angiotensin 0.5 0.0 0.0 2.0x105 4.0x105 6.0x105 8.0x105 1.0x106 1 2 3 4 5 6 7 8 9

Precursor Target number of injection events All 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.

PO64107-EN 0614S

Thermo Scientific Poster Note• PN-64107-ASMS-EN-0614S 5 Improved Electron Transfer Dissociation (ETD) Duty Cycle and Spectral Signal to Noise Ratio in a Dual Cell Linear Ion Trap

Christopher Mullen1, Lee Earley1, Jean-Jacques Dunyach1, John E.P. Syka1, Jeffrey Shabanowitz2, A. Michelle English2, Donald F. Hunt3 1Thermo Fisher Scientific, San Jose, CA; 2Department of Chemistry, University of Virginia, Charlottesville, VA

Unfortunately, certain combinations of axial well depth and RF pseudopotential can FIGURE 6. Comparison of the Angiotensin I ETD c and z ion fragment TIC for an The geometry of the Orbitrap Fusion Tribrid MS in conjunction with it’s ability to run Overview Results lead to ion excitation as the ions spend more time at greater radii. The increased up to 8-fills ETD approach versus a single fill. The multiple fill data was taken at scans in a parallel/pipelined fashion affords a significant decrease in scan acquisition kinetic energy in these situations causes fragmentation which generates significant three individual precursor targets per fill corresponding to 1e4 (blue squares), time per spectrum for the multiple fills approach (Figure 8) as the ETD fills are run in Purpose: Improve ETD Signal to Noise (S/N) ratio and scan duty cycle. Motivation for Employing Multiple Fills ETD 2 amounts of b and y series ions , and contributes to overall lower signal to noise (S/N) 5e4 (red circles), and 1e5 (green triangles) charges/fill. parallel with OT m/z analysis. Methods: Thermo Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer with the Performing the ETD reaction in the HPT of the Orbitrap Fusion Tribrid MS requires the ratio ETD spectra. We demonstrate that the precursor fragmentation is occurring Thermo Scientific™ EASY-ETD™ ion source. cation precursor population to be sequestered to the back section of the HPT, so that during the sequester events by monitoring the fragment TIC in the absence of ion-ion the reagent anion species can be injected into the trap prior to the charge sign 5 Results: Developed a methodology incorporating multiple fills of ETD products into a reaction time, Figure 4. 1.50x10 independent trapping (CSIT) and ETD reaction events. As a result, the maximum FIGURE 8. Scan acquisition time per spectrum for the ETD multiple fills storage cell followed by a single m/z analysis leading to improved spectral S/N ratio MultFills 1e4 Target/Fill number of cation precursor charges that can be employed per ETD reaction is a experiment and the uScans approach taken at a variety of Orbitrap transient and acquisition speed. MultFills 5e4 Target/Fill durations. function of the space charge capacity of the section employed for sequestration. FIGURE 4. a) Angiotensin I b and y ion formation observed at high precursor 5 1.25x10 MultFills 1e5 Target/Fill Figure 2 shows the potentials employed during the cation sequestration/reagent initial targets resulting from harsh sequestration conditions. b) Fragment yield Single Fill Introduction injection events and the relative locations of the cationic and anionic species. versus precursor target demonstrating the onset of b and y ion formation. 1.2 12 5 Electron transfer dissociation (ETD) has been demonstrated to be a useful tool for the AngioVsTarget_ETD_50msec #548 RT: 2.58 AV: 1 NL: 6.18E5 1.00x10 FIGURE 2. Cation sequestration and reagent injection conditions prior to the T: FTMS + p ESI Full ms2 [email protected] [150.0000-1500.0000] 32 msec analysis of polypeptide compounds including peptides with labile PTM’s, peptides with x2 ETD reaction in the high pressure cell of the dual cell linear trap 432.90 64 msec 100 many basic sites, and large proteins. One drawback of the approach is that it leads to a 128 msec reduction of both scan duty cycle and total MS2 ion signal, compared to conventional 95 4 1.0 10 Accumulation of Precursor Cations and Reagent Anions TIC ETD Fragement 7.50x10 256 msec 90 resonant CID, due to necessity to perform the ion-ion reaction and the fact that the a) 3+ 512 msec 85 130620 CaP4 Area vs. Precursor Target reaction consumes charge. This research explores the possibility to circumvent these Reagent = 2e5. Rxn Time = 70 msec 1024 msec 80 Precursor = Angio3+ 433.23 6 limitations by performing multiple ETD reactions per m/z analysis in a 2D linear ion trap 10 Analyzer = Orbitrap 4 75 0.8 8 on a three-analyzer hybrid instrument based upon a mass resolving quadrupole, b) 5.00x10 70 Orbitrap (OT), collision cell, and dual linear ion trap (Q-OT-LT) architecture. 5 65 10

60 2.50x104 0.6 6 Methods 55 104 50

HPT LPT I [M+3H] Angiotensin 513.28 45 The multi-fill per m/z analysis scan mode in the dual cell linear ion trap is accomplished Area 3

Relative Abundance 10 by using the high pressure trap (HPT) as a reaction vessel, and utilizing the low 40 0.00 0.4 4 Previously m/z Isolated Precursor Cations Held In Back Section 5 5 5 5 6 pressure trap (LPT) as an accumulation and m/z analysis cell. In this fashion, the 35 tic 0.0

2 2.0x10 4.0x10 6.0x10 8.0x10 1.0x10

multiple fills ETD scan cycle becomes a loop over n (the number of fills requested) 30 10 precursor

c and z - - -

ETD reaction, transfer and storage cycles, followed by a single m/z analysis in either +10 V - 25 b and y Precursor Target

20 1 0.2 2 the low pressure trap of the dual cell or, after the appropriate transfer, in the OT mass 10 (sec) Spectrum per Scan Time

- 3 4 5 6 analyzer. All experiments were performed on an Orbitrap Fusion Tribrid MS. 15 10 10 10 10 583.31 10 Precursor Target 0 V + + 619.37 784.44 514.77 647.37 1028.59 It is apparent (Figure 6) that the working range using the multiple fills approach is 5 785.45 500.77 569.31 592.30 649.37 434.72 756.45 786.45 1000.60 1030.60 0.0 0 0 much greater than that for a single fill, while preserving soft sequestration conditions. 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 0 2 4 6 8 10 0 2 4 6 8 10 m/z The fit to the data (Figure 7) represents the theoretical gains that could be realized if The charge capacity of the back section of the HPT is determined by the axial potential the S/N ratio increases linearly with the ion population and as a square root function well and radial pseudopotential, and can be measured by a number of methods. We ETD Fills uScans for scan averaging. FIGURE 1. Schematic of the Orbitrap Fusion Tribrid mass spectrometer choose to examine the product yield from the ETD reaction of Angiotensin I as a showing the location of the Easy-ETD reagent ion source within the overall ion function of the starting precursor ion population. Fragment TIC plotted as a function of ETD Multiple Fills Proof of Concept optics path. The exploded view shows how the reagent ion source is the axial potential in the back section of the high pressure trap during the ion The multiple fills ETD approach has been previously demonstrated3,4 and is incorporated into the S-Lens/Q00 region. sequester event (Figure 3) is characterized by a linear region where ETD products advantageous as it has the capability to provide higher S/N ratio ETD spectra, while increase with the initial precursor population, followed by a transition to a plateau providing facile ETD sequestration conditions. A flow diagram detailing the approach is FIGURE 7. Comparison of the S/N ratio of ETD c and z fragments from tetrapeptide MRFA [M+2H]2+ after reaction for 100 msec versus the number of Ion-routing multipole region where additional precursor does not yield additional product species. The presented in Figure 5. Figures 6, 7, and 8 compare and contrast the multiple fills Conclusions HCD Cell location of the plateau is dictated by the balance of space charge forces with that of approach to the scan averaging approach in terms of spectral S/N ratio scan injection events. For the case of multiple ETD fills this represents the number of 1 . Single fill ETD limited by space charge capacity of the back section of the ion the effective RF focusing forces, and has been well modeled in the literature. acquisition speed. precursor fills/ETD cycles before m/z analysis; uScans represents the number of scans averaged. trap in the Orbitrap Fusion Tribrid MS. Dual-pressure . linear ion trap FIGURE 5. Flow diagram describing the ETD multiple fills approach when the 4.0 The sequestration conditions during ETD need careful consideration in order to FIGURE 3. Angiotensin I ETD product ion yield vs. the starting precursor ETD reaction is conducted in the HPT and product accumulation is conducted minimize trap-like collision induced dissociation. population for a single ETD reaction. ETD ion source Orbitrap Mass in the LPT. Mass analysis can be conducted in either analyzer. . A multiple fills methodology has been presented based on ETD reaction in the Analyzer 3.5 Quadrupole 1.0x105 high pressure trap followed by accumulation of ETD products in the low pressure Mass Filter -0.5 Volts trap. -1 Volts 3.0 . The geometry of the Orbitrap Fusion Tribrid MS in conjunction with the ability to HPT LPT 4 -2 Volts 8.0x10 run both analyzers in parallel allows for acquisition of high S/N ratio spectra in a much shorter time than can be done with scan averaging alone. 2.5 6.0x104 References 2.0 ETD Fragement TIC ETD Fragement 1. Tolmachev, A. V., Udseth, H. R., Smith, R.D., IJMS 222 (2003) pg 155-174. 3+ MRFA_c2 4.0x104 MRFA_c3 2. Sannes-Lowery, K., Griffey, R.H., Kruppa, G.H., Speir, J.P., Hofstadler, S.A., 1.5 MRFA_z3 Rapid Comm in Mass Spec 12 (1998) pg 1957-1961. MRFA 3. Rose, C.M. et al., J Am Soc Mass Spectrom 24 (2013) pg 816-827. 4 2.0x10 S/N(uscans) / S/N(multFills) n/sqrt(n) 1.0 4. Earley, L. et al., Anal Chem 85:17 (2013) pg 8385-8390.

Angiotensin I [M+3H] Angiotensin 0.5 0.0 0.0 2.0x105 4.0x105 6.0x105 8.0x105 1.0x106 1 2 3 4 5 6 7 8 9

Precursor Target number of injection events All 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.

PO64107-EN 0614S

6 Improved Electron Transfer Dissociation (ETD) Duty Cycle and Spectral Signal to Noise Ratio in a Dual Cell Linear Ion Trap www.thermoscientific.com ©2014 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. All other 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 Thermo Fisher Scientific, infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are San Jose, CA USA is available in all countries. Please consult your local sales representative for details. ISO 9001:2008 Certified. Africa +43 1 333 50 34 0 Denmark +45 70 23 62 60 Japan +81 45 453 9100 Singapore +65 6289 1190 Australia +61 3 9757 4300 Europe-Other +43 1 333 50 34 0 Latin America +1 561 688 8700 Spain +34 914 845 965 Austria +43 810 282 206 Finland +358 9 3291 0200 Middle East +43 1 333 50 34 0 Sweden +46 8 556 468 00 Belgium +32 53 73 42 41 France +33 1 60 92 48 00 Netherlands +31 76 579 55 55 Switzerland +41 61 716 77 00 Canada +1 800 530 8447 Germany +49 6103 408 1014 New Zealand +64 9 980 6700 UK +44 1442 233555 China 800 810 5118 (free call domestic) India +91 22 6742 9494 Norway +46 8 556 468 00 USA +1 800 532 4752 400 650 5118 Italy +39 02 950 591 Russia/CIS +43 1 333 50 34 0 PN-64107-EN-0614S