Fitting of ETD Rate Constants for Doubly and Triply Charged Ions … · 2017. 7. 21. · ab e a a p...
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Fitting of ETD Rate Constants for Do bl and Tripl Charged Ions in a Linear Ion Trap Fitting of ETD Rate Constants for Doubly and Triply Charged Ions in a Linear Ion Trap Fitting of ETD Rate Constants for Doubly and Triply Charged Ions in a Linear Ion Trap Dirk Nolting Thermo Fisher Scientific Bremen Germany Dirk Nolting, Thermo Fisher Scientific, Bremen, Germany Table 1. Maximal product ion current and product ion numbers for Angiotensin I Overview Results Mapping of First and Second Generation ions calculated from the measured rate constants. Overview Results k k 2 k Th ETD t fA it i I b di id di t / ith fi t d calculated from the measured rate constants. Purpose: The rate constants characterize the ETD reaction Knowledge of the rate Modelling Consecutive Reaction Kinetics [F 1 ] 2+ k 1 k 2 [F ] + k 3 [ l] The ETD spectrum of Angiotensin I can be divided into m/z ranges with first and d ti i (Fi 4) A l lt i f d i i f d Maximum number of Maximum product ion current Purpose: The rate constants characterize the ETD reaction. Knowledge of the rate constants gives information on fragment current yields and fragmentation pathways Modelling Consecutive Reaction Kinetics [M 3H] 3+ [F 1 ] [F 3 ] + [neutral] second generation ions (Figure 4). A nearly complete series of c and z ions is formed f f f f f product ions (reaction time) (reaction time) constants gives information on fragment current yields and fragmentation pathways. ETD is a consecutive reaction where each reaction is a charge reduction: [M+3H] 3+ k 3 by singly charged first generation ions. Most fragments are formed after the first ETD 2+ 56% (66 ) 28% (66 ) Methods: ETD reaction kinetics were measured and fitted to a consecutive reaction k 3 [neutral] 2[F ] + k step. For most doubly charged fragments a charge reduced species is also present. 2+ precursor 56% (66 ms) 28% (66 ms) kinetics model. [neutral] 2[F 2 ] + k 1 3+ 86% (45 ) 45% (33 ) R lt B fitti t t t f diff tf t it i ibl t i ti ( 1) k 1 k 2 k 3 3+ precursor ~86% (45 ms) ~45% (33ms) Results: By fitting rate constants for different fragments it is possible to assign reaction th [M+nH] n+ [M+nH] ·(n-1)+ [M+nH] ··(n-2)+ ··· pathways. Each reaction pathway shows a characteristic reaction process, i.e. the corresponding For the triply charged precursor exist several pathways which makes it difficult to species have maximum abundance at different points in time and different peak FIGURE 4 ETD spectrum of triply charged Angiotensin I Different mass ranges For the triply charged precursor exist several pathways which makes it difficult to calculate the efficiencies as the branching ratio is not known Most product ions Introduction shapes (Figure 3). FIGURE 4. ETD spectrum of triply charged Angiotensin I. Different mass ranges show different kinetics Red areas are first generation fragments grey calculate the efficiencies as the branching ratio is not known. Most product ions (>95%) follow the main reaction pathway forming first a doubly and then a singly Introduction The reaction rates can be described by a system of differential equations: show different kinetics. Red areas are first generation fragments, grey fragments are second generation fragments hatched areas have mixed (>95%) follow the main reaction pathway forming first a doubly and then a singly charged ion Accordingly only those ions were taken into account Ion/ion reactions in the gas phase are a versatile tool for probing molecular properties . FIGURE 3. Characteristic reaction processes for different reaction pathways. The reaction rates can be described by a system of differential equations: fragments are second generation fragments, hatched areas have mixed kinetics Brackets denote complementary ions charged ion. Accordingly, only those ions were taken into account. Ion/ion reactions in the gas phase are a versatile tool for probing molecular properties. In mass spectrometry electron transfer dissociation has become a common FIGURE 2. Reaction process of the precursor and the charge reduced species FIGURE 3. Characteristic reaction processes for different reaction pathways. 3a) First generation ions show fast increase and a decay rate depending on kinetics. Brackets denote complementary ions. In mass spectrometry electron transfer dissociation has become a common fragmentation method as it often results in complementary information in comparison FIGURE 2. Reaction process of the precursor and the charge reduced species for a) doubly and b) triply charged Angiotensin I. Points are measured data and 3a) First generation ions show fast increase and a decay rate depending on their charge state (m/z 290 2 m/z 5912 8) Second generation ions start delayed fragmentation method, as it often results in complementary information in comparison to the widely used thermal dissociation methods by collisional heating for a) doubly and b) triply charged Angiotensin I. Points are measured data and solid lines are results from fitting Given are the fitted rate constants and the their charge state (m/z 290.2, m/z 5912.8). Second generation ions start delayed and have a slow decay rate (m/z 1183 6) 3b) Some fragments as m/z 1045 can (1) 432 90 to the widely used thermal dissociation methods by collisional heating. solid lines are results from fitting. Given are the fitted rate constants and the corresponding coefficient of determination and have a slow decay rate (m/z 1183.6). 3b) Some fragments as m/z 1045 can be formed after the first or second ETD step The resulting kinetics can be (1) 100 432.90 z=3 The maximum product ion current, and thus the sensitivity are determined by corresponding coefficient of determination. be formed after the first or second ETD step. The resulting kinetics can be modeled by the sum of first and second generation kinetics 100 consecutive reaction kinetics. As each reaction step involves charge reduction, it is modeled by the sum of first and second generation kinetics. (2) 90 important to stop the reaction at an early point where the number of remaining charges a) (2) 649 35 for product ions reaches is maximum. Both, the maximum product ion current and the a) 10 a) 80 649.35 z=2 optimal reaction time are determined by the rate constants. ETD reaction kinetics were 2.0x10 7 1.0 m/z 1183 6 (k =63 k =25 k =7) a) 70 e z2 optimal reaction time are determined by the rate constants. ETD reaction kinetics were measured and compared with the theoretical model. precursor ions m/z 1183.6 (k 1 =63, k 2 =25, k 3 =7) (3) 70 nce 1298.70 measured and compared with the theoretical model. fragment ions m/z 289.2 (k 1 =63, k 2 =10) (3) 60 nda 1298.70 z=1 Methods fragment ions k 24 R 2 0 999 08 m/z 591.8 (k 1 =63, k 2 =25) Abun Methods 1 5x10 7 k 1 =24, R 2 =0.9997 0.8 1 2 50 ve A S l P ti Solving the differential equations gives the chronological sequence for each 1.5x10 1 k 24 k 9R 2 0 9973 ] 40 lativ Sample Preparation charge state: k 1 =24,k 2 =9, R 2 =0.9973 . u. ity 40 Rel All experiments were carried out with a 1 pmol/μl solution of Angiotensin I 1 2 arb. 0.6 ens 30 1281 68 All experiments were carried out with a 1 pmol/μl solution of Angiotensin I. t [a nte 20 1281.68 z=1 640.84 c c A mixture of 50:50 methanol/water (0.1% acetic acid) was used as solvent. All solvents (4) 1.0x10 7 oun d In 20 z1 z=2 289 16 591 81 c 2 c 3 c 5 c 7 c 8 c 9 z 9 z 7 z 8 were HPCL grade from Fisher Chemical. Angiotensin was purchased from Sigma- co zed 10 289.16 z=1 591.81 z=2 1009.54 z=1 388.23 z=1 1183.63 z=1 910.47 z1 664 38 551 30 747 41 197 08 c 5 z 5 z 6 Aldrich (A9650) and used without further purification. on 0.4 mali 10 z2 z=1 z=1 z=1 z=1 664.38 z=1 551.30 z=1 747.41 z=1 197.08 z=? c 4 z 3 z 5 Mass Spectrometry i orm 0 z? 3 Mass Spectrometry 5 0x10 6 no 200 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z All measurements were done with an Thermo Scientific Orbitrap Elite hybrid mass (5) 5.0x10 m/z spectrometer (Figure 1). The sample was directly infused using an IonMax ESI ion 0.2 spectrometer (Figure 1). The sample was directly infused using an IonMax ESI ion source. For the analyte ions a target value of 1x10 5 was used and an anion target of source. For the analyte ions a target value of 1x10 was used and an anion target of 2x10 6 if not noted otherwise Orbitrap detection was used for all mass spectra to avoid The ETD spectrum comprises a series of complementary c and z ions which contain 2x10 if not noted otherwise. Orbitrap detection was used for all mass spectra to avoid saturation effects of the channeltron detectors Transients were recorded using build-in 0.0 00 most of the sequence information (Figure 4). Those ions are singly charged and are saturation effects of the channeltron detectors. Transients were recorded using build-in software features (6) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0 00 0 05 0 10 0 15 0 20 0 25 0 30 0.0 formed after the first ETD step. The optimal reaction for this reaction pathway is 35 ms software features. time [s] 6x10 7 0.00 0.05 0.10 0.15 0.20 0.25 0.30 which is close to time of the maximum product current. Data Analysis 6x10 [M+3H] 3+ b) time [s] Transients were extracted from the recorded raw files using Thermo Scientific Xcalibur [M+3H] 2+ b) 10 Transients were extracted from the recorded raw files using Thermo Scientific Xcalibur 2 1 Qual Browser The extracted transients were imported into Origin 8 and fitted using 5 10 7 [M+3H] 2+ b) 1.0 Fit for m/z 1183 6 (k =63 k =25 k =7) 2.1 Qual Browser. The extracted transients were imported into Origin 8 and fitted using it li fitti ti 5x10 7 [M+3H] 1+ Fit for m/z 1183.6 (k 1 =63, k 2 =25, k 3 =7) Fit for m/z 289 2 (k =63 k =10) Conclusion its non-linear curve fitting routines. k =62.7, R 2 =0.9999 Fit for m/z 289.2 (k 1 =63, k 2 =10) Conclusion Fitting of the Precursor and the Charge Reduced Species 7 k 1 62.7, R 0.9999 k 62 7 k 25 05 R 2 0 9992 08 Fit for m/z 1045 (mixed model) / 1045 Measured ETD reactions kinetics can be described by rate equations for Fitting of the Precursor and the Charge Reduced Species F th d bl h dA it i I t t t b fitt d i th 4x10 7 k 1 =62.7, k 2 =25.05, R 2 =0.9992 ] 0.8 m/z 1045 Measured ETD reactions kinetics can be described by rate equations for consecutive reactions assuming a first order reaction. FIGURE 1. Schematics of an Orbitrap Elite TM hybrid MS equipped with an ETD For the doubly charged Angiotensin I, rate constants can be fitted using the k 1 =62.7, k 2 =25, k 3 = 8.8, R 2 =0.9979 . u. consecutive reactions assuming a first order reaction. module. For ETD anions and analyte cations are trapped simultaneously in the precursor and the total fragment current. In order to obtain the first rate constant k 1 k 1 62.7, k 2 25, k 3 8.8, R 0.9979 arb. ty Different reaction pathways can be assigned by the reaction kinetics. module. For ETD anions and analyte cations are trapped simultaneously in the linear trap, allowing an electron transfer from the fluoranthene anion to the the precursor intensity (Figure 2a) was fitted to eq. 4. The result shows a very good 3x10 7 t [a 06 nsi The maximum fragment yield for Angiotensin I can be calculated from the fitted linear trap, allowing an electron transfer from the fluoranthene anion to the analyte cation causing charge reduction and fragmentation. correlation. The second rate constant k 2 was obtained by fitting the total fragment unt 0.6 nten The maximum fragment yield for Angiotensin I can be calculated from the fitted rate constants (56% / 86% for the 2+/3+ precursor) analyte cation causing charge reduction and fragmentation. current to eq. 5. To reduce the degrees of freedom, k 1 was taken from the previous co d In rate constants (56% / 86% for the 2+/3+ precursor). . 1 fit and kept constant, thus, only A 0 and k 2 of eq. 5 were fitted. 2x10 7 on zed Most sequence information containing ions are singly charged ions formed after 0 2 Fragments of triply charged precursors can origin from different reaction pathways io 0.4 aliz the first ETD step. Fragments of triply charged precursors can origin from different reaction pathways d h diff t h tt b i th dt t Th l 0.4 rma and may have different charge states obscuring the detector response. Thus, only th h d d i df fitti th t t t (Fi 4b) Thi 1x10 7 nor the charge reduced species were used for fitting the rate constants (Figure 4b). This id bi iti i th i t f th f t i t hi h ti 1x10 n avoids ambiguities in the assignment of the fragments, i.e. to which reaction f f 3 2 0.2 pathway the fragment current contributes. For fitting the [M+3H] 2+· species k 1 was 0 0.2 taken from the previous fit and kept constant, i.e. only A 0 and k 2 of eq. 5 were fitted. 0 00 0 05 0 10 0 15 0 20 0 25 0 30 0 Accordingly, [M+3H] +·· was fitted constant k 1 and k 2 from the previous fits and only 0.00 0.05 0.10 0.15 0.20 0.25 0.30 ti [] fitting k 3 and A 0 to eq. 6. All fitted curves show good correlation to the experimental time [s] 0.0 data. Possible Reaction Pathways for Fragmentation 0.00 0.05 0.10 0.15 0.20 0.25 0.30 Possible Reaction Pathways for Fragmentation F til h d it l ti th hi h l dt time [s] For a triply charged precursor exist several reaction pathways which can lead to f time [s] Maximum Fragmentation Limit fragmentation: Origin is a trademark of ORIGINLAB CORPORATION,NORTHHAMPTON, MA, USA; Si Ald i h i t d k f SIGMA ALDRICH CO LLC ST LOUIS MISSOURI USA Th ETD ffi i i th i b ff t d di l th 1 ETD forming one doubly charged fragment F The mixed model (Figure 3b) is a combination of eq. 5 and eq. 6, i.e. Sigma Aldrich is a trademark of SIGMA-ALDRICH CO., LLC, ST. LOUIS MISSOURI, USA All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries The ETD efficiency, i.e. the maximum number of fragments and accordingly the i d (T bl 1) i i b h F h il d 1. ETD forming one doubly charged fragment F 1 I m/z1045 (t)=0.5*eq. 5 + 0.5*eq. 6 . All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. Thi if ti i ti t d dt f th d t i th t i hti f i th maximum product current (Table 1) is given by the rate constants. For the triply and 2. ETD forming two singly charged fragments F 2 m/z1045 This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others higher charged precursor ions the time for the maximum number of product ions is 3 Two charge reduction steps forming one singly charged fragment F intellectual property rights of others. P bli h d tU Fb 2012 different from time for the maximal product current. 3. Two charge reduction steps forming one singly charged fragment F 3 Published at Uppcon, February 2012.
Fitting of ETD Rate Constants for Doubly and Triply Charged Ions … · 2017. 7. 21. · ab e a a p oduct o cu e t a d p oduct o u be s o g ote s calculated from the measured rate
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