Fitting of ETD Rate Constants for Doubly and Triply Charged Ions in a Linear Ion Trap

Transcription

1 Fitting of ETD Rate Constants for Doubly and Triply Charged Ions in a Linear Ion Trap Dirk Nolting and Andreas Wieghaus Thermo Fisher Scientific, Bremen, Germany

2 Overview Purpose: Characterize the rate constants of electron transfer dissociation (ETD) reactions. Knowledge of the rate constants provide information on fragment-current yields and fragmentation pathways. Methods: ETD reaction kinetics were measured and fitted to a consecutive reaction kinetics model. Results: By fitting rate constants for different fragments, it is possible to assign reaction pathways. Introduction Ion/ion reactions in the gas phase are a versatile tool for probing molecular properties. In the field mass spectrometry, ETD has become a common fragmentation method, because it often provides complementary information compared to widely-used thermal dissociation methods which use collisional heating. The maximum product ion current, and thus the sensitivity, are determined by consecutive reaction kinetics. As each reaction step involves charge reduction, it is important to stop the reaction at an early point when the number of remaining charges for product ions reaches is maximum. Both the maximum product ion current and the optimal reaction time are determined by the ETD rate constants. ETD reaction kinetics were measured and compared with the theoretical model. Methods Sample Preparation All experiments were carried out with Thermo Scientific Pierce LTQ Velos ESI Positive Ion Calibration Solution (88323) or a 1 pmol/µl solution of angiotensin I (Sigma-Aldrich A965). Angiotensin was used without further purification. A mixture of 5:5 methanol/water (.1% acetic acid) was used as solvent. All solvents were HPLC grade (Fisher Chemical). Mass Spectrometry All measurements were done with an Thermo Scientific Orbitrap Elite hybrid mass spectrometer (Figure 1). The sample was directly infused using a Thermo Scientific Ion Max ESI ion source. For the analyte ions a AGC target value of 1x1 5 was used and an anion AGC target of 2x1 6 if not noted otherwise. Orbitrap TM mass analyzer detection was used for all mass spectra to avoid the saturation effects of channeltron detectors. Transients were recorded using build-in software features. Data Analysis Transients were extracted from the recorded raw files using Thermo Scientific Xcalibur software version 2.1 with Qual Browser. The extracted transients were imported into Origin 8 software (OriginLab) and fitted using its non-linear curve fitting routines. FIGURE 1. Schematic of an Orbitrap Elite TM mass spectrometer equipped with an ETD module. For ETD, anions and analyte cations are trapped simultaneously in the linear trap, allowing an electron transfer from the fluoranthene anion to the analyte cation causing charge reduction and fragmentation.. Results Modeling Consecutive Reaction Kinetics ETD is a consecutive reaction where each reaction is a charge reduction: k 3 [M+nH] n+ [M+nH] (n-1)+ [M+nH] (n-2)+ 2 Fitting of ETD Rate Constants for Doubly and Triply Charged Ions in a Linear Ion Trap

3 The reaction rates can be described by a system of differential equations: (1) (2) (3) Solving the differential equations gives the chronological sequence for each charge state: (4) (5) Fitting of the Precursor and the Charge-Reduced Species For the doubly charged angiotensin I, rate constants can be fitted using the precursor and the total fragment current. In order to obtain the first rate constant, the precursor intensity (Figure 2a) was fitted to eq. 4. The result shows a very good correlation. The second rate constant,,was obtained by fitting the total fragment current to eq. 5. To reduce the degrees of freedom, was taken from the previous fit and kept constant, thus, only A and of eq. 5 were fitted. Fragments of triply charged precursors can originate from different reaction pathways, and may have different charge states obscuring the detector response. Thus, only the charge-reduced species were used to fit the rate constants (Figure 2b). This avoids ambiguities in the assignment of the fragments, i.e. to which reaction pathway the fragment current contributes. To fit the [M+3H] 2+ species, was taken from the previous fit and kept constant, i.e. only A and of eq. 5 were fitted. Accordingly, [M+3H] + was fitted with constant and from the previous fits and fitting k 3 and A to eq. 6. All fitted curves show good correlation to the experimental data. FIGURE 2. Reaction process of precursor and charge reduced species for a) doubly, and b) triply charged angiotensin I. Points are measured data and solid lines are fit results. The fitted rate constants and corresponding coefficient of determination are given. a) (6) Validation of the First Order Approxi For doubly charged (AGC targe rate constants were measured at differ the pseudo first-order approximation. T independent of the number of reagent higher than the number of analyte ions rate constants depends strongly on the is not valid in this range. For a high exc strongly reduced, and the first order ap FIGURE 3. Rate constants measured ratio of the initial number reagent io should not depend on the AGC targe for a high excess of reagent ions. rate constant [s -1 ] uction: ion count [arb. u.] 2.x1 7 precursor ions fragment ions =24, R 2 = x1 7 1.x1 7 5.x1 6 =24, =9, R 2 = time [s] targe Optimizing the Anion Inject Time of In real life, the time to generate the ma depends on the reaction time and the t require similar amounts of time and are Increasing the reagent ion injection tim versa. The optimal reaction time, the re yield, is inversely proportional to the nu and proportional to the injection time (F event is the sum of the anion inject tim line). It has an absolute minimum whic anion AGC target of 1x1 5. In this rang (Figure 3) and increasing the number o an increase in the rate constants. Thermo Scientific Poster Note PN63593_E 6/12S 3

4 b) ion count [arb. u.] 6x1 7 [M+3H] 3+ 5x1 7 4x1 7 3x1 7 2x1 7 1x1 7 [M+3H] [M+3H] =62.7, R 2 =.9999 =62.7, =25.5, R 2 =.9992 =62.7, =25, k 3 = 8.8, R 2 = time [s] Validation of the First Order Approximation For doubly charged (AGC target 5x1 4 ) and angiotensin I (AGC target 1x1 5 ), rate constants were measured at different reagent-ion AGC targets (Figure 3) using the pseudo first-order approximation. This assumes that the rate constant is independent of the number of reagent ions if the number of reagent ions is much higher than the number of analyte ions. For a low excess of reagent ions (<5x), the rate constants depends strongly on the AGC target, and the first order approximation is not valid in this range. For a high excess of reagent ions (>1x), the impact is strongly reduced, and the first order approximation can be considered valid. FIGURE 4 Time optimization of the ani similar to the ETD reaction time. While with the anion AGC target, the reaction proportional. Thus, there is an absolut maximum fragmentation yield x1 5 2x1 5 time /ms FIGURE 3. Rate constants measured at different anion AGC targets given as ratio of the initial number reagent ions and analyte ions. As the rate constants should not depend on the AGC target, the first order approximation is only valid for a high excess of reagent ions. 26 rate constant [s -1 ] target ratio reagent/analyte Optimizing the Anion Inject Time of an ETD Event In real life, the time to generate the maximum number of ETD fragment ions depends on the reaction time and the time to inject the reagent ions Both steps require similar amounts of time and are dependent on each other (Figure 4). Increasing the reagent ion injection time reduces the ETD reaction time and vice versa. The optimal reaction time, the reaction time with the highest fragmentation yield, is inversely proportional to the number of reagent ions (Figure 4, green line) and proportional to the injection time (Figure 4, red line). The total time of an ETD event is the sum of the anion inject time and the ETD reaction time (Figure 4, black line). It has an absolute minimum which is at the current measurement close to an anion AGC target of 1x1 5. In this range, the change in reaction rate is small (Figure 3) and increasing the number of reagent ions is no longer compensated by an increase in the rate constants. 4 Fitting of ETD Rate Constants for Doubly and Triply Charged Ions in a Linear Ion Trap

5 I (AGC target 1x1 5 ), rgets (Figure 3) using te constant is gent ions is much gent ions (<5x), the t order approximation x), the impact is sidered valid. targets given as s the rate constants ximation is only valid FIGURE 4 Time optimization of the anion AGC target. The anion inject time is similar to the ETD reaction time. While the inject time increases proportionally with the anion AGC target, the reaction time decreases is inversely proportional. Thus, there is an absolute minimum time for obtaining the maximum fragmentation yield. time /ms x1 5 2x1 5 3x1 5 4x1 5 5x1 5 Possible Reaction Pathways for Fragmentation For a triply charged precursor, there are several reaction pathways which can lead to fragmentation: 1. ETD forming one doubly charged fragment F 1 2. ETD forming two singly charged fragments F 2 3. Two charge reduction steps forming one singly charged fragment F 3 k k 1 2 [F 1 ] 2+ [M+3H] 3+ 2[F 2 ] + anion target k 3 [F 3 ] + [neutral] total time ETD event anion inject time optimal reaction time Each reaction pathway shows a characteristic reaction process. The corresponding species have maximum abundance at different points in time and different peak shapes (Figure 5). FIGURE 5. Characteristic reaction processes for different reaction pathways. First-generation ions show a fast increase and decay rate depending on their charge state (m/z 29.2, m/z ). Second-generation ions start delayed, and have a slow decay rate (m/z ). k 3 [neutral] Mapping First- and Second-Gener The ETD spectrum of angiotensin I c second-generation ions (Figure 6). A by singly charged first-generation ion step. For most doubly charged fragm FIGURE 6. ETD spectrum of triply show different kinetics. Red areas fragments are second-generation kinetics. Brackets denote comple Relative Abundance z=3 64 c 2 c 3 z z= z=? z 3 c The ETD spectrum comprises a serie most of the sequence information (Fig formed after the first ETD step m/z ( =63, =25, k 3 =7) m/z ( =63, =1) m/z ( =63, =25) ragment ions ions Both steps er (Figure 4). tion time and vice hest fragmentation igure 4, green line) tal time of an ETD time (Figure 4, black rement close to an n rate is small er compensated by normalized Intensity time [s] Thermo Scientific Poster Note PN63593_E 6/12S 5

6 on inject time is es proportionally rsely taining the Mapping First- and Second-Generation ions The ETD spectrum of angiotensin I can be divided into m/z ranges with first- and second-generation ions (Figure 6). A nearly complete series of c and z ions is formed by singly charged first-generation ions. Most fragments are formed after the first ETD step. For most doubly charged fragments a charge-reduced species is also present. FIGURE 6. ETD spectrum of triply charged angiotensin I. Different mass ranges show different kinetics. Red areas are first-generation fragments, grey fragments are second-generation fragments, hatched areas have mixed kinetics. Brackets denote complementary ions z=3 time ETD event inject time al reaction time 5 5x1 5 Relative Abundance z= c 2 c 3 z= c z z= z z=? z 3 c c 7 z 7 z 8 c 8 z 9 c m/z The ETD spectrum comprises a series of complementary c and z ions which contain most of the sequence information (Figure 6). Those ions are singly charged and are formed after the first ETD step. Conclusion Measured ETD reaction kinetics can be described by rate equations for consecutive reactions assuming a first order reaction. Different reaction pathways can be assigned using the reaction kinetics. The maximum fragment yield for angiotensin I can be calculated from the fitted rate constants (56% / 86% for the 2+/3+ precursor). Most sequence-information-containing ions are singly charged ions formed after the first ETD step. Origin is a registered trademark of OriginLab Corporation. 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. 6 Fitting of ETD Rate Constants for Doubly and Triply Charged Ions in a Linear Ion Trap

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