Enhanced Dissociation of Intact Proteins with High Capacity Electron Transfer Dissociation
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Electron transfer dissociation (ETD) is a valuable tool for protein sequence analysis, especially for the fragmentation of intact proteins. However, low product ion signal-to-noise often requires some degree of signal averaging to achieve high quality MS/MS spectra of intact proteins. Here we describe a new implementation of ETD on the newest generation of quadrupole-Orbitrap-linear ion trap Tribrid, the Orbitrap Fusion Lumos, for improved product ion signal-to-noise via ETD reactions on larger precursor populations. In this new high precursor capacity ETD implementation, precursor cations are accumulated in the center section of the high pressure cell in the dual pressure linear ion trap prior to charge-sign independent trapping, rather than precursor ion sequestration in only the back section as is done for standard ETD. This new scheme increases the charge capacity of the precursor accumulation event, enabling storage of approximately 3-fold more precursor charges. High capacity ETD boosts the number of matching fragments identified in a single MS/MS event, reducing the need for spectral averaging. These improvements in intra-scan dynamic range via reaction of larger precursor populations, which have been previously demonstrated through custom modified hardware, are now available on a commercial platform, offering considerable benefits for intact protein analysis and top down proteomics. In this work, we characterize the advantages of high precursor capacity ETD through studies with myoglobin and carbonic anhydrase.
KeywordsIntact proteins Electron transfer dissociation Ion–ion reactions Instrumentation
Interrogation of intact proteins via mass spectrometry (MS) has the potential to capture nearly all of the relevant information encoded in each protein, including primary sequence information, combinatorial patterns of post-translational modifications (PTMs), and protein gas-phase structure [1, 2, 3, 4, 5]. As molecular weight alone is largely insufficient for full protein characterization [6, 7, 8, 9], tandem MS (MS/MS) is the key component of these top down sequencing methods, revealing both the primary sequence and protein modification state [10, 11, 12, 13]. The emergence of new ion dissociation methods continues to drive top down proteomics [14, 15, 16] by offering valuable alternatives to traditional slow-heating methods (e.g., collision-activated dissociation, CAD). Electron transfer dissociation (ETD) leverages electron-driven radical rearrangements to promote cleavage of N–Cα bonds between amino acid residues, preserving labile post-translational modifications (PTMs) and providing extensive sequence-informative fragmentation of peptides and proteins [17, 18, 19]. Ideally suited for large, highly charged protein molecules, ETD has afforded important gains in top down proteomics, extending protein sequence coverage and enabling characterization of important PTMs and sequence variants [20, 21, 22, 23, 24].
Despite advancements in fragmentation methods and mass analyzers in the past decade, MS instrumentation remains a barrier to further progress in whole protein analysis [25, 26, 27, 28]. Realizing the full potential of top down proteomics, especially when applied to large proteins, requires robust and comprehensive protein fragmentation, which continues to be a challenging endeavor. By providing high resolution/accurate mass (HR/AM) measurements for precursor and product ions with good sensitivity, Fourier-transform (FT) instruments are a well-suited MS platform for top down proteomics [29, 30, 31, 32, 33]. FT-MS instruments are easily coupled with other ion trapping devices (i.e., hybrid systems) to offer a considerable array of fragmentation methods, including CAD, higher-energy collisional dissociation (HCD), photo-activation, electron capture dissociation (ECD), and ETD [34, 35, 36, 37, 38, 39, 40, 41]. A characteristic inherent to all ion trapping instruments, however, is that the number of ions that can be analyzed in a given scan is limited by a fixed number of charges that can be effectively contained and manipulated [42, 43]. The charge capacity of ion trapping devices becomes especially consequential for intact protein fragmentation, where product ion signal is often distributed amongst hundreds of potential fragment channels and increasingly complex isotopic distributions.
As protein mass increases, the efficacy of MS/MS on whole proteins notably diminishes; larger proteins carry more charge and have a greater number of dissociation channels. Not only does this increase spectral complexity, but it also limits precursor capacity (i.e., ion number) in ion trap reaction vessels [44, 45, 46, 47]. For example, an ion trap with a charge capacity of approximately 300,000 charges can store ∼ 30,000 precursor ions for the z = +10 charge state of ubiquitin (∼8.6 kDa) but only roughly 9400 precursor ions for the z = +32 charge state of carbonic anhydrase (∼29 kDa). Compounding this, charge from those initial ion populations is potentially distributed across product ions from 75 backbone bonds for ubiquitin compared with carbonic anhydrase’s 258 backbone bonds. To improve the S/N of product ion measurements, spectral averaging (summing signal from several individual scans) is often required for MS/MS of even modest sized proteins. The tradeoff for the increase in S/N, however, is a significant increase in acquisition times required for generation of high quality spectra, accordingly limiting the sampling depth achievable in a given experiment. We conclude that increasing the number of precursor ion charges prior to initiation of the dissociation event is a direct way to improve S/N without spectral averaging [27, 48, 49]. Herein we describe modifications to ion processing and storage that permit increased precursor ion populations for ETD experiments—we call this method high capacity ETD (also called ETD high dynamic range, or ETD HD).
For ETD on hybrid ion trap-Orbitrap systems, the size of the precursor population is limited by the precursor sequestration event in the dual cell quadrupole linear ion trap m/z analyzer (A-QLT) prior to the reaction [50, 51]. We have shown previously that a larger ETD reaction cell, called the multipurpose dissociation cell (MDC), can accommodate 6- to 10-fold larger initial populations of precursor ions, thereby alleviating the capacity restrictions imposed by using the A-QLT . With the MDC, we achieved better ion statistics and increased the intra-scan dynamic range for protein fragmentation, leading to higher quality spectra (i.e., increased product ion S/N) with less spectral averaging required, which ultimately enabled better top down analyses of complex protein mixtures.
Hunt and co-workers described a different approach enabled by the development of a front-end ETD reagent source . Here the A-QLT remained as the ion–ion reaction cell, but products from multiple rounds of ion–ion reactions were accumulated in the C-trap before a single mass analysis of all product ions in the Orbitrap, ultimately improving the S/N of MS/MS spectra. The promising results characterized in both the Hunt and Coon lab strategies have motivated us to develop an improved implementation of ETD on the newest generation of quadrupole-Orbitrap-linear ion trap Tribrid mass spectrometers .
Here we demonstrate that the ion capacity of the precursor accumulation event prior to the ETD reaction can be increased by changing where in the A-QLT precursor cations and reagent anions are stored. This new implementation of high capacity ETD on the newest generation of Orbitrap Fusion Lumos Tribrid platform allows use of larger precursor populations for ETD MS/MS scans, enabling higher product ion S/N over standard ETD, for a given spectral acquisition time. Ultimately this translates to more sequence-informative fragment ions and higher protein sequence coverage achieved with less spectral averaging in high capacity ETD.
Materials, Reagents, and Sample Preparation
Myoglobin [P68082] and carbonic anhydrase [P00921] were purchased as mass spectrometry grade standards from Protea Biosciences (Morgantown, WV, USA). Formic acid ampoules and acetonitrile were purchased from Thermo Scientific (Rockford, IL, USA).
Mass Spectrometry Instrumentation
High precursor capacity ETD was implemented using the existing dual pressure linear ion trap (A-QLT) on the Orbitrap Fusion Lumos (Thermo Fisher Scientific, San Jose, CA, USA). In standard ETD, the precursor sequestration event occurs by creating a DC potential well of approximately 2 V in the back section of the high pressure cell (HPC). This holds the precursor cations in the back section of the HPC, while the center section and front section voltages of the HPC are set to allow for reagent anion accumulation. To enable high capacity ETD, instrument control code was modified to allow transfer of precursor ions directly from the ion routing multipole to the center section of the HPC for storage using a DC potential well of approximately 4 V, omitting relocation of precursor ions to the back section prior to the ETD reaction (Figure 2a). Reagent accumulation is then achieved by holding the front section at a positive DC offset to establish the potential well for anions. Charge-sign independent trapping for the ion–ion reaction was then performed in the same fashion for both standard and high capacity ETD by setting all DC bias voltages to 0 V and applying axial confining rf voltages to the end lenses of the HPC.
Myoglobin (P68082) and carbonic anhydrase (P00921) were resuspended at approximately 10 pmol per μL in 49.9:49.9:0.2 acetonitrile/water/formic acid, infused via syringe pump into the mass spectrometer at 5 μL per min through a 500 μL syringe, and ionized with electrospray ionization (ESI) at +3.5 kV with respect to ground. For myoglobin, MS/MS scans were performed in the Orbitrap with unthresholded transient acquisition at a resolving power of 120,000 (full width at half maximum) at 200 m/z with a range of 200–2000 Th. Precursor ions were isolated with the mass selecting quadrupole with an isolation width of 10 m/z, and automatic gain control (AGC) target values ranging from 100,000 to 1,000,000 charges as indicated. Transient averaging began after data acquisition was started so that scans with one to 100 transients averaged could be analyzed. An AGC target of 800,000 charges was used for fluoranthene reagent anions (m/z 202, isolated by the mass selecting quadrupole) for ETD and EThcD experiments, reaction times varied as indicated in Supplemental Table 1, and a normalized collision energy of 10 was used for EThcD. Analyses were performed in intact protein mode with a pressure of 3 mTorr in the ion-routing multipole. For carbonic anhydrase, MS/MS scans were performed in the Orbitrap at a both 120,000 and 240,000 resolving powers (at 200 m/z) with precursor AGC target values of 300,000 and 1,000,000 and a m/z range 400–2000 Th. Transient averaging began after data acquisition was started so that scans with one to 200 transients averaged could be analyzed. The AGC target for fluoranthene reagent anions was set to 700,000 charges, reaction times varied as indicated in the text, and the pressure in the ion-routing multipole was set to 1 mTorr in intact protein mode.
MS/MS m/z spectra were deconvoluted with XTRACT (Thermo Fisher Scientific) using default parameters and a S/N threshold of two. ProSight Lite  was used to generate matched fragments using a 10 ppm tolerance. ETD spectra were matched with c-, z-, and y-type ions, and EThcD spectra were also matched with those fragment types in addition to b-type ions. N-terminal methionines were removed from the protein sequences before matching with ProSight Lite, and carbonic anhydrase was matched with an additional sequence modification of N-terminal acetylation (+42.01 Da). Supplemental Figure 5 compares signal from fragments seen with ETD and high capacity ETD, and those unique only to high capacity ETD.
Results and Discussion
Implementing High Capacity ETD in a Dual Pressure Linear Ion Trap
To address the limitations in ion capacity imposed by precursor sequestration in the back section of the HPC, we have employed a new implementation of ETD called high capacity ETD. Figure 2a illustrates how precursor storage differs between standard and high capacity ETD by showing the voltages employed relative to 0 V during the reagent accumulation period. The practical implications of this change in precursor spatial confinement is highlighted in Figure 2b, showing ETD spectra from both the standard and high capacity implementations for the z = +18 precursor of myoglobin.
For both the standard and high capacity schemes, precursor AGC target values of 100,000, 200,000, 400,000, 600,000, 800,000, and 1,000,000 were investigated. In standard ETD, a target value of 400,000 produced the highest number of matched fragments (71), whereas higher target values did not translate to an increase in sequence-informative fragment ions. This is in accordance to the estimated capacity of ∼200,000 to ∼500,000 charges discussed above. In high capacity ETD, however, the largest AGC target value of 1,000,000 produced the most fragments (136), nearly doubling the number of fragments observed following standard ETD using the same amount of spectral averaging (two transients averaged for each). For product ions identified in both conditions, S/N values are approximately 3-fold higher, sometimes more, in high capacity ETD. Additional fragment ion identifications were often due to the improved S/N, enabling confident charge state assignment and subsequent matching against theoretical values.
Note that the change in the precursor accumulation event necessitates changes in the reagent accumulation event. In standard ETD, reagent anions are accumulated in the center section of the HPC, whereas in high capacity ETD only the front section is used (Figure 1a). This now limits the capacity for reagent anion storage, although the ion capacity of the end section should be higher for the reagent anions than it is for the precursor cations. The higher relative capacity can be accounted for by both the significant difference in ion m/z between reagent anions (202 Th) and their precursor counterparts (especially in top down proteomics) and by the single charge of the reagent anions compared with highly charged protein precursors. Slightly smaller reagent anion populations may affect the pseudo-first order kinetics of the ETD reactions, as the reagent, which may no longer be in a large excess of the precursor population, could be depleted during the reaction. Indeed, loss of pseudo-first order kinetics due to reagent depletion was observed in these experiments, requiring longer reaction times to achieve sufficient fragmentation, even with the same reagent AGC target values (Supplemental Table 1). Using the spectra in part b of Figure 2 as an example, the standard ETD scheme using a precursor AGC target of 400,000 used a reaction time of 5 ms; the high capacity ETD with a precursor AGC target of 1,000,000 required a 25 ms reaction time to accrue a comparable level of progression of the ETD reaction.
Although this is a significant increase in reaction time, the resulting increase in total scan time is marginal. The standard ETD spectrum in Figure 2, with two averaged transients and an average precursor injection time of 4.3 ms, required 848 ms of total elapsed scan time for 71 fragments. The high capacity ETD spectrum, comparatively, with two averaged transients and a 12.4 ms average precursor injection time, required 904 ms of total elapsed scan time for 136 fragments. Thus, even though high capacity ETD required approximately 56 ms longer in total acquisition time, it nearly doubled the number of identifiable fragments generated. Even when averaging an additional transient (for a total acquisition time of 1271 ms), the number of fragments identified in standard ETD increased to only 82 fragments. Furthermore, increasing the reaction time for standard ETD did not provide any beneficial information. Standard ETD at both an AGC target of 400,000 with an 11 ms reaction time (6 ms longer) and an AGC target of 1,000,000 with a 25 ms reaction time (20 ms longer) yielded fewer matching fragments, 64 and 55, respectively, indicating degrees of over-reaction.
High Capacity ETD for a Moderate Size Protein (∼17 kDa)
High capacity ETD maps show a distinct pattern of darker colors (more matching fragments) for higher precursor targets (upper half), demonstrating that the new implementation of ETD permits reaction of larger precursor populations for improved product ion S/N and more sequence-informative fragments for a given acquisition time. Importantly, Figure 3 also demonstrates the improvements in fragment ion generation seen with high capacity ETD over standard ETD when considering a given number of transients averaged. Higher AGC target values—especially 800,000 and 1,000,000—provide as many, if not more, matched fragments in two averaged transients as standard ETD can provide in five, and the benefits are striking when considering similar numbers of averaged transients for the two conditions. This may also indicate that approximately 2- to 5-fold more precursor ions can be stored successfully in high capacity ETD, if not more. Additionally, to eliminate differences in reaction time as a cause of the improvements observed with the high capacity scheme, we reacted precursors in the standard ETD scheme (HPC back section sequestration) for the duration used in high capacity ETD (Supplemental Figure 1). The larger precursor AGC targets required longer reactions times in high capacity ETD, so the number of fragments generated from standard ETD at high capacity reaction times is noticeably decremented because of over-reaction and generation of internal fragments, confirming that the benefits seen in high capacity ETD are attributed to the reaction of larger precursor populations.
As noted previously, the increased precursor injection times and longer reaction times in high capacity ETD can increase total spectral acquisition time slightly, even when using the same number of averaged transients, but Supplemental Figure 2 shows that these increases are minor relative to total scan time. The spread in the curves for different AGC target values in high capacity ETD (red) is greater than the curves in standard ETD (blue); this difference further demonstrates that larger precursor populations are indeed being retained when precursor target values are increased in high capacity ETD, whereas the size of the precursor population plateaus in standard ETD despite elevated AGC target values. Overall, the high capacity ETD scheme greatly improves the protein sequence coverage that can be obtained per second of data acquisition, which makes high capacity ETD highly advantageous when spectral quality must be balanced with acquisition time, as is needed in high-throughput top down (i.e., using online chromatography) proteomics experiments.
Beyond traditional ETD fragmentation, we observed that high capacity ETD can aid hybrid fragmentation methods as well. EThcD, which uses beam-type collisional activation of ETD products after the ion–ion reaction [70, 71], can improve sequence coverage for precursor ions, especially those with low-charge density where precursor-to-product ion conversion efficiency is hindered by noncovalent interactions. We saw that the high capacity ETD scheme aided in fragment ion generation and protein sequence coverage with EThcD on the z = +15 precursor of myoglobin (Supplemental Figure 3). We surmised that high capacity ETD can be especially valuable for these hybrid fragmentation techniques, where secondary activation has to be carefully balanced since more fragmentation channels are being added to erode product ion signal. As expected, the best benefits to EThcD with the high capacity scheme were seen at the highest precursor targets.
High Capacity ETD for a Larger Protein (∼29 kDa)
High capacity ETD is well-positioned to provide pronounced gains for larger proteins, where the number of dissociation channels is significantly greater and even considerable degrees of spectral averaging cannot approach the increases provided by reaction of large precursor populations [72, 73]. To investigate this, we reacted the z = +34 precursor of carbonic anhydrase (∼29 kDa) using standard ETD (AGC target of 300,000) and high capacity ETD (AGC target of 1,000,000). To explore how the high capacity ETD and standard ETD schemes compare with higher resolution spectra, we also collected MS/MS spectra at two resolving powers (120 and 240 K). First, the best reaction times to use for each condition were determined experimentally (Supplemental Figure 4), and reaction times of 4 and 7 ms were used for standard and high capacity ETD, respectively.
We have enabled the accumulation and retention of 2- to 5-fold more precursors for ETD reactions by altering the region in the ion trap where precursor ions are stored during reagent ion injection. When holding precursor cations in the center section of the high pressure cell of a dual cell quadrupole linear ion trap, as many as 1,000,000 charges or more can be stored for subsequent ion–ion reactions. This increase in precursor ion capacity boosts the signal-to-noise of product ions, producing higher quality MS/MS spectra with only minor increases in acquisition time. High capacity ETD facilitates a more robust characterization of intact protein cations—a single scan can achieve fragment ion production and protein sequence coverage equivalent to approximately five averaged scans of standard ETD. Overall, high capacity ETD improves the compromise between S/N improvements and spectral acquisition speed while still enabling enhanced MS/MS data quality for intact proteins, regardless the degree of spectral averaging. Moreover, high capacity ETD has been implemented using commercially accessible hardware and is available on the newest generation of quadrupole-Orbitrap-linear ion trap Tribrid mass spectrometer (Orbitrap Fusion Lumos), giving it a distinct advantage over earlier approaches that required custom modified devices.
The improvements in MS/MS characterization of intact proteins with high capacity ETD will advance top down proteomics by providing more robust fragmentation on a chromatographic time-scale. This new implementation of ETD also benefits hybrid dissociation methods like EThcD, which are demonstrating promise as new methods to intact protein fragmentation approaches. Future work will focus on how high capacity ETD can benefit other hybrid dissociation techniques (e.g., ultraviolet photo-dissociation (UVPD)-ETD methods  and activated ion ETD (AI-ETD) [75, 76]) with an emphasis on how this improved approach to ETD can be employed in large-scale proteome characterizations. With the implementation of high capacity ETD, we present a straightforward strategy to improve tandem mass spectra of intact proteins; accordingly, this approach is implemented on the Orbitrap Fusion Lumos and maintains all of the benefits of conducting ion–ion reactions in the dual-cell quadrupole linear ion trap.
The authors gratefully acknowledge support from Thermo Fisher Scientific and NIH grant R01 GM080148. N.M.R. was funded through an NSF Graduate Research Fellowship (DGE-1256259). The authors also thank Graeme McAlister for helpful discussions.
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