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Integration of Ultraviolet Photodissociation with Proton Transfer Reactions and Ion Parking for Analysis of Intact Proteins Dustin D. Holden, William M. McGee, and Jennifer S. Brodbelt Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03911 • Publication Date (Web): 03 Dec 2015 Downloaded from http://pubs.acs.org on December 5, 2015

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Analytical Chemistry

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Integration of Ultraviolet Photodissociation with Proton Transfer Reactions and Ion Parking for Analysis of Intact Proteins Dustin D. Holden, William M. McGee, Jennifer S. Brodbelt* Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712 *

Corresponding author: Jennifer S. Brodbelt, [email protected]

Correspondence to: [email protected]

ABSTRACT We report the implementation of proton transfer reactions (PTR) and ion parking on an Orbitrap mass spectrometer. PTR/ion parking allows charge states of proteins to be focused into a single lower charge state via sequential deprotonation reactions with a proton scavenging reagent, in this case a nitrogen-containing adduct of fluoranthene. Using PTR and ion parking we evaluate the charge state dependence of fragmentation of ubiquitin (8.6 kDa), myoglobin (17 kDa), and carbonic anhydrase (29 kDa) upon higher energy collisional dissociation (HCD) or ultraviolet photodissociation (UVPD).

UVPD exhibited less charge state dependence, thus

yielding more uniform distributions of cleavages along the protein backbone and consequently higher sequence coverage than HCD.

HCD resulted in especially prominent cleavages C-

terminal to amino acids containing acidic side-chains and N-terminal to proline residues; UVPD did not exhibit preferential cleavage adjacent to acidic residues but did show enhancement next to proline and phenylalanine.

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INTRODUCTION Mass spectrometry has become a well-established method for proteomics, largely based on the use of MS/MS methods to fragment and thus sequence peptides.1 Proteolytic digestion of proteins into component peptides and subsequent LC-MS/MS analysis has been termed the “bottom-up” approach.1–4 Although bottom-up methods are effective for many applications, proteins are frequently identified based on a handful of constituent peptides due to the complexity of the proteolytic mixtures and variations in ionization efficiencies and chromatographic resolution of individual peptides. The gaps in sequence coverage, some which potentially contain post-translational modifications, have spurred the development of top-down methods which entail the analysis of intact proteins.5–8 Advances in the sophistication of topdown approaches have led to some impressive achievements, such as identification of over one thousand gene products from human cells corresponding to more than 3,000 proteoforms.9 Additionally, 347 mitochondrial proteins accounting for roughly 50% of the mitochondrial proteome below 30 kDa have been identified recently using top-down mass spectrometry.10 Although the analysis of intact proteins offers notable advantages for comprehensive characterization of sequences, post-translational modifications, improvements in sensitivity, sequence coverage, and dynamic range remain key goals for top-down mass spectrometry. Two strategies that offer great promise for accelerating top-down proteomics include ultraviolet photodissociation (UVPD)11–15 and charge manipulation reactions, namely proton transfer reactions (PTR) with ion parking.16–20 PTR/ion parking utilizes ion-ion chemistry and selective waveforms to concentrate highly dispersed charge states of proteins into single lower charge states, thus concentrating the ion signal. PTR/ion parking not only provides improvements in the analytical metrics of top-down protein analysis but also conveniently facilitates the exploration of the impact of charge state on protein fragmentation.16–20 UVPD provides broad and deep fragmentation of proteins, thus giving high sequence coverage, and is well-suited for highthroughput proteomics.11–15 Integration of PTR/ion parking methodologies with UVPD provides 2 ACS Paragon Plus Environment


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a compelling means to advance the field of top-down proteomics and allow assessment of the performance of UVPD for a broader range of protein charge states, as described in this report. The concept of proton transfer reactions for charge state manipulation entails reacting a population of multi-charged ions with a proton-scavenging anion, such as the nitrogen adduct of fluoranthene (a reactive ion of m/z 216). Upon interactions with multi-protonated proteins simultaneously stored in an ion trap, the anions extract protons from the proteins and thus reduce their charge states.21–24 It has also been shown that applying supplemental waveforms to the ion trap during PTR has been very effective at reducing the rate of charge reduction and thus can be used to “park” the proteins at a specific charge-reduced state.16 Ion parking during PTR has been successful for concentrating a broad population of charge states into one selected charge state.16 While conventional electrospray ionization (ESI) utilizing acidic solvents or native ESI utilizing buffered solvents generally provide high or low protein charge states, respectively, PTR with ion parking utilizes ion-ion chemistry and ion resonance capabilities of the ion trap to concentrate these ions to a desired charge state. The McLuckey group, who pioneered PTR with ion parking, has examined the charge-state-dependence of collision-induced dissociation (CID) for the protein ubiquitin.24 Activation and fragmentation of different charge states of ubiquitin followed the mobile proton model.25 Ions in lower charge states (ones with few or no mobile protons) displayed preferential cleavages C-terminal to acidic backbone locations. Cleavages N-terminal to proline and at regions containing basic residues were favored for the higher charge states.25 Additionally, PTR proved useful for reducing the charge states of the more highly charged fragment ions, thus allowing more confident charge state determination in mass spectrometers lacking high resolution capabilities.25


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193 nm UVPD has proven to be an effective, fast activation method for providing high sequence coverages for top-down protein analysis,11–15 and more recently has been used to provide insight about conformations26 and ligand binding interactions of native proteins and protein-ligand complexes.27,28 UVPD occurs via excitation of ions to excited electronic states, then fast dissociation from those excited states or via internal conversion and intramolecular vibrational energy re-distribution prior to dissociation.29–32 These processes account for the rich diversity of fragment ions produced by UVPD. We have previously reported that UVPD displays less dependence on precursor ion-charge state than exhibited by conventional collision and electron activation methods.11 Using PTR and ion parking we further investigate the charge state dependence of protein fragmentation as well as assess the utility of UVPD for low protein charge states that lack mobile protons. Here we report the successful coupling of UVPD with PTR and ion parking on a commercial high resolution, high mass accuracy Orbitrap mass spectrometer. The capability of concentrating ion current into selected charge states by PTR offers potential benefits for extending the range of top down applications on high performance mass spectrometers. The analytical metrics of top down analysis of proteins by UVPD and higher energy collisional dissociation (HCD) are compared as a function of charge state with the aim of establishing the changes (improvements or deterioration) of MS/MS performance for the lower charge states generated by PTR. EXPERIMENTAL SECTION Materials Bovine ubiquitin, horse myoglobin, and bovine carbonic anhydrase were purchased from Sigma-Aldrich (St. Louis, MO).

Solvents were purchased from Thermo Fisher Scientific

(Pittsburgh, PA).



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Mass Spectrometry Proteins were suspended in 50/49/1 methanol/water/formic acid (v/v/v) at a final concentration of 10 µM. They were infused directly into an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) customized for implementation of UVPD. Proton transfer reactions were performed in the high pressure linear ion trap, as shown in Supplemental Figure 1, using the nitrogen-adducted fluoranthene anion of m/z 216 as the PTR reagent.23 Custom ion trap control language (ITCL) was developed to achieve ion parking simultaneous with PTR by applying auxiliary voltages at specific resonance frequencies to the ion trap. Optimal ion parking resonance frequency offsets were determined using a custom ITCL tuning program as described below. UVPD in the HCD cell was achieved via a single 5 ns laser pulse from a Coherent ExciStar XS 500 (Santa Clara, CA) 193 nm excimer laser. HCD collision energy (normalized collision energy, NCE) values were optimized per charge state, and 0.10 ms activation time was used. All mass spectra were acquired in the Orbitrap mass analyzer using a resolving power of 240,000 at m/z 400. For UVPD and HCD experiments the HCD nitrogen collision gas pressure was reduced to an UHV chamber penning gauge δ measurement of 0.1 x 10-10 Torr and 0.3 x 1010

Torr respectively. To assure optimal MS/MS spectra for ubiquitin, myoglobin, and carbonic

anhydrase, the numbers of transients averaged per protein were 200, 250, and 500, respectively. Analyte and PTR reagent targets were set at a reaction ratio of 1:1 (3E5:3E5), and in order to achieve very low charge states a PTR reaction time of up to 250 ms was used. Ion Parking Conventional trapping conditions during ion-ion reactions, such as for electron transfer dissociation, typically use a relatively low radiofrequency trapping voltage to allow simultaneous trapping of the low mass fluoranthene reagent (m/z 202) and the analyte ion of interest. This setup typically corresponds to a Mathieu q value of 0.4 relative to the fluoranthene reagent ion. For conventional ETD experiments, this establishes a low mass cutoff (LMCO) of ~ m/z 90.33 In 5 ACS Paragon Plus Environment

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contrast, since PTR of intact proteins generates charge-reduced ions of much higher m/z values than m/z 216, the applied trapping voltage may be adjusted to a significantly greater value (i.e. corresponding to a Mathieu q value of 0.7 instead of 0.4). This adjustment allows satisfactory trapping of PTR reagent anions while simultaneously enhancing the ability to trap chargereduced protein ions at much higher m/z values. For ion parking, an auxiliary waveform was applied at a specific frequency to cause low energy excitation of a desired ion charge state simultaneously with PTR. McLuckey et al. have previously shown that the auxiliary frequency applied to perform resonance excitation of an ion in a specific charge state to park during PTR benefited by offsetting the frequency either slightly higher or lower than the theoretical secular frequency of the targeted ion in order to optimize the abundances of the parked ions.16 For the present work, a custom ITCL tuning program was developed to record the abundance of an ion targeted for parking as the applied resonance frequency offsets were adjusted, at a fixed amplitude, beginning lower than the theoretical secular frequency and scanning to a slightly higher secular frequency of the ion of interest (as illustrated for the 8+ charge state of ubiquitin in Supplemental Figure 2A). A resonance frequency offset of 0.0 kHz indicated the theoretical secular frequency of a specific m/z value (i.e. for a particular charge state of interest). During infusion of a protein the most abundant charge state was isolated for PTR (such as 10+ for ubiquitin). The resulting parked ion spectra are shown in Supplemental Figure 2B for which resonance frequency offsets of -0.5 kHz and 1.5 kHz produced the optimal parked ion spectra (blue and red traces, respectively), while resonance frequency offsets closer to and slightly above the theoretical m/z secular frequency caused ejection of the ions (ex: 0.5 kHz, Supplemental Figure 2B green trace). For PTR without ion parking, the resulting charge reduced species exhibit a broader Gaussian distribution, which is depicted by the black trace in Supplemental Figure 2B. The ITCL tuning program facilitated optimization of the frequency of the applied voltage for ion parking.



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Data Analysis Spectra were deconvoluted using Xtract (Thermo Fisher Scientific) with an S/N threshold of 3. For generation of histograms, sequence coverage values and number of identified fragment ions, deconvoluted spectra were interpreted manually by comparison to 10 ppm error from theoretical fragment masses using ProteinProspector v5.14.3 (http://prospector.ucsf.edu). For UVPD data, fragment ion types a, a+1, b, c, x, x+1, y, y-1, Y, and z were considered. For HCD data, fragment ion types a, b, and y were considered. Additionally, UVPD and HCD spectra were analyzed using ProSight Lite (Northwestern University) with a 10 ppm error tolerance, using UVPD mode and HCD modes for the appropriate spectra. RESULTS AND DISCUSSION This study focuses on implementing PTR/ion parking on a high performance Orbitrap mass spectrometer and evaluating the fragmentation patterns produced by UVPD and HCD as a function of charge state for three proteins. Conventional top-down proteomics utilizes acidic solvents to effectively ionize proteins using ESI. At the same time acidic conditions may cause a protein to become partially denatured allowing more protonation sites to become accessible and thus resulting in higher charge states. In general proteins are more highly protonated based on their size with a few exceptions such as proteins containing disulfide bonds and proteins containing low numbers of basic sites in their sequences. Conventional collisional activation methods including HCD are dependent on the mobility of protons for backbone fragmentation, resulting in mainly b- and y-type fragment ions, and thus the charge state of a protein modulates the associated fragmentation patterns.25 In contrast to HCD, UVPD is a fast, high energy excitation process that results in formation of a diverse array of N-terminal a, b, c, and Cterminal x, y, and z type fragment ions for proteins.11–15 UVPD has been previously shown to be less charge state dependent compared to other activation methods.11 This makes it well-suited for strategies that take advantage of charge-state manipulation to increase sensitivity or to decongest complex spectra, as well as studies that focus on native-like proteins in low charge states. The 7 ACS Paragon Plus Environment

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present study represents the first step towards developing a full suite of charge manipulation strategies integrated with UVPD for top-down proteomics. PTR was implemented in the high pressure linear ion trap of the Orbitrap Elite mass spectrometer via reactions with nitrogen-adducted fluoranthene as a deprotonating agent, generated by the onboard ETD source. Ion parking16 was accomplished by application of an auxiliary waveform at a frequency corresponding to a specific ion charge state to reduce the rate of charge reduction as a means to “park” the ion at a particular charge state as described in the Experimental section. Once the appropriate charge-reduced state was parked using an optimal resonance frequency offset, the ions were transferred to the Orbitrap for subsequent mass analysis. Two proteins ubiquitin (8.6 kDa) and myoglobin (16.9 kDa) were used for this purpose and representative spectra are shown in Figure 1. PTR was performed on the most abundant initial charge state for each protein: 10+ for ubiquitin and 16+ for myoglobin. For the 10+ charge state of ubiquitin, the 7+ to 4+ charge states were sequentially parked during a period of 200 ms, as shown in Figure 1A. For the 16+ charge state of myoglobin, the 14+ to 6+ charge states were sequentially parked during a period of 150 ms, as shown in Figure 1B. As expected the amplitude of the auxiliary voltage applied to the ion trap to park a particular charge state increased when parking higher charge states relative to lower charge states owing to the increased rate of charge reduction moving from low to higher charged analyte ions.

The

magnitude of this auxiliary voltage ranged from 1.7 to 3.2 Vpp depending on the m/z value of the targeted parked ion. Interestingly, generating charge-reduced ions from a selected myoglobin precursor ion (16+) consistently resulted in a higher signal intensity of the charge-reduced protein (10+ to 6+ charge states) relative to the original selected precursor ion. The cause for this increase in abundance is currently unclear. PTR/ion parking may also be undertaken on broad charge state envelopes instead of individual charge states, as illustrated in Supplemental Figure 3 for ubiquitin (13+ through 8+) and myoglobin (24+ through 14+). Appropriate conditions were established to reduce and park 8 ACS Paragon Plus Environment


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the broad range of charge states of ubiquitin into a single charge state (3+) (Supplemental Figure 3A). Since myoglobin is more highly charged than ubiquitin, more charge states were included in broadband isolation window (e.g. using a window size of 600 m/z for 13+ through 8+), which translated to a greater concentration of the ion current into the parked charge-reduced 6+ state (Supplemental Figure 3B). This broadband isolation/charge-reduction capability offers a particularly compelling way to focus the ion current of large proteins that are initially dispersed into many charge states upon ESI, thus enhancing sensitivity for top down analysis, a problem that is especially challenging in high throughput LC-MS applications. Signal to noise is not necessarily increased using this broadband method, but accumulation time required to fulfill target ion counts using automatic gain control (AGC) is reduced. To explore ion parking over a more extended m/z range, the myoglobin ion population was also parked at the 3+ charge state (m/z 5652). As shown in Supplemental Figure 3B the signal intensity of the parked 3+ charge state was not as high as that observed for the parked 6+ charge state. The reason for this apparent loss of signal intensity is attributed to the less efficient trapping conditions for the 3+ charge state (m/z 5652, q = 0.027) compared to the 6+ charge state (m/z 2826, q = 0.054). Correspondingly, the amplitude of the auxiliary voltage applied to park a particular charge state must be reduced as a function of the selected charge state to prevent destabilization of the ions and ejection from the trap. UVPD and HCD (each performed in the HCD cell) were used to examine the sequence coverages, fragment ion distributions, and impact of preferential (enhanced) backbone cleavages for ubiquitin, myoglobin, and carbonic anhydrase in different charge states before or after PTR/ion parking. The McLuckey group previously showed the charge state dependence of CID using ubiquitin as a model protein and noted a loss of sequence coverage as well an increase in preferential backbone cleavages C-terminal to acidic amino acids (Asp, Glu) for the lowest precursor charge states.24 They also noted that sequence coverage was low for higher charge states of ubiquitin, primarily owing to a dominant preferential cleavage between Glu-18 and Pro9 ACS Paragon Plus Environment

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19 (both residues are known to serve as preferential cleavage sites: C-terminal to Glu and Nterminal to Pro), while the highest sequence coverages were obtained for the middle charge states of ubiquitin.24 In the present study, to allow optimal control of total ion abundance and allow comparisons of charge states over a wide range, an individual charge state from each protein was isolated and subjected to PTR/ion parking to reduce its charge state. Ubiquitin (10+) was parked at the 8+, 6+, and 4+ states; myoglobin (16+) was parked at the 12+, 10+, 8+, 7+ and 6+ charge states, and carbonic anhydrase (22+) was parked at the 18+, 14+, and 10+ charge states prior to UVPD or HCD. The fragmentation patterns obtained by HCD and UVPD for ubiquitin and myoglobin were converted to histogram format based on summation of the Nterminal (a,b,c) and C-terminal (x,y,z) ions attributed to backbone cleavages between each adjacent pair of amino acids in the protein sequence (see Figures 2 – 4). The HCD results obtained for ubiquitin (4+, 6+, 8+, 10+, 12+) display the same charge-state dependent trends as noted by McLuckey et al., as shown in Figure 2. For the lowest charge state (4+), preferential cleavages C-terminal to Glu (residues16, 34, 51) and Asp (residues 21, 32, 39, 52, 58) were observed. As the charge state increased to 8+ and finally 12+, the relative abundances of these Glu and Asp C-terminal cleavages diminished, while cleavages localized between Glu-16 and Pro-18 became dominant.

These results aligned well with results previously reported by

McLuckey et al.24 In comparison to the HCD results, UVPD of ubiquitin (4+ to 12+ charge states) resulted in more uniform cleavages across the entire backbone and significantly less charge state dependence (Figure 3), although some enhanced fragmentation N-terminal to proline and C-terminal to acidic amino acids was observed for the lowest (4+) and highest (12+) charge states of ubiquitin upon UVPD (Figures 3A and 3E). Interestingly, enhanced backbone fragmentation in close proximity to two of the aromatic side-chains, Phe-45 and Tyr-59, was observed in the UVPD data, an outcome which is not surprising since Phe and Tyr have UV absorbing side-chains (and may reflect prompt dissociation from excited states).



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The charge state dependence of the fragmentation of myoglobin was similarly explored in conjunction with PTR/ion parking and HCD or UVPD. Figure 4 shows the HCD fragmentation of myoglobin for the 8+ charge state, a low charge state for this protein, and the higher 12+ and 20+ charge states. For the 8+ charge state, HCD caused preferential backbone cleavages Nterminal to Pro and C-terminal to Glu and Asp, similar to the pathways observed for the low charge state of ubiquitin and consistent with the pathways arising from a lack of abundant mobile protons (Figure 4A). For the 12+ charge state, HCD generated a greater portion of C-terminal fragments closer to the C-terminus and more N-terminal fragments close to the N-terminus (Figure 4B).

HCD of the 20+ charge state promoted predominantly N-terminal dominant

cleavages with a few notable proline cleavages and one highly enhanced cleavage C-terminal to Met-131 (Figure 4C). Interestingly, for the low 8+ charge state, the relative abundances of Nand C-terminal ions generated by HCD were more equally distributed, indicating more uniform cleavages along the backbone of myoglobin as shown in Supplemental Figure 4A. However, for higher charge states (12+ and 20+), fragmentation of the backbone was far more localized near the N- and C-termini upon HCD as showcased in Supplemental Figures 4B and 4C. In comparison to HCD, UVPD of myoglobin showed more uniform backbone cleavages across the protein for all three charge states (8+, 12+, 20+) as well as abundant fragments related to cleavage C-terminal to UV-absorbing phenylalanine residues, the latter of which seemed especially enhanced for the 8+ and 12+ charge states (Figure 5A and 5B). For the highest charge state (20+), the backbone cleavages adjacent to phenylalanine were not quite as prominent, and there was a more notable preference for formation of N-terminal ions closer to the N-terminus and C-terminal ions closer to the C-terminus (Figure 5C). As noted above for UVPD of ubiquitin, UVPD resulted in a greater array of fragment ions for all charge states compared to HCD. The variations in fragmentation of carbonic anhydrase (29 kDa) were also evaluated upon HCD and UVPD, as summarized in Supplemental Figures 5 and 6 and the corresponding 11 ACS Paragon Plus Environment

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sequence maps shown in Supplemental Figure 7. For all three charge states of carbonic anhydrase (14+, 22+, 28+), HCD generated relatively few backbone cleavages. For the 14+ charge state achieved by PTR/ ion parking, a dominant backbone cleavage occurred at Pro-193 upon HCD (Supplemental Figure 5A). HCD of the two higher charge states (22+ and 28+) yielded similar distributions of fragment ions (Supplemental Figure 5B and 5C) for which cleavages adjacent to Asp (residues 31, 33, 40)

and Pro (41,193,199,235) were notably

enhanced. The HCD trends for carbonic anhydrase were relatively independent of charge state, more so than for HCD of ubiquitin and myoglobin. Interestingly, although carbonic anhydrase has a relatively large number of acidic amino acids (11 glutamic acids and 19 aspartic acids), backbone cleavages C-terminal to these acidic residues was not consistently or significantly enhanced. UVPD of the 14+, 22+, and 28+ charge states of carbonic anhydrase resulted in a greater number of backbone cleavages than observed for HCD (Supplemental Figure 6). The fragmentation behavior showed relatively little dependence on charge state for UVPD, even for the lowest charge state, aside from minor changes in the extent of backbone cleavage between Leu-196 and Thr-197. The comprehensive HCD and UVPD fragmentation maps for carbonic anhydrase in Supplemental Figure 7 showcase the broader array of backbone cleavages upon UVPD compared to the more limited cleavages that are clustered closer to the N- and C-termini upon HCD. Sequence coverages and the number of identified fragment ions for ubiquitin (4+, 6+, 8+, 10+ and 12+), myoglobin (6+, 7+, 8+, 10+, 12+, 16+ and 20+), and carbonic anhydrase (10+, 14+, 18+, 22+, 26+ and 28+) upon HCD and UVPD are shown in Figure 6. In general, UVPD resulted in a modest but consistent increase in sequence coverage moving to higher charge states. In contrast, HCD generally showed highest sequence coverages for intermediate charge states with slight variations for each protein tested. In particular, the sequence coverages obtained by HCD diminished for the lowest charge states of myoglobin and carbonic anhydrase and for the 12 ACS Paragon Plus Environment


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highest charge states of ubiquitin and myoglobin. Although sequence coverages for carbonic anhydrase were higher for UVPD for all charge states, both HCD and UVPD exhibited minor decreases in coverage for the highest charge states. Additionally, the number of identified fragment ions for all protein charge states were higher for UVPD, an outcome attributed to the greater diversity of fragmentation channels for UVPD. These results suggest that although PTR/parking may concentrate the ion signal and has the potential to improve signal-to-noise, there is a trade-off in sequencing performance for HCD, less so for UVPD. A dotted line was added to each histogram in Figure 6 to differentiate PTR-ion parking/fragmentation (left side) from direct isolation/fragmentation (right side, no parking). At this interface a slight discontinuity in sequence coverage is observed. This is caused by a modest reduction in the PTR/ion parking efficiency as the charge state of the parked ion increases, observed in this situation as the ions closest to the original precursor charge state (such as the initial 16+ charge state of myoglobin or the initial 10+ charge state of ubiquitin). McLuckey et al. have previously reported that proton transfer reaction rates are greatest for high charge states, thus increasing the likelihood of further charge reduction during the PTR/ion parking periods.16 This effect is seen in the ion parking spectrum of ubiquitin (7+) in Figure 1A for which rather large abundances of the next few lower charge states are observed. Ion parking spectra for lower charge states of ubiquitin (Figure 1A, 6+, 5+ and 4+) resulted in progressively less prominent abundances of lower charge states. Since the signal-to-noise values of the parked ion mass spectra depend on the efficiency of converting higher charge states to a single reduced charge state, this ultimately influences the number of precursor ions available for subsequent HCD or UVPD. This variation in the number of parked precursor ions likely explains the slight drop in sequence coverage between the charge states directly on either side of the dotted line in Figure 6. The overall S/N of HCD mass spectra is generally higher than UVPD (largely because the ion current of UVPD is sub-divided into significantly more product ions), so these effects are more apparent for PTR/ion parking with UVPD than for PTR/ion parking with HCD. 13 ACS Paragon Plus Environment

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To further showcase the prominence of residue-specific backbone cleavages as a function of charge state, fragment ion distributions generated by UVPD and HCD are tabulated as stacked bar graphs in Figure 7 for various charge states of ubiquitin, myoglobin, and carbonic anhydrase. Pro-directed cleavages were consistently more dominant for HCD compared to UVPD for ubiquitin and carbonic anhydrase for the mid- or higher charge states. Cleavages Cterminal to glutamic and aspartic acids were more prominent upon HCD than UVPD for all charge states of ubiquitin and myoglobin, but not significantly different for carbonic anhydrase. The portion of preferential Pro, Glu-, and Asp-cleavages were collectively always more prominent upon HCD for all charge states, a result not unexpected due to the dependence of these cleavages on proton-modulated pathways. Phe-directed backbone cleavages were only notable for UVPD of each protein. CONCLUSIONS PTR assisted by ion parking is an effective method for controlling protein charge states and concentrating dispersed ion signals into single charge states.

With respect to overall

sequence coverage, UVPD showed a lower degree of charge state dependence compared to HCD and generally outperforming or showing equal performance to HCD for most charge states. HCD typically demonstrated optimal performance for intermediate charge states. Normalization of UVPD laser parameters will also likely improve efficiency and sequence coverage beyond that reported in this first study. Specific preferential backbone cleavages, such as those adjacent to Asp, Glu and Pro, were particularly prominent upon HCD, thus leading to accumulation of ion signal in just a few fragmentation channels.

The PTR/ion parking/UVPD strategy seems

particularly promising for extending the performance of LCMS analysis of intact proteins when ion signals are limited due to a highly dispersed series of charge states, an opportunity currently underway in our lab.



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ACKNOWLEDGMENTS We acknowledge the following funding sources: NSF (Grant CHE1402753) and the Welch Foundation (Grant F-1155). We thank Jae C. Schwartz and John E.P. Syka for their assistance with customizing mass spectrometer control coding (ITCL).

Supplemental Information: Additional figures include a schematic of the modified Orbitrap mass spectrometer, a tuning graph for optimization of ion parking, examples of ion parking for two proteins, histograms of the distribution of N-terminal and C-terminal fragment ions, HCD and UVPD cleavage maps of carbonic anhydrase, and fragmentation maps of carbonic anhydrase.


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Figure Captions: Figure 1: A) Isolation of 10+ charge state ubiquitin followed by PTR (200 ms) and ion parking at charge states 7+, 6+, 5+, and 4+ shown at the same scale. B) Isolation of myoglobin (16+) followed by PTR (150 ms) and ion parking at charge states 14+, 12+, 10+, 9+, 8+, 7+, and 6+, all shown on the same abundance scale. Figure 2: PTR (200 ms) of ubiquitin (10+) and HCD of the resulting parked charge states A) 4+, B) 6+, and C) 8+. HCD of charge states D) 10+ (not parked) and E) 12+ (not parked). HCD NCE values used were 33, 30, 30, 23, and 18, respectively. In D and E, the abundance of the backbone cleavage between E and P is off-scale, and thus the true percentages are listed. Figure 3: PTR (200 ms) of ubiquitin (10+) and UVPD of the resulting parked charge states A) 4+, B) 6+, and C) 8+. UVPD of charge states D) 10+ (not parked) and E) 12+ (not parked). One 3.5 ∗

mJ pulse was used for UVPD. ( Enhanced cleavages adjacent to Phe-45 and Tyr-59.) In E, the abundance of the backbone cleavage between E and P is off-scale, and thus the true percentages are listed. Figure 4: PTR (200 ms) of myoglobin (16+) and HCD of charge states A) 8+ and B) 12+, and C) HCD of charge state 20+ (not parked). HCD NCE values used were 33, 30, and 21, respectively. Figure 5: PTR (200 ms) of myoglobin (16+) and UVPD of charge states A) 8+ and B) 12+, and C) ∗

UVPD of 20+ (not parked). One 2.5 mJ pulse was used for UVPD. ( Enhanced cleavages adjacent to phenylalanine “F” residues). Figure 6: Sequence coverages (upper) and subsequent number of identified fragment ions (lower) for A) ubiquitin (4+, 6+, 8+, 10+ and 12+), B) myoglobin (6+, 7+, 8+, 10+, 12+, 16+ and 20+), and C) carbonic anhydrase (10+, 14+, 18+, 22+, 26+, and 28+). A dotted line differentiates PTR/ion parking fragmentation (left side, parked) from direct isolation/fragmentation (right side, not parked). Figure 7: Distributions of backbone cleavages as percentages, specifically N-terminal to proline (blue), C-terminal to phenylalanine (red), C-terminal to glutamic and aspartic acids (green), and all other backbone cleavages (purple) for UVPD and HCD. A) ubiquitin 4+, 8+, and 12+, B) myoglobin 8+, 12+, and 20+, and C) carbonic anhydrase 14+, 22+, 28+.



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Figure 1: A) Isolation of 10+ charge state ubiquitin followed by PTR (200 ms) and ion parking at charge states 7+, 6+, 5+, and 4+ shown at the same scale. B) Isolation of myoglobin (16+) followed by PTR (150 ms) and ion parking at charge states 14+, 12+, 10+, 9+, 8+, 7+, and 6+, all shown on the same abundance scale.


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Figure 2: PTR (200 ms) of ubiquitin (10+) and HCD of the resulting parked charge states A) 4+, B) 6+, and C) 8+. HCD of charge states D) 10+ (not parked) and E) 12+ (not parked). HCD NCE values used were 33, 30, 30, 23, and 18, respectively. In D and E, the abundance of the backbone cleavage between E and P is off-scale, and thus the true percentages are listed.



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Figure 3: PTR (200 ms) of ubiquitin (10+) and UVPD of the resulting parked charge states A) 4+, B) 6+, and C) 8+. UVPD of charge states D) 10+ (not parked) and E) 12+ (not parked). One 3.5 ∗

mJ pulse was used for UVPD. ( Enhanced cleavages adjacent to Phe-45 and Tyr-59.) In E, the abundance of the backbone cleavage between E and P is off-scale, and thus the true percentages are listed.


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Figure 4: PTR (200 ms) of myoglobin (16+) and HCD of charge states A) 8+ and B) 12+, and C) HCD of charge state 20+ (not parked). HCD NCE values used were 33, 30, and 21, respectively.



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Figure 5: PTR (200 ms) of myoglobin (16+) and UVPD of charge states A) 8+ and B) 12+, and C) ∗

UVPD of 20+ (not parked). One 2.5 mJ pulse was used for UVPD. ( Enhanced cleavages adjacent to phenylalanine “F” residues).


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Figure 6: Sequence coverages (upper) and subsequent number of identified fragment ions (lower) for A) ubiquitin (4+, 6+, 8+, 10+ and 12+), B) myoglobin (6+, 7+, 8+, 10+, 12+, 16+ and 20+), and C) carbonic anhydrase (10+, 14+, 18+, 22+, 26+, and 28+). A dotted line differentiates PTR/ion parking fragmentation (left side, parked) from direct isolation/fragmentation (right side, not parked).



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Figure 7: Distributions of backbone cleavages as percentages, specifically N-terminal to proline (blue), C-terminal to phenylalanine (red), C-terminal to glutamic and aspartic acids (green), and all other backbone cleavages (purple) for UVPD and HCD. A) ubiquitin 4+, 8+, and 12+, B) myoglobin 8+, 12+, and 20+, and C) carbonic anhydrase 14+, 22+, 28+.


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