(FIP) during Ultraviolet Photodissociation (UVPD)

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Implementation of Fragment Ion Protection (FIP) during Ultraviolet Photodissociation (UVPD) Mass Spectrometry Dustin D Holden, James D Sanders, Chad R. Weisbrod, Christopher Mullen, Jae C Schwartz, and Jennifer S. Brodbelt Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01723 • Publication Date (Web): 21 Jun 2018 Downloaded from http://pubs.acs.org on June 25, 2018

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Analytical Chemistry

Implementation of Fragment Ion Protection (FIP) during Ultraviolet Photodissociation (UVPD) Mass Spectrometry

Dustin D. Holden,1 James D. Sanders,1 Chad. R. Weisbrod,2 Christopher Mullen,2 Jae. C. Schwartz,2 Jennifer S. Brodbelt1* 1

Department of Chemistry, University of Texas at Austin, Austin, TX 78712

2

Thermo Fisher Scientific Inc., 355 River Oaks Pkwy, San Jose, CA 95134 *

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

Abstract Ultraviolet photodissociation (UVPD) is a non-selective activation method in which both precursor and fragment ions may absorb photons and dissociate. Photoactivation of fragment ions may result in secondary or multiple generations of dissociation which decreases the signalto-noise ratio (S/N) of larger fragment ions owing to the prevalent sub-division of the ion current into many smaller, often less informative fragment ions. Here we report the use of dipolar excitation waveforms to displace fragment ions out of the laser beam path, thus alleviating the extent of secondary dissociation during 193 nm UVPD. This fragment ion protection (FIP) strategy increases S/N of larger fragment ions and improves the sequence coverage obtained for proteins via retaining information deeper into the mid-section of protein sequences.

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Introduction Ultraviolet photodissociation (UVPD) has become a versatile activation method for characterization of small molecules, nucleic acids, lipids, peptides and proteins, as well as larger protein complexes.1–35 Amide bonds along the backbone of peptides and proteins act as chromophores which efficiently absorb 193 nm photons. This process allows access to excited electronic states and leads to a variety of fragment ions types.36 There are six dominant fragment ion types (a,b,c,x,y,z) produced upon 193 nm UVPD, some of which also occur in conjunction with hydrogen atom migration. The richness of the spectra arising from UVPD is one of its wellknown hallmarks and is a feature that has contributed to its success for localizing modifications1,14,18,23,27 and differentiating isomers.6,17 At the same time, the ion signal is extensively divided into many channels which diminishes product ion signal-to-noise. This factor is more significant for UVPD than collisional or electron activation methods, the latter which typically generate only two or three prominent types of product ions. In addition, fragment ions themselves may absorb photons and dissociate, a process termed secondary or consecutive dissociation and which may include multiple generations of fragmentation. Infrared multiphoton dissociation (IRMPD) has likewise been observed to cause secondary dissociation.37,38 Intact proteins contain an amide linkage per amino acid residue (N-1 amide bonds per protein/peptide where N is the number of residues) along the backbone which absorb in the deep UV range. UVPD at 193 nm exploits these chromophores and has proven to be a particularly compelling activation method for analysis of intact proteins20 and has been successfully integrated into high throughput top-down proteomic workflows.21,26 Top-down protein analysis poses special challenges associated with the large number of backbone cleavage sites and the range of potential charge states of the resulting fragment ions. In essence, as the number of


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fragmentation pathways increases, the signal-to-noise ratio of fragment ions decreases due to dispersion of the ion current into a variety of available product ion channels as recently underscored by Riley et al.39 Furthermore, the inverse relationship between protein size and the signal-to-noise ratio (S/N) of product ions, a relationship derived from the fact that the storage capacity of ion traps is defined by the total number of charges, not the number of ions,39 further exacerbates the S/N issue. UVPD is most often performed in ion trapping devices by exposing selected precursor ions to one or more laser pulses that align with the ion cloud. For analysis of intact proteins, the number of laser pulses or laser power is often restricted in order to limit the formation of difficult-to-assign internal, or extremely small, fragment ions, both of which are outcomes of secondary dissociation. Obtaining a balance between effective production of informative primary fragments and minimizing secondary dissociation often means that a substantial portion of the precursor ions may not be activated nor transformed into diagnostic product ions. To alleviate secondary dissociation during photodissociation, several innovative strategies have been reported. An early study by the McLafferty group proposed using stored waveform inverse Fourier transform (SWIFT) excitation of fragment ions to minimize secondary dissociation by moving them away from the path of an IR laser beam during FTICR experiments.37 Utilizing a quadrupole ion trap mass spectrometer, the Glish group reported an “axial expansion” method in which fragment ions were moved outside of the beam path of an IR laser by application of appropriate broadband waveforms.40 The Glish group also developed a method to resonantly excite precursor ions into a laser beam that was offset from the center of a quadrupole ion trap while accumulating product ions in the center region.41 These methods, all of which were proposed or implemented for infrared multiphoton dissociation (IRMPD) applications,



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demonstrated the ability to reduce the secondary photodissociation of fragment ions into smaller ions, allowing preservation of the larger and more informative fragment ions and at the same time permitting more efficient transformation of precursor ions into product ions. Here we apply dipolar excitation waveforms during UVPD to reduce the overlap of the spatial distribution of fragment ions with the laser beam, thus protecting them from secondary dissociation while optimizing the conversion of precursor ions into product ions.42 Exciting ions in a mass-selective way to minimize their reactions with other ions is a concept that was originally termed “ion parking”43 and also proves to be a key strategy in the present work. We demonstrate the performance gains obtained for UVPD of intact proteins performed in the dual-pressure linear ion trap and analyzed in an Orbitrap mass spectrometer for top-down proteomics. We refer to this method as fragment ion protection (FIP). Experimental Section Materials Bovine ubiquitin, equine myoglobin and cytochrome c were purchased from SigmaAldrich (St. Louis, MO). Solvents were purchased from Thermo Fisher Scientific (Pittsburgh, PA). All samples were diluted to 2 µM in 70:29.9:0.1 (v/v/v) methanol/water/formic acid and infused using gold-coated borosilicate emitters with an applied voltage ranging from 0.9 – 1.2 kV. Mass Spectrometry A Thermo Fisher Scientific Fusion Lumos Tribrid mass spectrometer (San Jose, CA) modified to allow UVPD via integration with a 193 nm ArF excimer laser (500 Hz Excistar, Coherent, Santa Clara, CA) in a manner described previously5 was used in the present study. Visualization of the main RF DAC, auxiliary waveforms, and laser pulse triggers was


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accomplished using a Tektronix MSO 2014B oscilloscope (Beaverton, OR). UVPD-FIP was performed by utilizing the built-in auxiliary RF power supply and waveform generator to generate broadband waveforms to resonantly excite, but not eject, fragment ions, without exciting the precursor ions in a manner analogous to conventional ion trap ion isolation. Custom instrument control code was written to dictate the precise timing, amplitude, slope, and notch size of the waveform output from the auxiliary RF power supply, as well as to allow precise control of the laser pulse triggering. To attenuate the laser beam diameter, a Thorlabs ringactuated iris diaphragm was mounted to a Thorlabs XY translator for precise placement (Newton, NJ). An automatic gain control (AGC) target of 5 x 105 charges was used. Spectra consisting of 6 µscans were collected at an Orbitrap resolving power of 120,000 at m/z 200, unless specified otherwise, to mimic the data acquisition parameters typically used for high throughput LCMS applications in top-down proteomics.26,30 The Lumos mass spectrometer was set to Intact Protein Mode with an ion routing multipole (IRM) pressure of 3 mTorr. Data Analysis Spectra were deconvoluted using the Xtract algorithm (Thermo Fisher Scientific) with a S/N threshold of 3 unless otherwise specified. For generation of histograms of fragment percentages, deconvoluted spectra were interpreted manually using ProteinProspector v5.20.0 (http://prospector.ucsf.edu) with an error tolerance of 10 ppm relative to calculated masses of fragment ion types a, a+1, b, c, x, x+1, y, y-1, and z. Sequence coverage plots, maps and fragment mass histograms were generated using ProSight Lite v1.344 in UVPD mode with a 10 ppm error tolerance. The changes in relative abundances of specific fragment ions as a result of FIP were calculated based on the total abundance of each fragment ion relative to the abundance of the residual precursor ion (e.g., fragmentation efficiency for production of large-size fragment



ions:

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Σ(abundances of large-size fragment ions)/(Σ(abundances of large-size fragment ions +

residual precursor)). Signal to noise values were calculated using the following values obtained from Xcalibur: (peak height - baseline)/(noise - baseline). Raw ion signal values were calculated also from Xcalibur values: (accumulation time x peak height). Results and Discussion UVPD is an effective method for generating rich protein fragmentation spectra, yet the simultaneous photoactivation of intact proteins and primary fragment ions contributes to the formation of unassigned secondary ions, including internal ions (ones containing neither the Nterminal or C-terminal residue which are typically key anchor points for identification of fragment ions) and uninformative small fragment ions. The array of secondary processes also contribute to a reduction in the signal-to-noise of UVPD mass spectra owing to the dispersion of ion current among many fragmentation channels. One general way to reduce the extent of secondary fragmentation is to minimize the overlap of the spatial distribution of primary fragment ions with the laser beam. Previous studies have proposed or shown the utility of applying auxiliary waveforms to modulate the trajectories of ions into41 and away from37,40 an infrared laser beam, thus increasing or decreasing ion overlap with the laser beam. These strategies were used to modulate the dissociation of fragment ions, and our approach draws on this previous founding work.37,40,41 Three proteins, ubiquitin, myoglobin, and cytochrome c, were used to develop the FIP strategy and evaluate its performance metrics. UVPD is performed in the low pressure trap (LPT) of a dual-pressure linear ion trap located at the back end of the Orbitrap mass spectrometer. The critical concept in protecting primary fragment ions from UV irradiation is to minimize their overlap with the laser beam, meaning there must be a region in the linear ion trap


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that does not overlap with the laser path. To help achieve this requirement, an iris was used to attenuate the laser beam diameter as shown in Figure 1A. The optimal iris aperture size was determined by performing conventional UVPD in the LPT (Figure 1B) without the application of auxiliary waveforms, while reducing the iris aperture to the point just prior to reducing the rate of precursor photodissociation. In this way, the optimal aperture diameter was determined to be ~0.9 mm, a size that minimized degradation of UVPD by attenuation of the laser beam. Fragment ions are protected from UVPD during application of the waveforms (Figure 1C). A key aspect of the strategy described herein is the ability to move ions away from the laser beam without causing ion ejection or collisional activated dissociation (CAD), two other processes that are also promoted by application of waveforms. As a first proof-of-principle, a resonant waveform was applied to a selected precursor ion (angiotensin I, 3+) during UVPD to evaluate the excitation amplitude that could be applied without causing ejection or CAD (Figure S1). At very low excitation amplitudes (0 – 7 normalized collision energy (NCE)), the selected precursor ion undergoes UVPD as normal. At very high excitation amplitudes (16 – 20 NCE), the precursor is converted to b/y sequence ion akin to conventional CAD. At intermediate excitation amplitudes (8-15 NCE), the precursor ion undergoes neither UVPD nor CAD. This range is considered the range of protection, and it convincingly demonstrates the ability to move the ions away from the laser beam path in a manner that neither induces CAD nor causes ejection of the precursors from the trap. Since this initial proof-of-principle was focused on exciting precursor ions to observe protection versus undesired CAD, further optimization was required to optimize a strategy for resonantly exciting and protecting fragment ions, not precursor ions, from successive laser pulses. Resonantly exciting fragment ions requires the use of a broadband waveform with multiple frequency components. A schematic of the waveform and additional



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details are shown in Figure S2. The waveform applied during UVPD-FIP had a repeat frequency of 500 Hz and was composed of frequency components spaced by 500 Hz ranging from 5 kHz to 600 kHz. A flat magnitude versus frequency profile (Figure S2B) was employed, but further refinement of the magnitude profile is an area of ongoing research. The timing of UVPD-FIP relative to the typical ion trap analytical scan sequence is shown in Figure 1D with the main RF DAC trace shown in yellow, auxiliary RF waveforms applied to the LPT in blue, and the laser triggers in purple. The pre-scan sequence is followed by the ion accumulation period (with accumulation waveforms applied) and ion trap isolation step, which is evidenced by the maximum applied auxiliary RF waveform amplitude in blue. Following the isolation step is the activation step during which the laser is triggered one or more times, and finally the ions may be mass analyzed in the ion trap, as exhibited by the ramp in the main RF DAC (yellow trace), or sent to the c-trap for high resolution Orbitrap analysis (not shown). Illustrated in Figure 1E is an expansion of the activation period for UVPD-FIP. In this example, the laser is triggered ten times (purple trace). The FIP process entails resonance excitation of fragment ions during the UVPD period through application of a low amplitude auxiliary waveform, repeating at 500 Hz, 2 ms period (Figure 1E, blue trace). Other than a short 0.21 ms period to initiate the waveform and 0.18 ms to stop the waveform (0.39 ms total), UVPD-FIP does not require any additional time during a scan sequence because it is applied simultaneously during the laser triggering period. The concept of fragment ion protection is demonstrated in Figure S3 for UVPD of myoglobin (19+). Figure S3A shows a cascade of mass spectra with increasing number of laser pulses without fragment ion protection enabled. Precursor ions are converted into product ions as evidenced by the broad distribution of ion current around the selected precursor ion.


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Moreover, the dispersion of the ion current density across the m/z range increases with the number of laser pulses, indicating the conversion of product ions into successively smaller m/z product ions. As these product ions undergo multiple generations of UVPD, the information content is reduced as more internal fragments are generated. The similar photoabsorption crosssections of precursor and product ions means that complete conversion of the precursor ion population into diagnostic fragment ions is possible only by sacrificing the observation of the early generation product ions. Figure S3B displays a similar cascade of mass spectra with FIP enabled during UVPD. Early generations of product ions survive exposure to multiple UV pulses as shown by the sustained concentration of ion current density in the m/z region around the selected precursor. Additional evidence of the striking impact of fragment ion protection is the observation of the “notch” in the series of spectra in Figure S3B which demonstrates nearly complete conversion of the precursor population into product ions. While no fragment ion assignments have been made in these spectra, the differences in the distribution of ion current and the relative dispersity of the resulting m/z distributions between the FIP-enabled and FIPdisabled spectral cascades illustrate the ability to dramatically reduce the probability of additional generations of photodissociation. Using appropriate experimental parameters, in theory 100% conversion of precursor ions into product ions should be possible while simultaneously arresting successive product ion dissociation and formation of internal fragment ions. A main focus of the present study is the assessment of the improvement in S/N of UVPD generated fragment ions from intact protein analytes, for which spectral averaging is inherently limited. For example, during high throughput LCMS runs, typically few spectral acquisitions are averaged in order to allow maximum sampling of eluting proteins, and this type of situation was



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mimicked in our study. The LPT (~4 x 10-4 Torr)45 is an ideal location to resonantly excite fragment ions formed during UVPD to reduce their overlap with the laser beam, while at the same time minimizing collisional activation that might otherwise occur in the higher pressure ion trap (HPT). To determine the maximum auxiliary RF waveform amplitude suitable for exciting the ions outside of the path of the laser beam while limiting collision activated dissociation and ion ejection, the auxiliary RF amplitude and activation period were varied iteratively, and the abundance of ubiquitin ions (12+, m/z 714) was monitored (Figure S4). To monitor effects from applying excitation waveforms, separate from photodissociation, the laser was turned off during this assessment. The impact on ion losses in the LPT was monitored as this set of parameters (activation time, amplitude of applied waveform, trapping q value) was varied. An activation time up to 50 ms represents an extreme case of up to 25 laser pulses (if the laser was on), a period during which ions would also experience resonance excitation and potentially experience slow-heating collisional activation effects from waveform resonance excitation. As summarized graphically in Figure S4, minimal loss of ions via ejection or fragmentation occurred when the waveform amplitude was held between 0.7 and 0.75 Vpp over a wide range of ion trapping parameters (q 0.1 – 0.8). Following this assessment, an amplitude of 0.75 Vpp was used for the remainder of the study, thus allowing ions to be moved away from the laser beam while simultaneously minimizing unwanted collisional activation of resonantly excited ions. It is possible to cause minimal off-resonance excitation of the precursor ions if the UVPD-FIP auxiliary waveform notch width (analogous to the isolation width) used for FIP is too narrow, a factor which would reduce conversion of precursor ions into UVPD generated fragment ions. Therefore, a conservatively wide notch width of 35 m/z was used to prevent precursor ions from being moved outside of the laser beam path.


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Comparisons of the level of sequence coverage, number of identified fragment ions, sequence coverage maps, signal-to-noise levels, types of fragment ions (a,b,c,x,y,z), and sizes of fragment ions were undertaken using UVPD with and without FIP. Low laser powers (0.25 mJ and 1.0 mJ) and a relatively high number of laser pulses (eight) were used for these experiments in order to maximize secondary dissociation and explore the impact of FIP. The results are illustrated in Figures 2 to 6 and S4 to S8. Results for ubiquitin (MW 8.6 kDa), a benchmark protein used in nearly every top-down MS study, are featured in Figures 2-5. Shown in Figure 2A are the UVPD (top) and UVPD-FIP (bottom) mass spectra and the resulting sequence maps for ubiquitin (12+).

The main

differences are the observation of fewer fragment ions in the lower m/z range and new fragment ions in the mid m/z range upon application of FIP, an outcome directly reflecting the alleviation of extensive secondary dissociation and enhanced survival of larger fragment ions during UVPD. The level of sequence coverage and number of fragment ions identified for ubiquitin (12+) using UVPD and UVPD-FIP for two pulse energies (0.25 mJ/pulse and 1.0 mJ/pulse are shown in Figure 2B and 2C. A maximum sequence coverage of 71% was obtained for UVPD-FIP (using 0.25 mJ per pulse) compared to 55% without FIP (Figure 2B). Sequence coverage increased with the number of laser pulses for both UVPD and UVPD-FIP (using 0.25 mJ per pulse), reflecting the identification of a greater number of fragment ions (Figure 2B). The maximum number of fragment ions identified was 71 for UVPD with FIP compared to 61 for UVPD alone (Figure 2C). The impact of FIP on the signal-to-noise levels for four key fragment ions observed upon UVPD of ubiquitin (12+) is illustrated in more detail in Figure 3. Among the large array of fragment ions produced upon UVPD, four of the larger fragments: including y5810+ (m/z 654,



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6527 Da), z639+ (m/z 788, 7083 Da), a7010+ (m/z 787, 7861 Da), and a7110+ (m/z 798, 7974 Da), were examined because these large fragment ions are likely to undergo secondary UVPD and produce smaller fragment ions. Shown in Figure 3A is the sequence map of ubiquitin with y5810+, z639+, a7010+, a7110+ ions highlighted in blue, red, and green boxes, respectively, and the corresponding ion profiles annotated with the S/N levels of each of the four fragment ions without and with FIP. The theoretical isotopic distribution of one of the ions (a7110+) is shown for comparison in Figure S5 as an example of an idealized isotopic profile in the absence of isobaric interferences and noise peaks. The increases in both S/N and raw ion signal (counts) upon application of FIP are evident for all four fragment ions, and this outcome is shown quantitatively in Figure 3B as the factor increase in S/N and raw ion signal. The factor gain for both S/N and raw ion signal ranges from 1.5 for the z63 ion to 3.5 for the a71 ion.

The

fragmentation efficiency for production of the four representative large fragment ions increased from 11% to 23% (Figure 3C), an approximate doubling of the ion current owing to FIP. These results underscore the protection of the larger fragment ions from exposure to the laser owing to strategic deployment of FIP, thus suppressing secondary dissociation. A more global examination of the variations in fragmentation of ubiquitin from deployment of the FIP strategy is shown in Figure 4. Figure 4A illustrates a histogram of the change in fragmentation along the protein backbone based on the compilation of all N-terminal (a,b,c) and C-terminal (x,y,z) fragment ions. For this display, the fragment abundances were normalized based on the percentage of total ion current for all identified fragment ions, and the values obtained for UVPD without and with FIP are shown as a difference plot. Bars above the zero line represent the enhancement of fragment ions for UVPD-FIP; bars below the zero line indicate greater abundances for UVPD without FIP. Across the board, abundances of larger


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fragment ions are increased for UVPD-FIP, along with significant suppression of very small fragments containing only a few amino acids (i.e. negative blue bars at the N-terminus) and Nterminal fragments arising from cleavages at proline (P19) or glutamic acid (i.e. notable negative blue bars at residues E16, E18). FIP also modulated the distribution of two key types of fragment ions (a and b) for UVPD, with relatively little impact on the other fragment ion types (c,x,y,z). As shown in Figure 4B and 4C, the total percentage and number of a ions increased substantially for UVPD-FIP. Interestingly, although the percentage of ion current attributed to b ions decreased significantly for UVPD-FIP relative to UVPD without FIP, the total number of different b ions dropped by a more modest amount. The reason for the change in a/b ion abundances and distributions is not known but may relate to their stabilities or mechanisms for formation by UVPD (whether produced directly from ions in excited electronic states or after intramolecular vibrational energy re-distribution or via consecutive pathways). The distributions of sizes of fragment ions produced upon UVPD with and without FIP of ubiquitin (12+) are displayed in Figure 5. All fragment ions were deconvoluted and grouped in 400 Da bins. The histogram format of Figure 5 reveals the shift to higher mass fragments upon application of FIP, and this result is underscored by the trend-lines which track the cumulative percentage of fragment ions going from low to high mass. The later rise of the trend-line for UVPD-FIP shows the shift in the distribution of fragment ions to larger sizes. For UVPD without FIP, only 10% of the identified fragments had masses greater than 5500 Da, whereas 30% of the identified fragments exceeded 5500 Da for UVPD-FIP. This result illustrates the successful application of FIP for curbing secondary dissociation, thus protecting larger primary fragment ions from decaying into smaller fragment ions.



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The detailed assessment of FIP described for ubiquitin was extended to two other proteins, cytochrome c (12.4 kDa) and apo-myoglobin (MW 16.9 kDa). Examples of UVPD mass spectra and sequence maps obtained with and without FIP are provided in Figure S6 for apo-myoglobin (21+), along with the variations in sequence coverage and number of identified fragments using up to ten 0.25 mJ laser pulses or up to six 1.0 mJ laser pulses. Similar to the results observed for ubiquitin, the number of fragment ions identified and the sequence coverage increase upon application of FIP. For myoglobin, six 0.25 mJ pulses provided higher sequence coverage and a greater number of fragment ion identifications compared to using multiple 1.0 mJ pulses for both UVPD and UVPD-FIP. Figure 6 displays histograms of the change in fragmentation along the backbone of myoglobin based on comparisons of the abundances of all N-terminal and C-terminal fragment ions for UVPD versus UVPD-FIP using eight 0.25 mJ pulses (Figure 6A) and UVPD-FIP using six 0.25 mJ pulses versus UVPD-FIP using two 1.0 mJ pulses (Figure 6B). The variations in fragmentation are again shown in a difference format similar to Figure 4A to showcase the increases and decreases in backbone fragmentation for each type of UVPD mode. Bars above or below the zero line represent the enhancement or inhibition of fragment ions, respectively. For Figure 6A, the abundances of a number of midand large-size fragment ions are increased for UVPD-FIP (positive bars), along with decreased abundances of a few smaller fragment ions (negative bars). The pattern of changes in the backbone fragmentation varied somewhat more dramatically upon comparison of UVPD-FIP using six low energy (0.25 mJ) pulses versus two higher (1.0 mJ) energy pulses (Figure 6B). Some of the small fragment ions were suppressed using 0.25 mJ pulses (e.g., negative bars near the N-terminus and C-terminus), and at the same time a substantial number of the mid-sized fragments covering the mid-section of the myoglobin


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sequence were significantly enhanced (e.g., positive bars corresponding to backbone cleavages between L32 and L104). More detailed analysis of the sizes of the fragment ions reveals that nearly 19% of the identified fragment ions for low energy (0.25 mJ) UVPD-FIP had masses greater than 7,000 Da, whereas only 2% of the fragment ions for higher energy (1.0 mJ) UVPDFIP had masses greater than 7,000 Da (Figure S7). The differences seen in Figures 6B and S7 demonstrate that a series of low fluence pulses is not equivalent to fewer pulses of higher fluence, a result that may reflect variations in the propensity for fragmentation directly from excited electronic states versus fragmentation after internal conversion and intramolecular vibrational energy re-distribution. The probabilities of multiphoton absorption events also differ for the low fluence versus higher fluence conditions. The performance of UVPD-FIP was also examined for cytochrome c (MW 12.4 kDa, 16+). Examples of the UVPD-FIP mass spectra and sequence coverage maps obtained using eight low energy (0.25 mJ) pulses or two higher energy (1.0 mJ) pulses are shown in Figure S8, along with the sequence coverages and number of identified fragment ions as the number of laser pulses was varied. Interestingly, both low and high energy UVPD-FIP returned similar sequence coverages and number of fragment ion identifications. However, in many cases the fragment ions and backbone cleavage sites that contribute to the absolute sequence coverages differ. For example, inspection of the UVPD mass spectra (Figure S8C,D) reveals lower abundances of fragment ions in the low m/z region and a lower degree of the loss of heme for low energy UVPD-FIP (Figure S8D). The abundance of the precursor is reduced 30% more using two higher energy pulses than using six low energy pulses. In addition, using eight low energy pulses caused an even greater reduction of the precursor ion (70%) (data not shown). Examination of the differences in the abundances of product ions (Figure S9A) indicated a variety of changes in



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the fragmentation across the backbone, thus illustrating the impact of the laser energy in conjunction with FIP. Deconvolution and binning of the fragment ions based on mass reveals the presence of several higher mass products observed for low energy (0.25 mJ) UVPD-FIP not detected for higher energy (1.0 mJ) UVPD-FIP (Figure S9B). Improvements in deconvolution programs are anticipated to have a notable impact on the ability to assign larger fragment ions possessing more complicated isotope patterns. Conclusion Resonant excitation was implemented as a means to protect fragment ions from exposure to UV laser pulses, favoring the observation of primary dissociation products. The application of FIP resulted in an increase in the signal-to-noise of higher mass fragment ions and an overall increase in the size of fragment ions compared to conventional UVPD. Using lower energy pulses rather than higher energy laser pulses during UVPD-FIP further enhanced the preservation of larger fragment ions. Absolute sequence coverages increased modestly using FIP or were similar for UVPD and UVPD-FIP; however, even in cases for which the total sequence coverages were similar, there were different backbone cleavage sites for UVPD versus UVPDFIP owing to the shift to larger fragment ions mapping the mid-section of proteins during FIP. Deployment of FIP via application of resonance excitation waveforms during the UVPD period added a negligible period to the scan sequence and caused no detectable CAD or ion losses over a broad range of ion trapping parameters, thus making it a practical strategy to consider for LCMS applications. For LCMS strategies, the number of laser pulses and pulse energy used for UVPD could be adjusted independently of the FIP for optimal performance for the type of analytes of interest.


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Acknowledgments We acknowledge the following funding sources: NSF (Grant CHE1402753) and the Welch Foundation (Grant F-1155). We thank John E.P. Syka for fruitful advice and assistance. SUPPORTING INFORMATION Supporting information includes a graphical proof-of-principle illustrating the application of a resonant excitation waveform to move an ion out of the path of the laser beam during UVPD and its impact on production of CAD and UVPD fragment ions; a schematic of a representative waveform applied during UVPD-FIP; ion profiles produced from UVPD of myoglobin with and without FIP; graphical display of the impact of activation time and waveform amplitude to cause loss of 15% of precursor ions; theoretical isotope distribution of a71 ion from UVPD of ubiquitin; graphical displays of sequence coverage and number of fragment ions generated from UVPD of myoglobin as a function of the number of laser pulses with and without FIP; distribution of deconvolved fragment ions from UVPD of myoglobin based on mass bins; and graphical displays of sequence coverage and number of fragment ions generated from UVPD of cytochrome c as a function of the number of laser pulses with and without FIP.

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Figure 1: A) Schematic of the set-up for UVPD in the low pressure linear ion trap, with a depiction of the laser beam intersecting the ion cloud, and B) conventional UVPD (B) versus UVPD-FIP (C) showing the intersection of multiple laser pulses with the fragment ions or displacement of the fragment ions, and D) oscilloscope traces during operation of main RF DAC (yellow), auxiliary RF waveforms (blue), and laser trigger (purple). E) Expansion of FIP auxiliary RF waveforms and laser main RF DAC (yellow), auxiliary RF waveforms (blue), and laser trigger (purple).


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Figure 2: UVPD of ubiquitin (12+): A) UVPD mass spectra and sequence maps without and with FIP, and B) sequence coverage, and C) number of fragment ions identified using 0.25 mJ/pulse (red) or 1 mJ/pulse (blue) upon application of 1 to 10 laser pulses (with solid lines for UVPD and dashed lines for UVPD-FIP). Error bars represent standard deviation of triplicate results. Average sequence coverage standard deviations were 3.4% and 3.0% for 0.25 mJ UVPD and UVPDFIP respectively, and 3.2% and 5.2% for 1 mJ UVPD and UVPDFIP respectively. Average identified fragment ion standard deviations were 4.3% and 3.8% for 0.25 mJ UVPD and UVPDFIP respectively, and 1.9% and 5.6% for 1 mJ UVPD and UVPDFIP respectively.



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Figure 3: UVPD of ubiquitin (12+) using eight 0.25 mJ pulses without or with FIP: A) Sequence coverage map and expansions of ion profiles and signal-to-noise levels for four large fragment ions, including y5810+ (m/z 654), a7010+ (m/z 787), z639+ (m/z 788), and a7110+ (m/z 798). The theoretical isotope distribution for one of the ions (a7110+) is shown in Figure S4. B) Histogram showing the factor of increase in S/N using UVPDFIP for y5810+, a7010+, z639+, and a7110+ ions. C) Histogram showing the efficiency of production of large fragment ions y5810+, a7010+, z639+, and a7110+. Results represent the average of three spectra (each consisting of 6 µscans) generated by Thermo Xcalibur Qual Browser v2.2.


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Figure 4: UVPD of ubiquitin (12+) using eight 0.25 mJ pulses with and without FIP. A) Histogram showing differences in fragment ion abundances between UVPDFIP and UVPD as a function of backbone cleavage site where a,b,c ions are grouped as N-terminal fragment ions and x,y,z ions are grouped as C-terminal fragment ions. The bars above the zero axis indicate ions with enhanced abundances for UVPD-FIP. B) Relative percentages of summed fragment ion types for UVPD versus UVPDFIP. C) Number of each fragment ion type for UVPD versus UVPDFIP. Ion types a were summed with a+1, x summed with x+1, and y summed with y-1. Error bars represent standard deviations of triplicate results.



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Figure 5: Distribution of deconvoluted fragment ions in 400 Da mass bins for UVPD and UVPD-FIP obtained using eight 0.25 mJ laser pulses for ubiquitin (12+). The trend-lines show the cumulative percentage of fragment ions from low to high mass. Results represent the average of triplicate experiments.


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Figure 6: UVPD of apo-myoglobin (21+). Histograms show differences in fragment ion abundances as a function of backbone cleavage site. A) UVPD versus UVPD-FIP using eight 0.25 mJ pulses. The bars above the zero axis indicate ions with enhanced abundances for UVPD-FIP. B) UVPD-FIP using six 0.25 mJ pulses versus two 1.0 mJ pulses. The bars above the zero axis indicate ions with enhanced abundances using six 0.25 mJ pulses. All a,b,c ions are grouped as N-terminal fragment ions, and all x,y,z ions are grouped as C-terminal fragment ions Results represent the average of triplicate experiments.



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ACS Paragon Plus Environment

(FIP) during Ultraviolet Photodissociation (UVPD)

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