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Improving Performance Metrics of Ultraviolet Photodissociation Mass Spectrometry by Selective Precursor Ejection Dustin D. Holden, and Jennifer S. Brodbelt Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03777 • Publication Date (Web): 06 Dec 2016 Downloaded from http://pubs.acs.org on December 7, 2016

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Analytical Chemistry

Improving Performance Metrics of Ultraviolet Photodissociation Mass Spectrometry by Selective Precursor Ejection Dustin D. Holden, Jennifer S. Brodbelt* Department of Chemistry, University of Texas at Austin, Austin, TX 78712 *

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

Abstract Confident protein identifications derived from high throughput bottom-up and top-down proteomics workflows depend on acquisition of thousands of MS/MS spectra with adequate signal-to-noise

and

accurate

mass

assignments

of

the

fragment

ions.

Ultraviolet

photodissociation (UVPD) using 193 nm photons has proven to be well-suited for activation and fragmentation of peptides and proteins in ion trap mass spectrometers, but the spectral signal-tonoise ratio (S/N) is typically lower than that obtained from collisional activation methods. The lower S/N is attributed to the dispersion of ion current among numerous fragment ion channels (a,b,c,x,y,z ions). In addition, frequently UVPD is performed such that a relatively large population of precursor ions remains un-dissociated after the UV photoactivation period in order to prevent over-dissociation into small uninformative, or internal fragment ions. Here we report a method to improve spectral S/N and increase the accuracy of mass assignments of UVPD mass spectra via resonance ejection of un-dissociated precursor ions after photoactivation. This strategy, termed precursor ejection UVPD or PE-UVPD, allows the ion trap to be filled with more ions prior to UVPD while at the same time alleviating the space charge problems that would otherwise contribute to the skewing of mass assignments and reduction of S/N. Here we report the performance gains by implementation of PE-UVPD for peptide analysis in an ion trap mass spectrometer.

1 ACS Paragon Plus Environment


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Introduction Ultraviolet photodissociation (UVPD) is a versatile ion activation method suitable for many classes of molecules, ranging from small molecules such as synthetic organic molecules, peptides, lipids, oligosaccharides, nucleic acids,1–15 to large biological molecules16–21 to even large protein complexes.22–26 UVPD has also proven to be a powerful MS/MS method for high throughput LCMS applications such as both bottom-up27–29 and top-down proteomics.30,31 Moreover, UVPD has been strategically employed for more specialized applications that have used specific wavelengths (such as 266 nm or 351 nm or 355 nm) to cleave specific bonds32–39 or to selectively target certain classes of molecules based on incorporation of chromophores matched to the wavelength of irradiation.40–45 UVPD using 193 photons is particularly effective at generating rich fragmentation patterns of peptides and proteins and distributing fragment ions across a wide m/z range. For many applications of UVPD, there is a trade-off between overall fragmentation efficiency and the optimal production of meaningful fragment ions. Very high photon fluxes typically result in conversion of more precursor ions into fragment ions but at the same time generate smaller product ions and more internal fragment ions that are difficult to assign. Both of these less desirable types of product ions typically originate from the secondary dissociation of primary fragment ions. Other activation methods such as electron transfer dissociation (ETD) and electron capture dissociation (ECD), which involve ion-ion or ionelectron reactions, also may be prone to loss of informative fragment ions via consecutive electron attachment reactions.46 For UVPD the generation of less informative product ions can be mitigated by using lower photon fluxes, thus leading to the tradeoff mentioned above: a reduction in overall fragmentation efficiency and lower S/N levels for UVPD compared to other conventional ion activation methods.


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As UVPD is applied to larger, more complex, and generally more highly charged ions, the signal-to-noise ratio (S/N) of fragment ion peaks becomes an increasingly important concern, especially considering the dispersion of ion current among numerous fragmentation channels and isotopic compositions. This issue is particularly confounding for trapping instruments which have finite capacity that is typically defined by total charge capacity rather than absolute ion capacity. Riley et al. described in detail the relationship between the number of charges trapped and the resulting signal-to-noise of the spectrum.47 S/N was found to be proportional to the number of charges, above a thermal noise threshold.47 Additionally, since the number of charges, as opposed to the absolute number of ions (which may have multiple charges), are used to fulfill target ion populations in commercial ion trapping mass spectrometers, a general rule was postulated that S/N is inversely related to the square root of molecular weight.47 In general this means that spectra acquired for more highly charged ions of larger molecular weight exhibit lower S/N compared to spectra obtained for correspondingly smaller, lower charged ions, even for cases in which similar number of charges are trapped. This is a key concept to consider in the context of development of strategies to improve the performance of UVPD for the most challenging applications like proteomics. Moreover, some of the commonly used proteomics database search algorithms, use S/N metrics to evaluate confidence of peptide or protein identifications.48,49 In sum, improving the S/N ratio of UVPD mass spectra is important for increasing overall sequence coverage and search score values in proteomics applications. Just as with other activation methods performed in ion trapping instruments, increasing the S/N for UVPD may be undertaken by increasing the number of charges accumulated prior to ion activation. However, this strategy is ultimately restricted by the spectral space charge limit50 during mass analysis. During mass analysis, space charge causes mass shifting and peak shape



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broadening (loss of resolution) owing to disruptive ion-ion interactions which distort the net potential of the trapping field.50–57 One way to relieve mass analysis space charge effects that occur when the number of stored ions exceeds the spectral space charge limit50 is to remove a selected population of ions prior to the analytical scan. However, if not performed carefully, informative ions may be lost as a consequence. In the specific case of UVPD, when laser power is regulated to optimize sequence information, the largest population of ions consists of undissociated precursors. Removal of these uninformative surviving precursor ions provides a route to alleviate space charge effects and thus improve spectral quality without significant loss of informative fragment ions. This concept serves as the basis for the present study and is accomplished via strategic application of selective waveforms to the ion trap. The addition of supplemental waveforms to ion traps provides many opportunities for manipulation of the ion population via modulation of the ion trajectories. Supplemental waveforms have been previously integrated with photodissociation for several applications.58–60 Little et al. first applied a voltage at the resonant frequency of targeted ions to move those ions away from a laser beam used for infrared multiphoton dissociation (IRMPD) in an FTICR cell.58 This enhanced the survival of product ions that would otherwise be continuously irradiated and undergo annihilation via secondary dissociation. Payne et al. implemented a similar strategy in a quadrupole ion trap, in this case using a broadband waveform to shift the trajectories of all product ions out of the path of an IR laser beam, a process that alleviated secondary dissociation.59 In a clever follow-up study, the position of the IR laser beam was axially offset away from the center of the ion trap, meaning that only ions selectively excited via an auxiliary waveform would intersect the beam.60 This strategy, termed selective broadband IRMPD, was used to enhance the photoactivation and dissociation of specific precursor ions while limiting


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activation of the diagnostic product ions which accumulated in the center of the trap.60 Each of these previously reported methods was aimed at controlling the extent of secondary fragmentation of ions during IRMPD and could be adapted for UVPD. Other methods could be devised to selectively irradiate precursor ions, protect product ions, or perform multiple trap fills utilizing concepts previously established to mass-selectively transfer ions from one ion trap to another in multiple ion trap mass spectrometers.61,62 However, the higher energy deposition of UV photons63 means that absorption of even a single photon may be sufficient to cause dissociation of peptides and fragment ions, making it particularly challenging to mitigate secondary dissociation. In the present study, we focus on utilizing selective waveforms to mitigate space charge effects that may occur when ion traps are loaded to their storage capacity during a UVPD experiment, ultimately improving S/N and resolution while minimizing mass shifts. The strategy described herein involves overfilling the ion trap to maximize the number of ions prior to UVPD, then implementing resonance ejection of the un-dissociated precursor ions prior to mass analysis. We refer to this method as precursor ejection ultraviolet photodissociation (PE-UVPD). This method offers the dual benefits of increasing the S/N of the resulting UVPD product ion mass spectra while also alleviating mass shifts caused by having excessive charges in the ion trap (space charge). This strategy is demonstrated for LC-MS/MS analysis of a complex tryptic digest of an E. coli lysate, resulting in improved search score values and successful identification of over 600 proteins. Experimental Section Materials



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Angiotensin I and iodoacetamide (IAM) were purchased from Sigma-Aldrich (St. Louis, MO). Dithiothreitol (DTT) was purchased from MP Biomedicals, LLC (Solon, OH). Mass spectrometry grade Trypsin Gold was purchased from Promega (Madison, WI). Solvents were purchased from Thermo Fisher Scientific (Pittsburgh, PA). Peptide Preparation A whole cell lysate of wild-type E. coli was prepared as previously described.64 20 µg of the lysate was placed into 50 mM ammonium acetate buffer and disulfide bond reduction commenced through a 30 minute incubation with DTT at a final concentration of 5 mM at 55°C. Alkylation was then performed through the addition of a final concentration of 15 mM IAM and incubated for 30 minutes at room temperature in the dark. Digestion was performed using Trypsin Gold at a 1:50 (protease:protein) ratio and incubated for 12 hours at 37°C. Using Pierce C-18 spin columns (Thermo Fisher Scientific (Rockford, IL), digested peptides were purified using three wash steps with 2% acetonitrile: 0.5 % formic acid to remove excess DTT and IAM. Samples were then dried under vacuum. Chromatography Peptides were separated using a Dionex UltiMate 3000 RSLCnano system (Sunnyvale, CA) at a flow rate of 300 nL/min. An injection of approximately 1 µg of digest was first passed through a 1 cm New Objective IntegraFrit 100 µm I.D. trap column (Woburn, MA) packed inhouse with 3.5 µm Waters Xbridge BEH C18 media (Milford, MA) for 6 minutes at a flow rate of 4 µL/min with a 2% acetonitrile: 0.1% formic acid mobile phase composition. The peptides were then passed through a New Objective PicoFrit 75 µm I.D. (15 µm tip I.D., 15 cm long) column (Woburn, MA) packed in-house with 3.5 µm Waters Xbridge BEH C18 media (Milford, MA). Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of


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0.1 formic acid in acetonitrile (ACN). The applied gradient increased the mobile phase B from 2% to 10% for 5 min and then from 10% to 35% over 80 minutes. The column was flushed for 10 minutes with 90% mobile phase B. The column was allowed to equilibrate to the 2% mobile phase B for an additional 15 minutes prior to the next injection. Mass Spectrometry and Data Analysis Mass analysis was performed on a Thermo Fisher Scientific (San Jose, CA) Velos Pro dual linear ion trap mass spectrometer.

A 193 nm excimer laser was used for UVPD

experiments, and the mass spectrometer was modified as described previously.4,65 Infusion and LC-MS/MS data-dependent spectra were collected using 3 and 1 µscans, respectively. PE-UVPD was assessed and optimized through infusion of angiotensin I using automatic gain control (AGC) targets ranging from 1E+4 to 1E+5 charges while using one 193 nm laser pulse at 2 mJ. All S/N values were calculated manually by dividing the intensity of the most abundant isotopic peak of the observed fragment ion by the average of all peak intensities 50% or less of the base peak intensity, excluding base peak isotopic peaks, within an m/z range defined for each figure. Bottom-up LC-UVPD-MS analysis of the digest of E. coli lysate was performed using the Velos Pro mass spectrometer, with PE-UVPD enabled or disabled. AGC targets of 1E+4, 2E+4, and 3E+4 were used in conjunction with application of one 193 nm laser pulse at 3.5 mJ to accommodate a wider variety of peptides and precursor charge states. Custom instrument control programming was undertaken to enable UVPD to be performed in the low pressure trap (LPT) following initial accumulation and isolation of precursor ions in the high pressure trap (HPT) of the dual-pressure linear ion trap.66 The precursor activation q value was maintained at 0.25 during laser irradiation. Following UVPD, and prior to mass analysis, the m/z of the precursor was resonantly ejected from the LPT during



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data dependent LC-MS/MS via a customized instrument control program modification. To enable resonance ejection for a 0.2 m/z window centered at the precursor m/z, the main RF amplitude was dithered while single frequency supplemental AC resonance ejection (q value = 0.25) was applied. This comprised the process of PE-UVPD. Following PE-UVPD, the remainder of the scan function including mass analysis with a conventional resonance ejection (q value = 0.88)50 was unaltered. Bottom-up protein identification was performed using Proteome Discoverer 1.4 (Thermo Fisher Scientific, Waltham, MA) with the SEQUEST search algorithm being used to match peptide MS/MS spectra with proteins in the E.coli proteome. Fragment types a, b, c, x, y, z were searched with a 0.2 Da tolerance, with precursor tolerance set to 0.8 Da, and a false discovery rate (FDR) of 1% used to determine false positives through randomized decoy spectra. In addition, the influence of un-dissociated peptide precursor on scoring was negated by removing a 2 m/z window centered at the activated precursor m/z value from the search parameters. Results and Discussion While 193 nm UVPD has been an effective method for characterizing proteins and peptides, one hallmark is the generation of a diverse array of fragment ions along the entire m/z range. This means the ion current is distributed over many different dissociation channels and may also remain as un-dissociated precursor ions. As noted earlier, using high pulse energies or a large number of pulses not only increases the dissociation efficiency but also causes more extensive secondary fragmentation that may produce internal fragment or very small fragment ions. This means that the ability to optimize fragmentation efficiency and S/N of the spectra, along with the interplay of trap capacity and space charge effects, can have a particularly high impact for overall UVPD-MS performance.


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The relationship between trap capacity (and the affiliated automatic gain control (AGC) procedure), S/N of the UVPD mass spectra, and mass shifts merits further discussion. AGC is the process by which the mass spectrometer controls the number of precursor ions based on scaling the accumulation time according to the concentration of charges assessed in a sampling prescan.66 This function greatly improves the ability to control the number of charges in the ion trap and avoid the deterioration of resolution and m/z assignments that are exaggerated by overfilling the trap (space charge condition). Shown in Figure 1 are a series of UVPD mass spectra acquired for angiotensin (3+) that illustrate the impact of the ion fill setting (AGC target). The informative yet low abundance fragment ions x7 and y7 (m/z 952.5 and 926.5, respectively), found in the m/z 900 – 1000 range, were observed. Slight variations in isotopic peak intensities, partially due to stochastic variability67 and low fragment intensities, were observed and expected when averaging only three scans per spectrum (3 µscans). Decreasing the AGC target from 1E+5 charges (Figure 1A) to 5E+4 (Figure 1B) to 1E+4 (Figure 1C) results in a reduction in the level of S/N and loss of detection of some fragment ions. S/N values of the spectra were estimated based on the section of the spectrum surrounding the x7 and y7 fragment ions for a range of AGC targets based on spectra collected in the low pressure linear ion trap (Figure 1D). The S/N increased to a certain extent with the AGC target, a parameter directly associated with the precursor ion accumulation time. The S/N of a mass spectrum is restricted by the spectral space charge limit50 and under the current parameters showed leveling off near an AGC target of 7.5E+5 charges. In sum, increasing the AGC target increases the number of precursor ions, leading to a greater population of ions subject to UVPD and resulting in higher S/N values for the resulting UVPD mass spectra.



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The consequence of increasing the AGC target value is the potential drawback of reaching or exceeding the spectral space charge limit of the trap. This space charge limit occurs when there are sufficient charges in the trapping device during mass analysis to cause perturbations of the expected frequencies of motion of ions resulting in mass shifts, loss of resolution, and even ion losses due to their ejection from the trap. This effect during UVPD is depicted in Figure 2A for which a large number of precursor ions are accumulated, followed by photoirradiation, and finally m/z analysis (via ejection of ions from the trap in order of increasing m/z). Space charging can cause detrimental effects during each of the steps of ion manipulation, including storage, isolation, and more importantly in this case during mass analysis. As depicted by the green circles in Figure 2A, the ions of lower m/z experience the most significant deleterious space charge effects during mass analysis. As an ion trap performs a mass scan, ions are sequentially ejected from lower m/z to higher m/z, and thus the lowest m/z ions are analyzed when the trap is still overfilled with charges. Since UVPD mass spectra often contain a large portion of un-dissociated precursor ions, the m/z region between the fragment ion of lowest m/z value and the precursor is prone to the most significant space charge effects. The companion UVPD mass spectra acquired with AGC targets of 1E+4, 5E+4, and 1E+5 are shown in Figures 2B, 2C, and 2D respectively, for angiotensin (3+). As the AGC target is increased, the relative abundances of fragment ions with m/z values lower than the precursor ion appear diminished owing to broadening of the ion peak widths. Moreover, the m/z assignments of the fragment ions shifted because of the same space charging effects. At the same time, fragment ion peak shapes and m/z assignments for those ions greater than the precursor remained largely unaffected as a function of the AGC target. The interplay between m/z value and space charge-induced mass shifts from theoretical values for fragment ions y1, y2, z3, b72+, a82+, a5, b6, b8 (theoretical m/z


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values: 132.10, 269.16, 400.21, 441.24, 500.77, 619.36, 784.41, and 1028.53, respectively, all from UVPD of angiotensin I (3+)) are graphically displayed as mass errors in Figure 2E for four different AGC targets, represented as the green (1E+4 AGC target), blue (3E+4 AGC target), red (5E+4 AGC target), and purple (1E+5 AGC target) lines. In addition, mass errors for fragment ions generated by CID in the HPT (NCE value 21, AGC target of 1E+4) are shown with black triangles. As the AGC target was progressively increased, the degree of mass error also increased in the UVPD mass spectra. Mass errors for a low m/z fragment ion (m/z 269, y2) and a higher m/z fragment ion (m/z 664, c5) produced upon UVPD of angiotensin (3+) at varying AGC targets up to 3E+5 are illustrated in Figure S1A. The mass error increased enormously for the low m/z ion as the AGC target increased above 1E+4, whereas there was little change in the mass error for the higher m/z fragment ion. Comparison of the mass errors for UVPD and CID using the same 1E+4 AGC targets revealed that the mass errors for ions in the lowest m/z range were somewhat greater for UVPD than CID (Figure 2E). In order to retain the potential gain in S/N and mitigate the extent of mass shifts when using higher AGC targets, the confounding space charge effects must be relieved. Our strategy to address this issue, precursor ejection UVPD, is depicted in Figure 3A. In this method, precursor ions are accumulated and isolated as normal in the high pressure linear ion trap (HPT). Ions are then transferred to the lower pressure linear ion trap (LPT) and irradiated with 193 nm photons. After conclusion of the laser irradiation period (5 ns per pulse, typically one pulse used), and prior to mass analysis, un-dissociated precursor ions are ejected during a 1.3 ms resonant excitation period. In order to effectively eject precursor ions and to allow this to be an automated process during LC-MS/MS, the frequency of the resonance ejection pulse is applied as a narrow band that corresponds to a window of 0.2 m/z centered on the m/z of the precursor ion and with



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resonance excitation amplitudes ranging from 1.7 to 6.1 Vpp for precursor ions in the m/z range of 200 to 1500. The benefit of performing PE-UVPD in the LPT is that the lower pressure reduces collisional effects (activation or deactivation) that might otherwise contribute to formation of fragment ions from collision induced dissociation (CID) or impede the fast and efficient ejection of the precursor. A graphical depiction of the effect of the magnitude of the applied resonant excitation amplitude (Vpp) on the mass error of two fragment ions (y2, m/z 269, and c5, m/z 664) produced upon PE-UVPD of angiotensin (3+) is shown in Figure S1B. The mass error for the low m/z y2 fragment ion was notably reduced when a resonant excitation amplitude of 1.4 to 2.2 Vpp was used to eject the un-dissociated precursor, with the mass error reduced by a factor of 4 to 331 ppm (0.09 Da above theoretical), from 1260 ppm, at an amplitude of 2.2 Vpp. Although CID is a possibility whenever a precursor ion is resonantly excited in an ion trap, this was not a goal in our strategy, and thus the use of a high excitation amplitude (such as 2.2 Vpp) in the LPT offered the most compelling option for efficient ejection of the precursor and alleviation of mass error of the product ions under space charge conditions. The mass spectra obtained without and with precursor ion ejection (2.2 Vpp resonant excitation) are compared in Figure 3B and 3C for UVPD of angiotensin (3+). The most notable difference is the increase in the abundances of fragment ions in the highlighted region below the m/z of the precursor ion in Figure 3C. The reason for this increase is through peak sharpening attributed to alleviation of the most serious space charge effects that occur at lower m/z. Shown below Figure 3C are expanded sections (m/z 125 – 350) from the resulting spectra without and with PE-UVPD respectively. The abundances and peak widths of the fragment ions higher in m/z than the precursor are largely unaffected by the precursor ion ejection mode. Depicted in Figure 3D is the reduction in mass error (ppm) for measurement of fragment ions y1, y2, z3, b72+, a82+, a5,


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b6, and b8 (theoretical m/z values: 132.10, 269.16, 400.21, 441.24, 500.77, 619.36, 784.41, and 1028.53, respectively, all from UVPD of angiotensin I (3+)) when implementing PE-UVPD using AGC targets of 3E+4 and 5E+4 (light blue and light red lines) versus conventional UVPD (dashed blue and red lines). PE-UVPD using a 3E+4 AGC target resulted in lower mass errors for fragment ions below m/z 600 compared to PE-UVPD using a AGC target of 5E+4. In addition, the mass error of the y2 ion (m/z 269) was similar to the level of error obtained for conventional UVPD using a standard 1E+4 AGC target (Figure 2D). The effect of PE-UVPD upon the total ion current (TIC) using a 3E+4 AGC target is shown in Figure S1C. The initial TIC level was 1.33E+6 without any resonance amplitude applied (entire precursor population intact) and dropped to 6.32E+5 at the maximum applied resonance amplitude of 2.2 Vpp, representing a 52% reduction in TIC (corresponding to ejection of the entire precursor population and only retention of UVPD fragment ions in the trap). The estimated number of charges remaining in the trap was calculated by using the 3E+4 AGC target setting and applying a 52% reduction, resulting in 1.4E+4 charges remaining. The additional 4E+3 charges could account for the increased mass error (1121 ppm, 0.15 Da above theoretical mass) for the y1 fragment ion (m/z 132) of angiotensin I. Overall mass errors were lower using an AGC target of 3E+4 versus 5E+4 for PE-UVPD (Figure 3D dashed blue and dashed red lines, respectively). This means that the implementation of PE-UVPD is most effective for relieving space charging via removal of undissociated precursor ions following laser irradiation, but does not compensate for space charge problems caused by creation and storage of excessive fragment ion current. In essence, if the fragment ions themselves exceed the spectral space charge limit of the ion trap, then space charging effects will still be observed. For this reason a maximum AGC target of



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3E+4 was used for the rest of the study since elevated mass errors for a broader range of fragment ions were observed using PE-UVPD with a 5E+4 AGC target. Successful demonstration of the PE-UVPD method for a standard peptide (Figure 3) motivated the scale-up of the method for LC-MS/MS analysis of a tryptic digest of an E. coli cell lysate. The UVPD mass spectra for two representative tryptic peptides eluting around 30 minutes (sequence GITINTSHVEYDTPTR, MW 1.8 kDa, from the protein Elongation Factor Tu 1 OS) and 60 minutes (sequence TTDVTGTIELPEGVEMVMPGDNIK, MW 2.5 kDa, from the protein Elongation Factor Tu 1 OS) acquired without and with precursor excitation are displayed in Figure 4. The spectra were acquired using identical conditions (1 laser pulse, 3.5 mJ, 1 µscan, 3E+4 AGC target) and are shown individually scaled for UVPD and PE-UVPD. The peak profile for one representative fragment ion from each peptide (y4, nominal m/z 474, theoretical m/z 474.2671 from GITINTSHVEYDTPTR; and y6, nominal m/z 643, theoretical m/z 643.3410 from TTDVTGTIELPEGVEMVMPGDNIK) are expanded in Figure 4B and 4D to allow closer inspection of the peak shapes and mass assignments. For each of the two fragment ions, the monoisotopic peaks in the PE-UVPD spectra are sharper and more closely aligned with the accurate mass assignment (e.g. the peak centroids are shifted by -0.08 Da for y4 (GITINTSHVEYDTPTR peptide) or 0.09 Da for y6 (TTDVTGTIELPEGVEMVMPGDNIK peptide) relative to the same fragment ion detected without PE). Full widths at half maximum (FWHM) are also shown in Figure 4B and 4D. Average results from triplicate repeats returned FWHM values of 0.36 ± 0.04 for UVPD (m/z 474.34) and 0.30 ± 0.02 for PE-UVPD (m/z 474.26) for the first fragment ion (y4 from peptide GITINTSHVEYDTPTR) and 0.33 ± 0.04 for UVPD (m/z 643.43) and 0.30 ± 0.03 for PE-UVPD (m/z 643.34) for the second fragment ion (y6 from TTDVTGTIELPEGVEMVMPGDNIK peptide). Peak areas were calculated manually, based on 14 ACS Paragon Plus Environment

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area = (∆ peak height / 2) x (∆ m/z), resulting in average areas of 35.7 ± 1.5 for UVPD and 31.0 ± 2.0 for PE-UVPD for the first fragment ion (y4) and 35.3 ± 4.0 for UVPD and 30.4 ± 1.1 for PE-UVPD for the second fragment ion (y6). All outcomes from both peptides are related to the alleviation of space charge effects by ejecting undissociated precursor ions using PE-UVPD. Triplicate LCMS runs of the E. coli lysates were performed for conventional UVPD and PE-UVPD using AGC targets of 1E+4 (“standard” AGC value), 2E+4, and 3E+4. The results for the LC-UVPD-MS analysis of the tryptic digests are summarized in Figure 5. With respect to peptide identifications, the XCorr score for peptide spectrum matches (PSMs) progressively increased with increasing AGC target for PE-UVPD (Figure 5A). This trend diverged from the one observed for conventional UVPD in which the PSM XCorr score increased going from an AGC target of 1E+4 to 2E+4 but decreased beyond 2E+4. The latter decline in the PSM XCorr score for conventional UVPD is attributed to a skewing of mass assignments and a reduction in the abundances of fragment ions due to space charging effects; this is the same type of deterioration of performance exhibited in Figure 2. Conventional UVPD resulted in PSM XCorr scores comparable to that of PE-UVPD at an AGC target of 1E+4, but at AGC targets above 1E+4 PE-UVPD consistently outperformed conventional UVPD. For data acquisition using an AGC target of 3E+4, the improvement in average XCorr score from 5.8 for conventional UVPD to 6.6 for PE-UVPD (Figure 5A) is a significant gain. These trends are also reflected in average XCorr values of three tryptic peptides from with E. coli elongation factor Tu 1 OS with sequences

TTDVTGTIELPEGVEMVMPGDNIK

NMITGAAQMDGAILVVAATDGPMPQTR

(MW

(MW

2.5

kDa

(3+)

2.7

kDa

(3+)

Figure

Figure 5C),

5B), and

GITINTSHVEYDTPTR (MW 1.8 kDa (3+) Figure 5D). For each peptide, the XCorr score



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increased for PE-UVPD relative to conventional UVPD, and the improvement was more marked as the AGC value increased. The average number of PSMs and number of proteins identified for the tryptic digest by UVPD-MS and PE-UVPD-MS are shown in Figure S2A and S2B, respectively, ranging from 4300-4800 PSMs and 620-650 proteins. The duty cycle decreased with increasing AGC target, and this resulted in a corresponding decrease in the total number of PSMs and proteins identified for both UVPD and PE-UVPD. An additional 1.3 ms per scan is required for the PE step, a factor which explains the slight decrease in PSMs and proteins identified for PE-UVPD relative to conventional UVPD. Average protein sequence coverages varied only slightly with AGC value, and there was little if any change in protein coverage for conventional UVPD compared to PEUVPD, as shown in Figure S2C. Overall, sequence coverage did not improve but XCorr confidence values improved using PE-UVPD. The PE-UVPD mass spectrum of one representative tryptic peptide from the E. coli lysate, NMITGAAQMDGAILVVAATDGPMPQTR (MW 2.7 kDa, 3+), is illustrated in Figure 6A. The PE-UVPD mass spectrum displays a rich array of diagnostic a, b, c, x, y, and z fragment ions, with expansions of three ions, c3, y6, and b15, shown in the insets of Figure 6A. The average S/N and average mass error (in ppm) for each of these three fragment ions in the conventional UVPD and PE-UVPD mass spectra are displayed in Figures 6B, 6C, and 6D, respectively. Overall, as AGC target increased so did S/N values for each of the three fragment ions, with particularly significant gains in S/N for y6 and b15 for the PE-UVPD method. The average ppm errors for the c3 fragment ion, displayed in the lower section of Figure 6B, showed the greatest change as a function of AGC target for the conventional UVPD data. As shown, the average mass error increased substantially during conventional UVPD (red line) as the AGC


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target increased, an outcome that emphasized the impact of increased space charging which resulted in the deleterious mass shifts for the c3 ion (and presumably other low m/z ions). The corresponding trend in average mass error for the PE-UVPD data (blue line) showed little variation for the c3 ion. The average mass errors for the y6 ion (lower section of Figure 6C) were near zero for the PE-UVPD data collected using 1E+4 and 2E+4 AGC targets. At a target of 3E+4 both PE-UVPD and UVPD produced similar average mass errors. Lastly, for the b15 ion, an ion which has a higher m/z value than the precursor peptide, the average mass errors were lowest using conventional UVPD (bottom section of Figure 6D). For high accuracy mass spectra, S/N values are typically utilized to enhance the calculation of confidence scores as currently enabled, for example, with ProSight PTM software for top-down protein analysis.48 Complete integration of PE-UVPD with a high accuracy multiplexing mass spectrometer such as an Orbitrap platform should fully exploit the gains in performance anticipated for analysis of intact proteins. In addition, database search programs do not currently incorporate S/N factors into their scoring algorithms for low resolution spectra, but this might be a compelling option for future consideration for high resolution analysis. Conclusions The S/N of UVPD mass spectra are often lower than typical MS/MS spectra acquired by other conventional ion activation methods, such as collisional activation. The lower S/N is readily rationalized by the dispersion of precursor ion current into the numerous fragmentation channels characteristic of UVPD, creating a,b,c,x,y, and z ions. Moreover, UV photoactivation is typically not pushed to the limits of laser power in order to avoid production of very small fragment ions and internal ions that arise from excessive energy deposition and prove to be uninformative or not easily interpreted. These factors contribute to a substantial population of



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un-dissociated precursor ions, particular for UVPD of intact proteins. PE-UVPD provides an innovative solution through a combined strategy of increasing the number of ions trapped and ejection of un-dissociated precursor ions after photoactivation. This approach was implemented in the low pressure trap of a dual-pressure linear ion trap mass spectrometer to allow precise and efficient ejection of un-dissociated precursor ions without causing collisional activation that would otherwise offset the alleviation of space charge effects. Utilization of the PE-UVPD method for bottom-up analysis of a tryptic digest of E. coli proteins resulted in higher average XCorr values compared to conventional UVPD. Integration of PE-UVPD into a multiplexing mass spectrometer should alleviate the slight decrease in duty cycle owing to the 1.3 ms required for the precursor resonant ejection step, thus further improving the performance metrics of the PE-UVPD method. Moreover, PE-UVPD would be a natural fit for top-down analysis in a high accuracy mass spectrometer because intact proteins typically produce extremely rich mass spectra with the ion current not only divided into un-dissociated precursor species but also numerous highly charged fragment ions that may span a large m/z range. The production of hundreds of different fragment ions reduces the overall S/N of top-down UVPD mass spectra, thus emphasizing the need for strategies that maximize ion current and alleviate space charge effects.

Acknowledgments The authors declare no competing financial interests. We acknowledge the following funding sources: NSF (Grant CHE1402753) and the Welch Foundation (Grant F-1155). We thank Chad R. Weisbrod, Jae C. Schwartz and Alexander Makarov for their helpful advice and suggestions. We also thank James D. Sanders for his assistance with MATLAB data processing.


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Supplemental Information Available: Additional figures include assessment of mass error (ppm) versus AGC target and resonance amplitude; and average PSMs, identified proteins, and sequence coverages at various AGC targets for LC-MS/MS analysis of a tryptic digest of E. coli lysate. This material is available free of charge via the Internet at http://pubs.acs.org.



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Figure 1: UVPD mass spectra (1 pulse, 2 mJ) of angiotensin (3+) obtained while varying the AGC target A) 1E+5, B) 5E+4, and C) 1E+4. D) S/N (left axis) of the x7 fragment ion (m/z 952.5, with a noise window range from m/z 930 – 980) is shown with the blue line, S/N (left axis) of the y7 fragment ion (m/z 926.5, with a noise window range from m/z 918 – 935) is shown with the red line, and the precursor accumulation time (right axis) is shown with the purple line as the AGC target value (1E+4, 5E+4, and 1E+5) is varied. All were collected using three spectrum averages, and spectra are shown with similar scaling.



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Figure 2: A) Schematic representation of events for UVPD in the ion trap beginning with ion accumulation, followed by UVPD, and the resulting space charging that occurs at high AGC targets. UVPD mass spectra (1 pulse, 2 mJ) for angiotensin (3+) obtained using an AGC target of B) 1E+4, C) 5E+4, and D) 1E+5, all with similar scaling. Highlighted region represents the m/z range where space charging effects are most apparent. E) Mass error (ppm) of UVPD fragment ions ranging from m/z 130 – 1030 using AGC targets 1E+4 (green line), 1E+4 CID with 21 NCE (black triangles), 3E+4 (blue line), 5E+4 (red line), and 1E+5 (purple line). The m/z value of the precursor ion is indicated by a solid black vertical line. The zero ppm error value is shown with a horizontal gray line.


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Figure 3: A) Schematic representation of a sequence of events to relieve space charge effects beginning with ion accumulation, then UVPD, followed by resonance ejection applied to the remaining un-dissociated precursor (PE-UVPD). UVPD mass spectra (1 pulse 2 mJ) of angiotensin (3+) obtained using an AGC target of 3E+4 B) without PE-UVPD, and C) with PE-UVPD. Zoomed sections within m/z range 125 – 350 are shown below for each spectrum without and with PEUVPD. D) Mass error (ppm) of fragment ions ranging from m/z 130 – 1030 using AGC target 5E+4 without PE-UVPD (light red line), with PE-UVPD (red dashed line), 3E+4 without PE-UVPD (light blue line), and with PE-UVPD (blue dashed line). The m/z value of the precursor ion is indicated by a solid black vertical line. The zero ppm error value is shown with a horizontal gray line.



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Figure 4: Spectra collected during LC-MS/MS UVPD analysis of a tryptic digest of an E. coli lysate. Eluting around 30 minutes is a peptide from Elongation Factor Tu 1 OS (sequence GITINTSHVEYDTPTR, MW 1.8 kDa, 3+) with the resulting UVPD spectra shown in A) standard UVPD on the top and PE-UVPD on the bottom. The region around the abundant y4 ion of m/z 474 (theoretical m/z 474.2671) is expanded in B) to show the mass shift and resolution using standard UVPD (top) and using PE-UVPD (bottom). Eluting around 60 minutes is a peptide from Elongation Factor Tu 1 OS (sequence TTDVTGTIELPEGVEMVMPGDNIK, MW 2.5 kDa, 3+) with the resulting UVPD spectra shown in C) standard UVPD on the top and PE-UVPD on the bottom. The region around the abundant y6 ion of m/z 643 (theoretical m/z 643.3410) is expanded in D) to show the mass shift and resolution using standard UVPD (top) and using PE-UVPD (bottom). All spectra consisted of 1 µscan using a 3E+4 AGC target and were acquired using 1 laser pulse at 3.5 mJ.


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Figure 5: Summary of database search results for LC-UVPD-MS analysis of a tryptic digest of an E. coli lysate (1 pulse, 3.5 mJ). A) XCorr values for all PSMs for each AGC target are represented through box-and-whisker plots. Red boxes represent results obtained using conventional UVPD, and blue boxes represent results obtained using PE-UVPD. X-axis values represent replicate number and AGC target used. Average XCorr values for three peptides derived from Elongation Factor Tu 1 OS, including B) peptide TTDVTGTIELPEGVEMVMPGDNIK (MW 2.5 kDa, 3+), C) peptide NMITGAAQMDGAILVVAATDGPMPQTR (MW 2.7 kDa, 3+), and D) peptide GITINTSHVEYDTPTR (MW 1.8 kDa, 3+). Error bars represent the standard deviations between averaged values for triplicate runs.



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Figure 6: Summary of database search results for LC-UVPD-MS analysis of a tryptic digest of an E. coli lysate (1 pulse, 3.5 mJ) Elongation Factor Tu 1 OS (sequence NMITGAAQMDGAILVVAATDGPMPQTR, MW 2.7 kDa, 3+). A) PE-UVPD spectrum using 3E+4 AGC target with zoom insets of fragments c3 (theoretical m/z 376.20), y6 (theoretical m/z 729.37) and b15 (theoretical m/z 1486.74). Average S/N (top) and average mass error in ppm (bottom) are shown for three fragment ions: B) c3 in m/z range 372 – 380, B) y6 in m/z range 700 – 750, and D) b15 in m/z range 1472- 1510. Red bars represent results obtained using conventional UVPD, and blue bars represent results obtained using PE-UVPD. Error bars represent the standard deviations between averaged values for triplicate runs.


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UVPD

Space Charging PE-UVPD

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