Use of Ultraviolet Photodissociation Coupled with Ion Mobility Mass

Sep 15, 2016 - The smaller conformer of melittin has fewer cleavage sites along the peptide backbone than the larger conformer suggesting considerable...
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Use of Ultraviolet Photodissociation Coupled with Ion Mobility Mass Spectrometry To Determine Structure and Sequence from Drift Time Selected Peptides and Proteins Alina Theisen,† Bin Yan,† Jeffery M. Brown,‡ Michael Morris,‡ Bruno Bellina,*,† and Perdita E. Barran*,† †

Michael Barber Centre for Collaborative Mass Spectrometry, Manchester Institute of Biotechnology, and Photon Science Insitute, University of Manchester, 131 Princess Street, Manchester, M1 7DN, United Kingdom ‡ Waters Corporation, Stamford Avenue, Altrincham Road, Wilmslow, SK9 4AX, United Kingdom S Supporting Information *

ABSTRACT: We demonstrate the capabilities of a laser-coupled ion mobility mass spectrometer for analysis of peptide sequence and structure showing ultraviolet photodissociation (UVPD) spectra of mass and mobility selected ions. A Synapt G2-S mass spectrometer has been modified to allow photointeraction of ions post the mobility cell. For this work, we have employed a single wavelength laser, which irradiates at 266 nm. We present the unique capabilities of this instrument and demonstrate several key features. Irradiation of luteinizing hormone releasing hormone (LHRH), growth hormone releasing hexapeptide (GHRP-6), and TrpCage (sequence NLYIQWLKDGGPSSGRPPPS) yields extensive b- and y-type fragmentation as well as a- and c-type ions. In addition, we observe side chain losses, including the indole group from tryptophan, and immonium ions. For negatively charged ions, we show the advantage of using collision-induced dissociation (CID) post-UVPD: radical ions are produced following irradiation, and these fragment with higher efficiency. Further, we have incorporated ion mobility and subsequent drift time gating into the UVPD method allowing the separate analysis of m/z-coincident species, both conformers and multimers. To demonstrate, we selectively dissociate the singly charged dimer or doubly charged monomer of the peptide gramicidin A and conformers of the [M + 5H]5+ form of the peptide melittin. Each mobility selected form has a different “fingerprint” dissociation spectrum, both predominantly containing b and y fragments. Differences in the intensities of various loss channels between the two species were revealed. The smaller conformer of melittin has fewer cleavage sites along the peptide backbone than the larger conformer suggesting considerable structural differences. For gramicidin, a single laser shot UVPD discriminates between primary photodissociation and subsequent fragmentation of fragments. We also show how this modified instrument facilitates activated electron photodissociation. UVPD-IM-MS analysis serves both as a method for peptide sequencing for peptides of similar (or identical) m/z and a method for optical analysis of mobility separated species.

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method, collision-induced dissociation (CID), is implemented on all commercially available tandem mass spectrometers,7 it has some drawbacks. These are predominantly a result of the mode of energy deposition and preferential cleavage of the weakest bond, manifesting in loss of post-translational modifications, noncovalent interactions, and at times limited sequence coverage.8 Electron-based methods such as electroncapture dissociation (ECD) or electron-transfer dissociation

ass spectrometry is commonly used for analysis of complex mixtures and identification of proteins and peptides, making it a central technology in the field of proteomics.1,2 In many proteomic experiments, fragmentation of mass selected peptide ions is used to identify proteins with reference to genomic databases. Many ion activation techniques have been developed over the past 30 years, to provide better sequence coverage and/or more rapid analysis.3,4 For proteomic applications, fragmentation data should be reproducible as well as diagnostic of the primary sequence; this can also be used to provide information on the three-dimensional structure or conformation of the peptide or protein under examination.5,6 While the most commonly used fragmentation © 2016 American Chemical Society

Received: April 30, 2016 Accepted: September 15, 2016 Published: September 15, 2016 9964

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of ion mobility as an orthogonal method of separation. Typically, IMS involves pulsing ions into a chamber filled with neutral buffer gas to which a weak electric field is applied. Ions are pulled through this chamber by the field but are retarded by collisions with the buffer gas. The time it takes an ion to reach the end of the drift tube, that is its mobility, is dependent on its physical characteristics such as size and charge. In traveling-wave ion mobility (TWIMS), ions are moved through a buffer gas filled series of stacked ring electrodes by a wave-like DC potential.27 This technique allows the separation of species that populate the same m/z such as mass-coincident monomer and dimer, as well as of molecules that adopt a variety of different conformations. In order to explore and further develop the application of laser-coupled MS to the area of structural research, we have modified a TWIMS-enabled Q-ToF mass spectrometer to enable photodissociation within the instrument.17 While we previously demonstrated UVPD and mobility selection in this setup using a model system (flavin mononucleotide), here, we report enhanced capabilities of the instrument using a variety of peptide systems including photodissociation of both mass and conformer-selected ions.

(ETD), while usually less efficient, are often complementary to CID and as such can overcome some of its limitations.4,9 While these electron-based methods allow retention of post-translational modifications (PTMs) and produce greatly enlightening fragmentation patterns, they are highly dependent on the charge density of the analyte ion and only applicable on some instruments.7 The coupling of mass spectrometry with laser systems has long been available and is used widely for spectroscopic investigation where mass separation is required. Many lasercoupled MS applications use photodissociation to interrogate the ion, which can take place if ions and photons are colocated long enough for absorption of one or more photons to occur.1 Fragmentation then proceeds via two pathways; either the internal energy introduced into the ion by absorption of photons is converted into vibrational modes and distributed by intramolecular vibrational relaxation and fragmentation then occurs in the ground state or dissociation occurs directly in the excited electronic state.1,10 The first mechanism produces CIDlike fragmentation and is accessed in infrared multiphoton dissociation (IRMPD) which requires absorption of multiple or many photons,11 while the second one is usually attributed to short-wavelength photons (less than 200 nm) and produces fragments due to single photon processes.1 By adjusting the wavelength, ion exposure time, and laser power, the energy deposited into the ion can be modulated, and this approach is gaining popularity in application to peptide and protein analysis.12 Fragmentation at longer wavelengths (200−400 nm) requires the presence of chromophores with suitable absorption cross sections; for example, with a fixed wavelength laser at 266 nm, dissociation of a peptide will only occur if the sequence contains aromatic amino acids (tyrosine, tryptophan, phenylalanine),1 although this wavelength has also been shown to be capable of cleaving disulfide bonds leaving two radical ions.13,14 The need for suitable chromophores is lifted at 193 nm and below as the backbone amide group begins to absorb.8 At this wavelength, a single photon deposits enough energy into a protein ion to cause fragmentation irrespective of sequence or length, making it an amenable technique for top-down analysis of intact proteins.13 Unlike CID, which exhibits better sequence coverage at the termini of proteins, ultraviolet photodissociation (UVPD) can produce cleavage throughout.13 Since it is not necessarily the most labile bonds that are broken first, UVPD allows retention of post-translational modifications.13 Another advantageous characteristic of UVPD is the ability to dissociate side chains which allows discrimination between leucine and isoleucine.1,15 Overall, PD is well suited to application in proteomics and an increasing number of instruments have been modified to implement photodissociation.16−24 A notable example of this is in the recent work by Cammarata and Brodbelt who demonstrated UVPD at 193 nm of myoglobin and related fragmentation yields to the flexibility or rigidity of the structural elements in which cleavage occurred.25 Reilly and co-workers used 157 nm photons to investigate fragmentation of proline-containing peptides, and this allowed discrimination between the two conformations of proline even when multiple prolines were present in the sequence.26 This has since been extended to whole proteins with the example of ubiquitin, for which specific fragmentation depending on proline isomerization was observed.12 Mass spectrometry alone is unable to distinguish between isobaric species; however, this can be achieved by incorporation

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EXPERIMENTAL SECTION Materials. HPLC-grade water and methanol (>99.9% purity) were purchased from Sigma-Aldrich (UK). Gramicidin A (sequence HCO-VGALAVVVWLWLWLW-NHCH 2 CH2OH) and luteinizing hormone releasing hormone [DTrp6]-LH-RH (sequence pE-HWSYWLAPG-NH2, also known as LHRH or GnRH) were purchased from Sigma-Aldrich with a purity of 90% and 99%, respectively. Growth hormone releasing hexapeptide GHRP-6 (sequence HWAWFK-NH2) was purchased from GeneCust (Luxembourg) as a lyophilized powder with a purity of 99%. Melittin from honey bee venom (sequence GIGAVLKVLTTGLPALISWIKRKRQQ-NH2) was purchased from Sigma-Aldrich (UK) with a purity of 97%. TrpCage (sequence NLYIQWLKDGGPSSGRPPPS) was synthesized by FMOC solid state peptide synthesis, purified, and lyophilized. Gramicidin A, GHRP-6, and LHRH were prepared in 50/50 water/methanol to a peptide concentration of 50 μM while melittin was prepared to a concentration of 20 μM. TrpCage was dissolved in 50 mM ammonium acetate to a peptide concentration of 50 μM. Ion Mobility Mass Spectrometer: Achieving UVPD on m/z and Conformer Selected Ions. Several modifications have been made to a Waters Synapt G2-S to permit injection of a laser beam23 into the transfer cell, post mobility separation. A CaF2 window has been incorporated into the upper vacuum flange of the time-of-flight region. The push plate assembly has been machined to accommodate a custom size mirror (12.7 × 5 × 1.52 mm) coated with UV-enhanced aluminum (Thorlabs) mounted at a 45° angle to guide the laser beam collinear to the ion beam through the entire setup. In order to facilitate laser entrance into the transfer cell, the internal diameter of the transfer cell’s exit plate has been increased from 2 to 3.3 mm. Finally, to perform selection of ions based on their mobilities, the exit plate of the mobility cell is grounded. This plate is then acting as a defocusing lens allowing only a defined drift-time window to enter the transfer cell region.17 The instrument’s software (MassLynx) and the Waters Research Enabled Software (WREnS) are used in tandem to achieve ion transmission through the instrument as well as 9965

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Figure 1. UVPD at 266 nm of LHRH and GHRP-6. (a) Doubly charged LHRH was trapped and irradiated for 1 s (10 laser shots), yielding a series of b and y ions as well as a-, c-, and x-type fragments. (b) Singly charged GHRP-6 was trapped and irradiated for 500 ms (5 laser shots). UVPD of both peptides produces a plethora of fragments, of which the dominant ions, mainly b and y, have been annotated. H refers to the histidine immonium ion while W represents the tryptophan side chain. The laser produces noise peaks in the lower m/z region which have been annotated with an asterisk (∗).

trapping and conformer selection prior to detection. WREnS controls ion trapping and extraction to the time-of-flight by applying sequences of DC potentials to the stacked ring electrodes of the TWIMS region (Scheme S1). The look-up table function of MassLynx is used to define the drift-time window of the chosen conformer. An electromechanical shutter triggered by the WREnS trapping signal via an Arduino Uno board28 synchronizes laser irradiation with trapping of the ions. Alternatively to the trapping approach, conformer-selective UVPD has been carried out by using the ion mobility cycle to trigger laser irradiation. A Stanford delay generator is used to match the laser activation with the drift time of a specific conformer (Scheme S2). The location within the TWIMS region where photodissociation occurs can be varied. Due to the collinear geometry of laser beam and ion beam, a delay of 0 equals photodissociation in the trap cell/at the very beginning of the IMS cell before separation occurs, whereas setting the delay to the drift time of a certain conformation means photodissociation of this molecule occurs in the transfer cell after ion mobility separation has taken place. Experimental Workflow. Samples were ionized using a nanoESI source in positive ion mode with a capillary voltage in the range of 1.2 kV, sampling cone ranging from 30 to 80 V, and a source temperature of 25 °C. The desired species was mass selected using the quadrupole, trapped in the transfer cell, and activated using the UV laser. Optionally, specific conformations were selected by imposing a gate at the end of the mobility cell defocusing ions other than those arriving in the chosen drift time window. Typical trap fill times were 200 ms; typical activation times ranged from 500 ms to 1 s. Ions were irradiated with a Q-switched, Nd:YAG Continuum Minilite II operating at a wavelength of 266 nm and a repetition rate of 10 Hz. The average pulse energy was measured at 1 mJ. Typical acquisition times for the spectra shown here ranged from 10 min to a maximum of 1 h, although data of sufficient intensity, depending on the analyte and chromophore, can be obtained in 20 s which would be compatible with an HPLC workflow. Data was analyzed using

MassLynx v4.1 (Waters Corporation, USA), OriginPro 9.1 (OriginLab Corporation, USA), and Microsoft Excel 2010 (Microsoft, USA).

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RESULTS AND DISCUSSION UVPD of Peptides and a Miniprotein. The peptides that we have initially examined all contain the aromatic residues tryptophan (W) and tyrosine (Y) and are therefore amenable to UV fragmentation at 266 nm. The doubly charged monomer of a D-Trp6 analogue of luteinizing hormone releasing hormone (LHRH), calculated Mw of 1311.9 Da and measured at m/z 656.5, was mass selected in the quadrupole and trapped to allow UV irradiation. The resulting UVPD spectra can be seen in Figure 1a; the CID and nonactivated spectra can be found in Figure S1. UVPD results in a spectrum with a wealth of features, and an overall fragmentation yield of 0.57 (defined as the sum of the fragment ions’ intensity divided by the intensity of all ions) compared to 0.2 when trapping without laser irradiation. Spontaneous fragmentation of trapped ions without further activation is occasionally observed mainly for multiply charged ions. This is likely to be due to a combination of collisional activation with the argon buffer gas and the presence of mobile protons.29 The UVPD fragments are predominantly b- and y-type ions as well as some a ions and a few other loss channels. UVPD at 266 nm has produced ions b2−9 and y1−9 indicating that total sequence information is contained in the spectra following cleavage at every peptide bond. In addition to these dominant b and y loss channels, loss of a small molecule (−18 Da attributed to loss of H2O) is apparent. The dissociation data is comparable to CID (Figure S1a) although the intensities of different loss channels vary. There are notable differences in the low m/z region, where UVPD produces the histidine immonium ion (H) at m/z 110 in comparatively high abundance. This is in accordance with previous studies, in which immonium ions from histidine, tryptophan, tyrosine, and phenylalanine were found to be produced abundantly in photodissociation but only to a lesser extent in low-energy CID.30,31 The histidine immonium in LHRH as an internal 9966

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Analytical Chemistry fragment is most likely produced by a combination of y- and atype cleavage, in this case y-type cleavage of the a2 fragment also visible in the obtained spectrum.30 While the a2 ion is produced by both CID and UVPD, the conversion from a2 into H appears increased by UVPD, even though the overall intensity of the a2 ion remains higher than H. Again, this has been seen previously and could be attributed to a high stability of the a2 ion.32,33 The histidine residue in LHRH has been shown to be involved in many interactions, with the C-terminus among others, giving the molecule a ring-like shape in which the histidine side chain sits inside.34 It is remarkable that this residue which appears important for the three-dimensional structure of the ion is lost readily. The UVPD spectrum of LHRH also reveals a small amount of [M + H]+ originating from charge stripping of the precursor ion; however, this is also observed without laser irradiation (Figure S1b) and may be due to proton loss during the trapping. UVPD displays a rather prominent peak at m/z 130 attributed to the tryptophan side chain which could be explained by it being the site of photon absorption. Similar results are shown for GHRP-6 (Figure 1b), again the dominant fragments are b and y ions, but there is an increase in intensity of the histidine immonium ion and tryptophan side chain peak from the UVPD compared to CID (Figure S2a). The terminal histidine immonium ion in this case may have been produced by a rearrangement of the oxazolone ring of the corresponding b ion.30 Compared to LHRH, the immonium ion is more abundant in the spectrum of GHRP, and this may be attributed to two reasons: GHRP has only 6 residues (compared to 10) and therefore has less vibrational modes available to it which could dissipate energy. Second, it has been shown that the closer the precursor residue to the N-terminus, the more abundant is the immonium ion.30,35 In addition to b, y, and a ions, photodissociation of GHRP-6 also yields the internal sequence fragments WAW and AWF-28. Interestingly, UVPD of this peptide also shows the unique fragment v6, a result of side chain cleavage of the histidine residue, which is not found in the CID spectrum. Typically, vtype fragments are associated with fragmentation at high energies and are found in higher-energy collisional dissociation (HCD) and vacuum UVPD spectra, although they have not previously been reported with 266 nm photons to our knowledge. Following the experiments on small peptides described above, we then applied our UVPD methodology to a miniprotein termed TrpCage.36 The [M + 2H]2+ ion (measured at m/z 1085.8, calculated Mw of 2169.4) was mass selected, trapped, and irradiated; a wealth of fragments were produced showing extensive sequence coverage of this large molecule (Figure 2). The overall fragment yield was 0.26 compared to 0.15 without irradiation, and as for the peptides above, the fragment channels are comparable to those obtained with CID with some distinction in intensities (Figure S3b). A notable loss channel that is significantly enhanced in UVPD is the neutral loss of 44 Da attributed to CO2. The most distinct difference between UVPD (Figure 2) and CID (Figure S3a) is a significant increase in the [M + H]2•+ peak at m/z 1084.8 corresponding to the loss of hydrogen from the precursor ion. Previous studies on UVPD of amino acids and peptides have also observed this dissociation channel.37,38 Sobolewski et al. explained this fragmentation channel in terms of crossing from the ππ* state to the πσ* state, transferring an electron from the

Figure 2. UVPD spectrum with an irradiation time of 1 s (10 laser shots) of TrpCage. UV activation yields a full series of b and y ions as well as loss of small neutral molecules such as CO2 (−44) and H2O (−18). The inset shows the isotopic distribution at the precursor m/z for CID and UVPD, revealing an increase in [M + H]2•+ upon laser activation (highlighted in blue). The most dominant fragments have been labeled on the spectrum.

aromatic ring of a residue to protonated N−H and subsequent loss of H.37 Upon close examination, we also are able to identify loss of hydrogen in the UVPD spectra of singly charged LHRH (data not shown) and gramicidin A; however, no loss of hydrogen occurred upon UVPD of doubly charged LHRH and GHRP-6. It has previously been suggested that H-loss might be dependent on charge location and net charge among other factors.38 Drift Time Selected UVPD of Peptides. Gramicidin A. After successfully achieving fragmentation of mass-selected peptides on our setup, we chose gramicidin A to demonstrate UVPD on a mass and conformer-selected ion using the drifttime gating method described. Gramicidin A is a 15 amino acid peptide which associates to a homodimer and forms channels in membranes.39 It has been shown that the dimerization is favored in solvents with low dielectric constant.40 Following IM-MS from a solution of 50/50 methanol/water, we observe it as a doubly charged monomer [M + 2H]2+ at m/z 941.7 and also a peak at m/z 1883.4 which is attributable both to the singly charged monomer [M + H]+ and the doubly charged dimer [2M + 2H]2+. IM-MS and isotope distribution analysis confirms that both are present at similar intensities; these species can be well separated in the drift cell (Figure 3a). Figure 3b shows the UVPD spectrum acquired following m/z selection of the species at 1883 with the complementary CID in Figure S4 and the trapped with laser off spectrum in Figure S5. Following UVPD of the m/z selected species, the fragments are of course a combination of fragments from [M + H]+ and [2M + 2H]2+. [M + H]+ and [2M + 2H]2+ appear to preferably fragment into b ions; the complete sequence (excluding discrimination between leucine/isoleucine) could be identified from this series alone. In contrast, CID (Figure S4) does not produce fragments smaller than b5, and UVPD, in this case is able to give complementary sequence information. 9967

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Figure 3. Drift-time selected UVPD with a trapping time of 500 ms (5 laser shots) of gramicidin A. (a) Mobility profile of m/z 1883 reveals masscoincident species. Isotopic distribution around m/z 1883 after mobility selection of drift time 6−8.1 ms corresponds to doubly charged dimer while the isotopic distribution around m/z 1883 after mobility selection of drift time 8.8−10.9 ms corresponds to singly charged monomer. The calculated isotopic distribution is shown as red dots. (b) UVPD without drift time selection reveals a complete series of b ions as well as dissociation into monomer. There is a distinct difference in intensity between lower and higher b ions. (c) UVPD of mobility-selected dimer shows a complete series of b ions with no distinct change in fragment intensity between b3−8 and b9−14. (d) UV activation of mobility-selected monomer shows a complete series of b ions with a clear change in intensity between lower and higher b ions. (e) Single laser shot UVPD of gramicidin A dimer yields dissociation into monomeric units as well as some b- and y-type ions. (f) Single laser shot activation of gramicidin A monomer produces a series of b and y ions.

studies on gramicidin A which has been shown to adopt various conformations depending on its environment and forms a functional ion channel as a head-to-head single stranded helical dimer or a nonfunctional conformation as a double-stranded helical dimer.39 UVPD of the gated monomer or dimer produces a distinctive fragmentation pattern containing the complete series of b ions in both cases with some y ions and neutral losses (Figure 3c,d). This effectively deconvolutes the data in Figure 3b, giving a better indication of the parent for each fragment.

For some fragments, it is possible to infer what the parent ion was; for example, the peak at 941.7 m/z is attributable to [M + 2H]2+ which is most likely to have arisen from the loss of a neutral peptide chain from the [2M + 2H]2+ parent. Other fragment ions are harder to assign, and the absence of any doubly charged fragments and of fragments that contain a partially sequenced monomer plus an intact monomer also prevents easy assignment of the parents of each loss channel. It should be noted that the arrival time distribution of the dimer presents with a shoulder indicating the presence of at least two conformers. This is in agreement with previous 9968

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Figure 4. Drift-time selected UVPD of 5+ melittin with an irradiation time of 2 s (20 laser shots). (a) UVPD of the compact conformer C produces cleavage between seven residues producing b and y fragments while (b) UVPD of the more extended conformer E yields more extensive fragmentation with cleavage between 17 residues. The black trace represents the trapped only spectrum without laser irradiation; the red dotted trace represents laser on. The main fragments produced or enhanced by UVPD are labeled on the spectra.

weakest interaction in the molecule and therefore first to dissociate. Interestingly, UVPD of the dimer produces a different b ion pattern (Figure 3c) in comparison to the monomer. While the lower b ions are at a similar high intensity, a drop between b8 and b9 is again visible. b9 and b10 present in equal intensities; however b11−13 are much more abundant in comparison, and b14 in turn is at a low intensity again. These results suggest that either the b11−13 ions are subject to some kind of protection from further fragmentation when originating from the dimeric precursor and could indicate structural differences between “free” monomer and “UVdissociated” monomer, or since the precursor is not depleted over the time frame of our experiment, they might still be produced in higher abundance than they are fragmented at subsequent laser shots. However, if the latter was the case, this would also be reflected in the UVPD spectrum of the monomer. Despite the lack of fragments that preserve the dimer, we can infer from the differences in Figure 3c,d that the dimeric monomer is indeed different from the monomeric monomer with respect to the environment of the tryptophan chromophore. Melittin. In addition to gramicin A, mass and drift time selected UVPD was performed on the 2.8 kDa peptide melittin. When sprayed from a solution of 50% methanol, it is observed predominantly as [M + nH]n+ where n = 4 and 5. Species corresponding to n = 3 and 6 are also observed at much lower abundance (see Figure S7). [M + 5H]5+ is found to have two conformational families as can be seen from the two well resolved peaks in the arrival time distribution (see Figure 4 top) and as found in previous studies.41,42 Following gating as discussed above, UVPD spectra of each conformer of [M + 5H]5+ were acquired and are shown in Figure 4 (a zoom on each region of the spectra can be found in Figure S9). During the trapping process, before laser irradiation, both [M + 5H]5+ conformers of melittin lose protons to generate [M + 4H]4+ ions; the extended conformer (E) does this more readily, indicating that the energy barrier for proton loss is much lower than for the compact conformer (C). For C, it can be seen that, apart from the proton loss pathway, all UVPD fragments are either b ions or y ions. Two of the three b fragments are singly

UVPD of [M + H]+ (Figure 3d) produces a complete series of b ions whose intensity differs. The b2−b8 present with a similar intensity, and then, a clear drop in intensity for the b9 ion occurs with b9−b14 also at lower intensity. This can be explained with reference to the primary sequence of gramicidin A, which contains four tryptophan residues toward the Cterminus which are the chromophores that absorb 266 nm photons. Fragments containing these chromophores are able to dissociate further with each additional laser shot, terminating in the b ions that do not contain tryptophan (b8 and below). In further support of this, the single shot UVPD spectrum of monomer (Figure 3f) does not exhibit a distinct difference in the intensities of all found b ions. This also provides an explanation for the very low intensity of y ions as all fragments from the C-terminus will contain tryptophan (as the C terminal residue) and be amenable to further fragmentation. The dimer dissociates predominantly into a singly as well as doubly charged monomer with loss of a charged and a neutral peptide, respectively; there is also evidence of an uneven break in the form of doubly charged monomer +39 Da at m/z 961.2. We have not been able to establish the identity of this additional 39 Da, although we also observe the loss of 39 Da from a fraction of the dissociated, singly charged monomeric species. Both monomer and dimer precursors experience loss of small neutral molecules upon UVPD; for example, the loss of 18 Da attributable to H2O. In the dimer UVPD spectrum, these molecules have been lost from the dimeric precursor (as evidenced by isotope distributions) and also after dissociation into singly charged monomer. The neutral losses from the dissociated monomer tend to be at a higher intensity than corresponding loss from the dimer suggesting that the dissociation to a doubly charged monomer happens first in most cases. We also did not find any singly charged fragments with a higher m/z than the doubly charged dimer, again indicating that dissociation of the dimer into monomeric units is preferred over production of fragments that leave the dimer intact. Further evidence of the main fragmentation channel of the dimer being monomerization is given by the single laser shot spectrum (Figure 3e), which presents monomeric species, large b ions, and tryptophan side chain loss. Our data suggests that the noncovalent bond between the monomers is the 9969

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Analytical Chemistry charged (b12, b13) while only one is doubly charged (b92+). Among the y fragments, most of them are doubly charged (y72+ and y12−y152+). The fragments y7 and y13 are also found as triply charged ions. Especially for y13, both its 2+ and 3+ ions are enhanced dramatically by UVPD. This can be explained by the effect of proline,43 in which cleavage N-terminal to proline leads to the production of the most dominant fragment. Two 4+ y fragments (y174+ and y244+) are also observed. Of the fragments from C, y73+ is unique to UV irradiation. For the extended conformer E, the UVPD fragmentation yield is much higher (Figure 4b), and the sequence coverage is also greater. While cleavage is only observed between 7 out of 25 residues for C, this increases to 17 for E. Apart from the dominant b and y fragments, two c-type fragments are also observed for the more extended conformer (c5 and c6). The b fragments consist of four singly charged ions (b5 and b11−13) and four doubly charged ions (b9−112+ and b162+) while the y fragments are composed of charge states from 1+ to 4+. The three singly charged y fragments (y6, y8, and y9) are observed with a mass shift of −17 Da attributed to loss of NH3. This can be correlated to the presence of a C-terminal amide group in the peptide sequence. Similar to C, the primary y fragments in E are also doubly charged (y72+ and y9−152+). Six 3+ (y133+, y15−173+, y223+, and y243+) and four 4+ (y19-NH34+, y20-NH34+, y224+, and y244+) y fragments are produced by UV irradiation. Other fragmentation channels include loss of water and internal fragmentation. Compared to only a single fragment unique to UVPD from C, more fragments (c5, c6, b162+, y243+ and several y fragments with NH3 loss) from E are produced by UV irradiation only and not observed in the control spectra. Compared with the CID fragments from the [M + 5H]5+ melittin (see Figure S8), the UVPD spectra are quite distinct, indicating that UVPD is sampling the different gas phase conformation of each form of this ion. Activated EPD. For negatively charged ions, a dominant loss channel is the detachment of one electron termed electron photodetachment (EPD).44 We exploited this behavior by using an IM synchronized laser shot to produce radical species before ion mobility separation and then increasing the collision energy in the transfer cell after mobility separation to allow comparison of fragmentation between the even-electron and odd-electron species (activated EPD). CID of [M − 2H]2− yields only a limited range of fragments (Figure S6) with dominant loss channels being the loss of water and internal sequence fragments. Elucidation of the full peptide sequence is therefore not possible from this approach alone. In comparison, CID of the EPD-produced radical provided an extensive range of fragmentation and therefore sequence information (Figure 5). Activated EPD yields a plethora of c-, z-, and x-type fragments, as observed in previous studies,45 as well as y-NH3 and side chain losses, mainly in the form of w-type ions. UVPD of TrpCage in positive ion mode (Figure 2) showed an overall low intensity of fragment ions, whereas activated EPD of the radical yields a very intense, albeit complicated fragmentation spectrum (Figure 5). UVPD and activated EPD therefore give complementary information which in combination can be used to maximize sequence information.

Figure 5. Activated EPD of TrpCage in negative mode. CID of the radical species [M − 2H]•− produced by electron photodetachment of the [M − 2H]2− yields a very extensive range of fragments of predominantly c-, z-, and x-type as well as y-NH3 type. Dominant fragments have been labeled on the spectrum.

of m/z coincident species. Overall, we observed very good sequence coverage following UVPD at 266 nm and some differences in the preference for a given loss channel compared with CID. We report UV-exclusive fragments, indicating that UVPD of chromophore-containing peptides, even at this wavelength, can provide complementary information to other fragmentation methods by accessing higher energy excited electronic states. Incorporation of ion mobility separation as well as electrode manipulation enables UV photodissociation of drift-time selected species which effectively deconvolutes the fragmentation spectrum in cases where mass-coincident monomer/dimer or different conformations are present and not separable by mass selection. We showed the viability of this approach using gramicidin A monomer/dimer and two conformers found for the 5+ charge state of melittin as examples and obtained UVPD spectra for each of the selected species. Differences in fragmentation patterns between the two were observed and present a possible avenue for conformer-selected UVPD in the analysis of structural differences. In summary, we have significantly enhanced the capabilities of this instrument and enabled several different experimental modes which allow mobility separated ions to be interrogated with a UV laser.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b01705. Control spectra without laser irradiation and CID spectra (PDF)

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CONCLUSION AND OUTLOOK We have shown UVPD of chromophore-containing peptides at 266 nm in a modified, commercial ion mobility instrument and obtained a wealth of fragmentation information from 5 peptides, where possible we have performed drift time selection

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. 9970

DOI: 10.1021/acs.analchem.6b01705 Anal. Chem. 2016, 88, 9964−9971

Article

Analytical Chemistry Notes

(27) Giles, K.; Pringle, S. D.; Worthington, K. R.; Little, D.; Wildgoose, J. L.; Bateman, R. H. Rapid Commun. Mass Spectrom. 2004, 18, 2401−2414. (28) De Souza, A. R.; Paixao, A. C.; Uzeda, D. D.; Dias, M. A.; Duarte, S.; Amorim, H. S. Rev. Bras. Ensino Fis. 2011, 33, 01. (29) Wysocki, V. H.; Tsaprailis, G.; Smith, L. L.; Breci, L. A. J. Mass Spectrom. 2000, 35, 1399−1406. (30) DeGraan-Weber, N.; Ashley, D. C.; Keijzer, K.; Baik, M.-H.; Reilly, J. P. J. Am. Soc. Mass Spectrom. 2016, 27, 834−846. (31) Kelkar, D. A.; Chattopadhyay, A. Biochim. Biophys. Acta, Biomembr. 2007, 1768, 2011−2025. (32) Bythell, B. J.; Maître, P.; Paizs, B. J. Am. Chem. Soc. 2010, 132, 14766−14779. (33) El Aribi, H.; Orlova, G.; Rodriquez, C. F.; Almeida, D. R. P.; Hopkinson, A. C.; Siu, K. W. M. J. Phys. Chem. B 2004, 108, 18743− 18749. (34) Barran, P. E.; Roeske, R. W.; Pawson, A. J.; Sellar, R.; Bowers, M. T.; Morgan, K.; Lu, Z.-L.; Tsuda, M.; Kusakabe, T.; Millar, R. P. J. Biol. Chem. 2005, 280, 38569−38575. (35) Hohmann, L. J.; Eng, J. K.; Gemmill, A.; Klimek, J.; Vitek, O.; Reid, G. E.; Martin, D. B. Anal. Chem. 2008, 80, 5596−5606. (36) Neidigh, J. W.; Fesinmeyer, R. M.; Andersen, N. H. Nat. Struct. Biol. 2002, 9, 425−430. (37) Sobolewski, A. L.; Domcke, W.; Dedonder-Lardeux, C.; Jouvet, C. Phys. Chem. Chem. Phys. 2002, 4, 1093−1100. (38) Park, S.; Ahn, W.-K.; Lee, S.; Han, S. Y.; Rhee, B. K.; Oh, H. B. Rapid Commun. Mass Spectrom. 2009, 23, 3609−3620. (39) Bouchard, M.; Benjamin, D. R.; Tito, P.; Robinson, C. V.; Dobson, C. M. Biophys. J. 2000, 78, 1010−1017. (40) Chitta, R. K.; Gross, M. L. Biophys. J. 2004, 86, 473−479. (41) Florance, H. V.; Stopford, A. P.; Kalapothakis, J. M.; McCullough, B. J.; Bretherick, A.; Barran, P. E. Analyst 2011, 136, 3446−3452. (42) Zinnel, N. F.; Pai, P.-J.; Russell, D. H. Anal. Chem. 2012, 84, 3390−3397. (43) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1993, 65, 425−438. (44) Gabelica, V.; Tabarin, T.; Antoine, R.; Rosu, F.; Compagnon, I.; Broyer, M.; De Pauw, E.; Dugourd, P. Anal. Chem. 2006, 78, 6564− 6572. (45) Larraillet, V.; Antoine, R.; Dugourd, P.; Lemoine, J. Anal. Chem. 2009, 81, 8410−8416.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by BBSRC grants BB/L002655/1 and BB/L016486/1, which is a studentship award to A.T., with additional financial support from Waters Corp. We are very grateful to Professor Tony Stace (University of Nottingham) for donating the Minilite laser and to the Photon Science Institute (University of Manchester) for the loan of the 266 nm crystal and for supporting our ongoing photo-IM-MS work.

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REFERENCES

(1) Ly, T.; Julian, R. R. Angew. Chem., Int. Ed. 2009, 48, 7130−7137. (2) Aebersold, R.; Mann, M. Nature 2003, 422, 198−207. (3) Yates, J. R.; Ruse, C. I.; Nakorchevsky, A. Annu. Rev. Biomed. Eng. 2009, 11, 49−79. (4) Syka, J. E. P.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 9528−9533. (5) Reinwarth, M.; Avrutina, O.; Fabritz, S.; Kolmar, H. PLoS One 2014, 9, e108626. (6) Polfer, N. C.; Haselmann, K. F.; Langridge-Smith, P. R. R.; Barran, P. E. Mol. Phys. 2005, 103, 1481−1489. (7) Reilly, J. P. Mass Spectrom. Rev. 2009, 28, 425−447. (8) Shaw, J. B.; Li, W.; Holden, D. D.; Zhang, Y.; Griep-Raming, J.; Fellers, R. T.; Early, B. P.; Thomas, P. M.; Kelleher, N. L.; Brodbelt, J. S. J. Am. Chem. Soc. 2013, 135, 12646−12651. (9) McLafferty, F. W.; Horn, D. M.; Breuker, K.; Ge, Y.; Lewis, M. A.; Cerda, B.; Zubarev, R. A.; Carpenter, B. K. J. Am. Soc. Mass Spectrom. 2001, 12, 245−249. (10) Yeh, G. K.; Sun, Q.; Meneses, C.; Julian, R. R. J. Am. Soc. Mass Spectrom. 2009, 20, 385−393. (11) Brodbelt, J. S.; Wilson, J. J. Mass Spectrom. Rev. 2009, 28, 390− 424. (12) Warnke, S.; Baldauf, C.; Bowers, M. T.; Pagel, K.; von Helden, G. J. Am. Chem. Soc. 2014, 136, 10308−10314. (13) Brodbelt, J. S. Chem. Soc. Rev. 2014, 43, 2757−2783. (14) Agarwal, A.; Diedrich, J. K.; Julian, R. R. Anal. Chem. 2011, 83, 6455−6458. (15) Thompson, M. S.; Cui, W.; Reilly, J. P. Angew. Chem., Int. Ed. 2004, 43, 4791−4794. (16) Zucker, S. M.; Lee, S.; Webber, N.; Valentine, S. J.; Reilly, J. P.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 2011, 22, 1477−1485. (17) Bellina, B.; Brown, J. M.; Ujma, J.; Murray, P.; Giles, K.; Morris, M.; Compagnon, I.; Barran, P. E. Analyst 2014, 139, 6348−6351. (18) Vonderach, M.; Ehrler, O. T.; Weis, P.; Kappes, M. M. Anal. Chem. 2011, 83, 1108−1115. (19) Vasicek, L. A.; Ledvina, A. R.; Shaw, J.; Griep-Raming, J.; Westphall, M. S.; Coon, J. J.; Brodbelt, J. S. J. Am. Soc. Mass Spectrom. 2011, 22, 1105−1108. (20) Fort, K. L.; Dyachenko, A.; Potel, C. M.; Corradini, E.; Marino, F.; Barendregt, A.; Makarov, A. A.; Scheltema, R. A.; Heck, A. J. R. Anal. Chem. 2016, 88, 2303−2310. (21) Redwine, J. G.; Davis, Z. A.; Burke, N. L.; Oglesbee, R. A.; McLuckey, S. A.; Zwier, T. S. Int. J. Mass Spectrom. 2013, 348, 9−14. (22) Valle, J. J.; Eyler, J. R.; Oomens, J.; Moore, D. T.; van der Meer, A. F. G.; von Helden, G.; Meijer, G.; Hendrickson, C. L.; Marshall, A. G.; Blakney, G. T. Rev. Sci. Instrum. 2005, 76, 023103. (23) Kim, T.-Y.; Schwartz, J. C.; Reilly, J. P. Anal. Chem. 2009, 81, 8809−8817. (24) Maître, P.; Le Caër, S.; Simon, A.; Jones, W.; Lemaire, J.; Mestdagh, H.; Heninger, M.; Mauclaire, G.; Boissel, P.; Prazeres, R.; Glotin, F.; Ortega, J.-M. Nucl. Instrum. Methods Phys. Res., Sect. A 2003, 507, 541−46. (25) Cammarata, M. B.; Brodbelt, J. S. Chem. Sci. 2015, 6, 1324− 1333. (26) Kim, T.-Y.; Valentine, S. J.; Clemmer, D. E.; Reilly, J. P. J. Am. Soc. Mass Spectrom. 2010, 21, 1455−1465. 9971

DOI: 10.1021/acs.analchem.6b01705 Anal. Chem. 2016, 88, 9964−9971