Article pubs.acs.org/ac
Fragmentation of Positively-Charged Biological Ions Activated with a Beam of High-Energy Cations Konstantin Chingin,† Alexander Makarov,‡ Eduard Denisov,‡ Oleksii Rebrov,† and Roman A. Zubarev*,†,§ †
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles väg 2, SE-17177 Stockholm, Sweden ThermoFisher Scientific, 28199 Bremen, Germany § Science for Life Laboratory, 17121 Stockholm, Sweden ‡
S Supporting Information *
ABSTRACT: First results are reported on the fragmentation of multiply protonated polypeptide ions produced in electrospray ionization mass spectrometry (ESI-MS) with a beam of high-energy cations as a source of activation. The ion beam is generated with a microwave plasma gun installed on a benchtop Q Exactive mass spectrometer. Precursor polypeptide ions are activated when trapped inside the collision cell of the instrument (HCD cell), and product species are detected in the Orbitrap analyzer. Upon exposure to the beam of air plasma cations (∼100 μA, 5 s), model precursor species such as multiply protonated angiotensin I and ubiquitin dissociated across a variety of pathways. Those pathways include the cleavages of C−CO, C−N as well as N−Cα backbone bonds, accordingly manifested as b/y, a, and c/z fragment ion series in tandem mass spectra. The fragmentation pattern observed includes characteristic fragments of collision-induced dissociation (CID) (b/y/a fragments) as well as electron capture/transfer dissociation (ECD, ETD) (c/z fragments), suggesting substantial contribution of both vibrational and electronic excitation in our experiments. Besides backbone cleavages, notable amounts of nondissociated precursor species were observed with reduced net charge, formed via electron or proton transfer between the colliding partners. Peaks corresponding to increased charge states of the precursor ions were also detected, which is the major distinctive feature of ion beam activation.
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The most commonly used MS/MS approach is referred to as collision-activated or collision-induced dissociation (CAD/ CID). In CAD/CID, accelerated precursor ions undergo multiple collisions with neutral gas molecules, resulting in gradual vibrational heating followed by the ultimate dissociation of the weakest bonds. Polypeptide precursor ions preferentially dissociate across backbone C−N bonds into b (N-terminal) and y (C-terminal) fragments, respectively. The advantages of CAD are the relatively short time needed to generate abundant fragmentation (on the order of milliseconds) and easy technical implementation. One of three key limitations of CAD relevant to biological analyses is its poor sensitivity to the presence of post-translational modifications (PTMs). Small PTMs, such as phosphate or sulfate groups, are often weakly bound to the polypeptide backbone and tend to be easily lost during
ass spectrometry (MS) has by now become a central tool in many fields of bioanalytical science, such as proteomics and metabolomics.1−3 The power of MS is steadily increasing with regard to sensitivity, mass accuracy, resolving power and precision of detection. With modern instruments, biological samples can be analyzed in a broad dynamic range (ca. 4 orders of magnitude) and mass-overcharge (m/z) range (∼200−4000) with high resolution (>100 000) and acquisition rates (>10 Hz).4,5 One of the main limiting factors for direct mass analysis is the sequencing capability of MS.6,7 Specificity and accuracy of analyte identification often suffer from the insufficient capacity of mass spectrometers to generate informative fragmentation patterns, which would be characteristic for the precursor ions. The yield of tandem MS (MS/MS) is particularly low for large proteins, which is among the major reasons preventing wider application of top-down approaches, especially to protein−molecule complexes obtained by “native” MS.8 © 2013 American Chemical Society
Received: February 15, 2013 Accepted: November 8, 2013 Published: November 15, 2013 372
dx.doi.org/10.1021/ac403193k | Anal. Chem. 2014, 86, 372−379
Analytical Chemistry
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shotgun proteomics, in which most of analyzed proteolytic peptides are doubly charged. Thus, increasing the charge state of precursor ions prior to electron transfer would enhance the yield of subsequent activation by ECD/ETD, enabling the analysis of even singly charged precursors. Such a “supercharging” of analyte cations can be achieved via electron ionization of trapped ions (electron ionization dissociation, EID).23 However, the difficulty of dealing with electron beams entering a radiofrequency multipole prevented such a method from being widely applied.24,25 The idea of replacing electrons with electronically excited gas cations for the activation of positively charged biological ions may appear somewhat irrational, given the low collision cross section of the reacting species. Nevertheless, ion−ion reactions can be expected to occur provided the ion energy exceeds the coulomb barrier, which is normally about a few electronvolts. The outcomes of such interaction are, however, rather difficult to predict a priori. It is therefore useful to experimentally explore the feasibility and characteristic products of ion−ion activation, particularly in reference to other electronic activation methods: with radical anions, electrons or metastable atoms. In the present study, we explored the possibility of activating ESI-produced polypeptide ions using a high-energy beam of air cations generated with a microwave plasma source. The accelerated ion beam (up to 1−2 keV) contains a large variety of ionic, electronically excited (metastable) and vibrationally excited species within a broad range of kinetic energies. Many of these species are nonreactive, but some react with the target precursors through a variety of collisional, proton-, atom-, and electron-transfer reactions. The goal of the study was to test the complex network of reaction pathways available in the experimental setup that has been based on commercially available instrumentation. The corresponding tandem MS analysis of trapped peptide ions revealed a plethora of reactions, including charge reduction, charge increase, as well as polypeptide backbone cleavages along each of the inter-residue bond types (C−CO, C−N, N−Cα). The observed fragmentation patterns are compared to reference CAD and ETD MS/ MS analyses, and the revealed features of the method are discussed.
activation, which prevents their observation in tandem MS. Second, the efficiency of sequencing based on CAD MS/MS commonly suffers from incomplete fragmentation along the peptide backbone.9 Finally, CAD is rather inefficient for large proteins because the energy supplied during the activation dissipates across the large number of vibrational modes. As a result, only a few peptide bonds fragmentthose that receive vibrational heating sufficient for their dissociation. Orthogonal to CAD/CID are electron capture/transfer dissociation (ECD/ETD) techniques, in which precursor ions receive an electron.10,11 Electron addition to closed-shell molecular cations converts them to unstable radical cations of the “hydrogen-abundant” type.12 In addition, 4−7 eV of recombination energy is deposited,12 and it adds a degree of vibrational heating. Electron transfer to multiply protonated polypeptides preferentially induces fast, perhaps even nonergodic, cleavage of N−Cα backbone bonds, yielding c (Nterminal) and z (C-terminal) types of fragments. In many cases, the high speed of the primary ECD process prevents significant redistribution of the recombination energy among vibrational modes prior to dissociation. As a result, loosely bound functional groups, such as those found in protein−molecule complexes and in proteins with labile PTMs, can “survive” dissociation and be localized in the generated c and z fragments.13 The sequence preferences in ETD/ECD and CAD/CID are complementary,14 and the combination of their MS/MS data can be applied to help spectral interpretation and reduce the rate of misidentifications in proteomics analyses. In high-resolution Fourier transform mass spectrometry (FTMS), the combined use of ECD and CAD has been demonstrated to improve the validity of the database search data by 20−100 times compared to CAD-only analysis.15 The combined approach also resulted in substantially higher number of proteins identified by a minimum of two peptides with a Mascot score above the significant level of 34.15 The cross section of electron capture rapidly increases with the number of protons on the polypeptide, making it particularly suitable for the fragmentation of highly protonated species. For instance, the cross section of electron capture for cytochrome c +15 ions measured at typical ECD conditions has been shown to exceed the cross section of ion-neutral collision by 2 orders of magnitude.16 Alongside with ECD and ETD, a number of tandem MS techniques have been introduced that employ electron activation.17 A popular approach to activate trapped polypeptide ions is with the use of electronically excited, metastable atoms of noble gas. This method, sometimes referred to as metastable-induced dissociation (MIDI) or metastable atomactivated dissociation (MAD), generates fragmentation patterns similar to ECD/ETD, although CAD-type ions (e.g., b and y) can be also observed.18−20 In electron detachment dissociation (EDD), deprotonated polypeptides are charge-reduced through the collisions with free electrons.21 Reduced radical species dissociate along the Cα−C backbone giving rise to x and a fragments. Alternatively, electron transfer to polypeptides can also be induced via high-energy collisions with alkali metal vapors.22 The yield of ECD/ETD, i.e., the ratio of the product ion abundances to the precursor ion abundance, strongly depends on the charge state of precursor species. At low charge states, the yield of these techniques tends to be limited, especially for 2+ precursors, for which one of the fragments is by necessity neutral. The limitation represents a serious problem for
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EXPERIMENTAL SECTION Experiments were carried out on the benchtop Q Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany).26 The ion gun was mounted at the back of higher-energy collision dissociation (HCD) cell of the instrument (Figure 1).
Figure 1. Graphical layout of experimental setup for the activation of ions trapped inside the HCD cell of a Q Exactive Orbitrap mass spectrometer with a beam of high-energy cations: magnetron source (1), water cooling (2), gas leak valve (3), electrical feedthrough (4), ion gun flange (5), MS adaptor interface (6), turbomolecular pump (7), anode (8), cathode (9), ion extraction lens (10), HCD flange (11), HCD back lens (12), HCD cell (13), C-trap (14), Orbitrap (15). 373
dx.doi.org/10.1021/ac403193k | Anal. Chem. 2014, 86, 372−379
Analytical Chemistry
Article
Figure 2. Tandem MS analysis of doubly (A and B) and triply (C and D) protonated angiotensin I ions activated with a beam of high-energy cations for 5 s. For each charge state, both full-scale (A and C) and zoomed (B and D) mass spectra are shown. Angiotensin I sequence with cleavage sites is shown in panel E. The cleavages of C−CO, C−N, and N−Cα backbone bonds are shown as gray, green, and red dashed lines, accordingly.
Precursor ions isolated by the selective quadrupole mass filter were trapped in the HCD cell (13) and exposed to plasma beam irradiation over the desired time interval. Generated ionic fragments were transferred to the Orbitrap mass analyzer (15) for detection. Experimental details are described below. Ion Gun. The ion beam was produced using a filamentless IonEtch sputter gun (Tectra, Frankfurt, Germany) that utilizes a microwave plasma discharge. In IonEtch, microwave energy is pumped into an alumina chamber, and plasma is ignited in the gas introduced externally into the chamber via a leak valve (3). Plasma density is enhanced via the effect of cyclotron resonance caused by a quadrupole magnetic field around the chamber. Positive ions are extracted out of the chamber using two-grid extraction optics (8, 9). The voltage ranges for the first lens (anode) and the second lens (cathode) were from 100 to 1000 V and from −600 to −1400 V, accordingly. The magnetron
current was varied from 15 to 20 mA, and the beam current was measured ∼5 cm away from the cathode lens was in the range of 50−200 μA. MS/MS Interface. The sputter gun was mounted on the back flange of HCD cell using a dedicated interface constructed in-house (6) and evacuated by an additional turbopump (7) to improve the background vacuum inside the plasma chamber. The pressure was measured in close proximity to the gun. The measured value was ca. 9 × 10−6 mbar when the gas leak valve was fully closed. The feed gas was supplied at the flow rate of ca. 1−5 sccm, which resulted in the pressure increase up to 5 × 10−5 through 5 × 10−4 mbar. The ion beam from the gun entered the interior of HCD cell via a 2.5-mm i.d. orifice in the back lens (12). The distance from the end of the gun to the HCD cell was ca. 2 cm. An additional ion lens (−2000 V) was 374
dx.doi.org/10.1021/ac403193k | Anal. Chem. 2014, 86, 372−379
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HCD). On the basis of the measured m/z values and isotopic distributions, most abundant signals were identified as metals, e.g., Cr+, Fe+, Ni+, Ag+, Bi+, and polyaromatic hydrocarbons (PAHs), e.g., C5H7+, C6H7+ or C7H7+ (Figure S-1A). Metal ions are likely to be desorbed from the surfaces of the plasma cavity and electrode coatings. For example, Ag, which yielded the highest-abundance signals in the mass spectrum, was used as the coating on the extraction lens. PAHs are, in turn, wellknown combustion products. Rather surprisingly, even though the ion current had positive magnitude (∼+100 μA), some gunrelated signals were detected in the negative ion detection mode, suggesting the presence of anionic species (Figure S-1B). Most abundant isotopic cluster was identified as MoO3−. MoO3 is a volatile oxidation product of Mo, which is the major component of the plasma chamber. However, the majority of ionic beam species, such as oxygen, nitrogen, hydrogen, etc., cannot be directly revealed by the ion-trap type of MS detection due to limitations of electronics, and their presence can only be manifested via the interaction with trapped polypeptide cations. Figure 2 summarizes the results of tandem MS analyses for the multiply protonated angiotensin I ions activated with the beam of high-energy air cations. Doubly (Figure 2A,B) and triply charged (Figure 2C,D) precursor species were selected by the quadrupole and subjected to plasma irradiation over the time interval of 5 s. While the yield of ion fragments and other product species was relatively low under the experimental conditions employed (≤10%), abundant MS/MS pattern could still be revealed upon zooming the generated mass spectra. Some signals were observed as doublets or triplets due to the presence of homologous species that only differ by the number of hydrogen atoms. This difference originates from simultaneous reactions that involve H· transfer (− H· or + H·) and those in which H· transfer does not occur. If one particular isotopic cluster within a multiplet is considerably more abundant than the others, it is highlighted in the spectrum with large font and/or bold type (e.g., c4+• or c4+′). Irradiation with the ion beam breaks all the three bond types of polypeptide backbone in angiotensin I cations: C−CO, C−N and N−Cα. The cleavage of C−N bonds solely proceeds via intramolecular H· transfer from N- to C-terminus, giving rise to y′ and b fragments. This fragmentation scenario is common in tandem MS analyses based on vibrational heating such as CAD/CID, infrared multiple-photon dissociation (IRMPD)29 and blackbody irradiation (BIRD).30 Conversely, the dissociation of N−Cα bonds proceeds via homolytic as well as heterolytic channels, manifested by c•/c′/c″ and z/z•/z′ fragment ion series. Dissociation via the cleavage of N−Cα bonds is highly characteristic for ion activation by electron transfer, e.g., in ECD and ETD analyses. The cleavage of the C−CO bond is only reflected by a series of a/a• ions, while the complementary x ions were not revealed. This scenario is similar to conventional CAD, suggesting that a ions in our experiments are likely to be produced indirectly, from b fragments, rather than via direct dissociation of C−CO bond. Besides backbone cleavages, notable amounts of nondissociated precursor angiotensin I species are observed with reduced charge, formed via electron or proton transfer between the colliding partners without dissociation or with hydrogen atom desorption. Dissociation-free electron attachment appears to be the dominant charge-reduction mechanism based on the much higher abundance of the reduced species having the same mass as the precursor ions (highlighted with bold type in Figure 2).
installed in between the cathode and the HCD back lens to facilitate ion beam extraction (10). Peptides. Angiotensin I (DRVYIHPFHL) and ubiquitin were bought (Sigma, Madison, Wisconsin) and used without further purification. Polypeptides were dissolved in water/ methanol/acetic acid mixture (49:49:2 v/v/v) at a concentration of 10−5 M and were electrosprayed using a standard IonMax ionization source. MS Operation. Experiments were performed in the MS/ MS mode on Q Exactive instrument, and the plasma source was constantly switched on. Source installation and operation required removal of the charge detector used for the ion current control in routine MS experiments; therefore, automated gain control was not fully operational in our experiments. Precursor ions were injected inside the HCD cell with near zero kinetic energy to avoid fragmentation via collisions with buffer gas. Ion injection was followed by a 3-ms time delay, after which the axial DC voltage gradient along the HCD cell was inverted (from +20 to −20 V). This voltage inversion allowed focusing of precursor ions near the gun exit, where the cation beam density was higher. Trapped ions were irradiated for no longer than 5 s, after which the DC voltage gradient was changed back to normal, and the product ions were transferred to the C-trap. MS detection was done in the Orbitrap analyzer using standard instrument settings. Fragmentation spectrum was normally accumulated from ten consecutive scans. Safety Considerations. Described experiments involve potential health threats, including high electrical currents and voltages, strong microwave fields, and plasma discharges, and must not be conducted without appropriate technical training. Spectral Peak Notation. In the present study, ion peaks corresponding to the fragmentation of polypeptides are annotated in accordance with the conventional nomenclature.27 N- and C-terminal fragments are denoted as a and x if they are produced via the cleavage of C−CO backbone bond, b and y for C−N, and c and z for N−Cα cleavage, accordingly. Fragments generated via homolytic bond cleavage are marked with a dot (e.g., b· and y·). If intramolecular transfer of hydrogen atom (H·) occurred during dissociation, the hydrogen-accepting fragment (+ H·) is labeled with a prime sign (e.g., y′), and hydrogen-donating (− H·) remains unlabeled (e.g., b). Peak assignment is done using MS-Product utility of online ProteinProspector proteomics tool.28 MS/MS pattern induced in silico included both internal and terminal types of fragmentation (a, b, c, d, v, w, x, y, z) as well as NH3 and H2O neutral losses with 20 ppm mass tolerance.
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RESULTS AND DISCUSSION In this pilot study, we deliberately chose to use air as reagent gas to generate the ion beam. First, air plasma is much more stable and easier to ignite than pure argon, hydrogen, methane, and so forth. Second, the use of air enables very safe and convenient operation of the experimental setup without chemical contamination of the gun chamber. Finally, air is a multicomponent mixture, which affords more constituents in the beam that can potentially react with trapped ions. Reactive species in the air plasma may include both positive and negative ions, as well as electrons, metastables, radicals, and clusters thereof. Many of these species have a low mass (
