Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex
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Manganese oxidation is an important biogeochemical process that is largely regulated by bacteria through enzymatic reactions. However, the detailed mechanism is poorly understood due to challenges in isolating and characterizing these unknown enzymes. A manganese oxidase, Mnx, from Bacillus sp. PL-12 has been successfully overexpressed in active form as a protein complex with a molecular mass of 211 kDa. We have recently used surface induced dissociation (SID) and ion mobility-mass spectrometry (IM-MS) to release and detect folded subcomplexes for determining subunit connectivity and quaternary structure. The data from the native mass spectrometry experiments led to a plausible structural model of this multicopper oxidase, which has been difficult to study by conventional structural biology methods. It was also revealed that each Mnx subunit binds a variable number of copper ions. Becasue of the heterogeneity of the protein and limited mass resolution, ambiguities in assigning some of the observed peaks remained as a barrier to fully understanding the role of metals and potential unknown ligands in Mnx. In this study, we performed SID in a modified Fourier transform-ion cyclotron resonance (FTICR) mass spectrometer. The high mass accuracy and resolution offered by FTICR unveiled unexpected artificial modifications on the protein that had been previously thought to be iron bound species based on lower resolution spectra. Additionally, isotopically resolved spectra of the released subcomplexes revealed the metal binding stoichiometry at different structural levels. This method holds great potential for in-depth characterization of metalloproteins and protein–ligand complexes.
KeywordsNative mass spectrometry High resolution mass spectrometry Protein complex Surface induced dissociation Metalloprotein Protein–ligand interaction
Mass spectrometry (MS) has been extensively used for characterization of proteins . Most of the routine analyses of proteins are carried out at the peptide level from enzymatically digested proteins. However, there is a growing interest in analyzing proteins using a combination of analytical techniques to gain more insight into higher order structure, especially with respect to how the different proteins interact with other proteins . In native MS [3, 4], proteins are ionized from volatile aqueous buffer at physiological pH to preserve the native-like state in the gas phase, where noncovalent interactions are largely maintained. Subunit stoichiometry and protein ligand/metal interactions can be studied by measuring the mass of the intact protein–protein or protein–ligand/metal complexes [5, 6]. The ability of MS for simultaneously detecting coexisting populations of a protein with various binding partners at different masses has provided critical insights for understanding the dynamics of heterogeneous protein complexes [7, 8, 9, 10] and allosteric mechanisms of bound ligands . Recent developments in high resolution MS instrumentation [12, 13, 14] have significantly expanded the structural details that can be obtained because even relatively small differences in ligands/modifications on high mass native proteins can be detected with high mass accuracy [15, 16, 17]. Spectra collected on high resolution mass spectrometers such as Orbitrap and Fourier transform ion cyclotron resonance (FTICR) MS for membrane protein assemblies have also been reported with promising results [18, 19, 20, 21]. In addition, intact protein complexes can be activated in the gas phase to release subcomplexes for mapping the subunit connectivity and characterizing the quaternary structure [4, 22, 23].
Multicopper oxidases (MCOs) have been implicated as the Mn oxidase in several Mn-oxidizing bacteria. These and other microbes contribute significantly to Mn redox cycling in a range of terrestrial and aquatic environments, including soils, sediments, freshwater, and marine systems . However, the roles of manganese oxidases in the geochemical processes of transforming soluble forms of Mn to minerals remain uncharacterized. An active Mn oxidase from Bacillus sp. PL-12, Mnx, has been successfully heterologously overexpressed and purified from E. coli. The overexpression construct contained four of the genes in the polycistronic mnx operon . Previous bottom-up and top-down liquid chromatography- mass spectrometry (LC-MS) experiments have shown that the protein complex consists of three subunits: MnxE (12.2 kDa), MnxF (11.2 kDa), and MnxG (138 kDa) [25, 26]. The presence of accessory proteins was surprising because no MCO has been previously purified as a heteromultimeric protein complex . While MnxE and MnxF both have homologs in other sporulating bacteria, they lack homology to any proteins with known structures or predicted function. Mnx is unusual in that it mediates a two-electron oxidation of a metal whereas all known MCOs with metal substrates only oxidize those metals by one electron . Furthermore, other known MCOs do not require accessory proteins to function. The Mnx complex has not been successfully crystallized, and thus its high-resolution structure remains elusive.
Although the intact mass of the Mnx complex determined by native MS (211.2 ± 0.8 kDa)  restricts the stoichiometry to a maximum of one MnxG and six copies of MnxE and/or MnxF, the exact copy numbers of MnxE and MnxF cannot be confidently determined because they have similar mass. The mass difference between MnxE and MnxF represents only about 0.5% of the mass of the intact complex, and the broad peak detected for the native complex generates about 1% of uncertainty. Additionally, the protein is known to bind 6–15 copper atoms, depending on preparation conditions, as determined by inductively coupled plasma optical emission spectrometry (ICP-OES) . This variation further contributes to the uncertainty of the composition assignment. Breaking down the intact complex into smaller subunits that can be better resolved by MS would allow confident determination of the stoichiometry and metal binding ratios.
We recently performed collision induced dissociation (CID) and surface induced dissociation (SID) of Mnx  on a modified ion mobility-time-of-flight (IM-TOF) mass spectrometer (Waters Synapt G2s, Manchester, UK), an instrument type that has been extensively used for studying protein quaternary structure [22, 29]. In contrast to the commonly used CID, which often causes subunit unfolding [4, 22, 30], SID typically generates folded subcomplexes that better represent the native structure [22, 29]. CID of Mnx resulted in ejection of the MnxE and MnxF monomers from the complex, providing limited information on the inter-subunit connectivity within the protein complex. Instead, at the available collision energy, SID yielded a variety of species, primarily the MnxE/F monomers, MnxE3F3 hexamer, and MnxG. Based on the pattern of the SID products, and specifically the MnxE/F multimers, we proposed the symmetrical structure of MnxE3F3 with alternating MnxE and MnxF subunits. This discovery led to a first plausible structural model for the uncharacterized Mnx complex, preceding any successful characterization by conventional structural biology techniques . In addition, MnxE is shown to bind strongly to at least one copper, while MnxF binds 0-2 Cu atoms with weaker affinity than MnxE. Metal binding by the two accessory proteins suggest they may play a more active role (e.g., serving as Cu chaperones) rather than being simply a structural part of the enzyme. However, several questions remained. Particularly, many suspected MnxE and MnxF peaks could not be confidently assigned due to unidentifiable extra mass based on expected combinations of different subunits and numbers of Cu bound. Because Mnx is known to bind other metal ions, these peaks could correspond to different metal bound MnxEF species . Our ability to confidently identify detected species was limited by the resolution achievable with TOF mass analyzer. However, FTICR MS, with added SID capability , provided the high resolution needed to resolve the uncertainties in peak assignment. The results demonstrate the unique potential of SID combined with high resolution MS for characterization of large heterogeneous protein complexes.
The SID data presented in this work were collected on a modified Bruker (Billerica, MA, USA) SolariX XR 15T FTICR  equipped with a dynamically harmonized cell (ParaCell) . Briefly, the original collision cell was replaced with a custom collision cell, which consist of an SID device followed by a short rectilinear quadrupole for trapping (Ardara Technologies, Ardara, PA, USA). The voltages on the electrodes of the custom cell were controlled both by an external power supply (static DC voltages) and by the existing power supply connections in the instrument (RF and pulsed DC voltages). The instrument performance has been examined in several experiments with well characterized protein complexes, which yielded SID data similar to previous studies in terms of types of fragments but with higher resolution and mass accuracy .
The wild-type Mnx protein was expressed in E. coli with the mnxDEFG operon following methods described previously . The purified protein complex was buffer exchanged into 100 mM ammonium acetate with a Micro Bio-Spin 6 column (Bio-Rad, Hercules, CA, USA) twice before native MS analysis. The protein was sprayed at 2.6 mg/mL (12 μM of Mnx complex) from a glass capillary pulled in-house, with a platinum wire inserted at ground potential. A voltage of –1.25 kV was applied in the inlet capillary for electrospray with dry gas flowing at 4 L/min at 180 °C. The spectra shown in the figures were collected from m/z 500–20,000 at 8 M resolution (transient length 9.2 s), with 300 spectrum averages each at an ion accumulation time of 0.5 s; rf voltages were set to 1.4 MHz, 2000 V in the collision cell, and 1 MHz, 450 V on the transfer guide. The collision cell gas flow was set to 100% for increasing trapping efficiency. In source fragmentation was set to 70 V for optimal desolvation and transmission for the intact Mnx complex. Mass calibration was performed by using perfluoroheptanoic acid (PHFA) up to the m/z of 8000. The absorption mode processing was calibrated with 0.1 mg/mL sodium formate and lithium formate, and the baseline was corrected using the CUSUM method with negative intensity trimmed in the spectra shown.
SID spectra of protein complexes under 150 kDa have been successfully collected with FTICR by using voltages similar to the previously established SID conditions in TOF mass spectrometers. However, using similar conditions on the modified FTICR, SID of larger complexes suffered from low sensitivity. An unconventional tuning was developed to generate SID spectra that are consistent with previous SID data acquired on a Q-IM-TOF for serum amyloid P pentameric complex (data not shown, precursor charge +16 ~ +19, m/z = 7000–8000 similar to the m/z of Mnx precursor). In this tuning, the surface target is at ground with two steering lenses (attractive voltages on the front top and the middle bottom deflectors) bending the ion trajectory off the central axis. We suspect that this tuning causes the precursor ions to collide with a stainless steel electrode on the path, instead of colliding on the gold surface target coated with self-assembled fluorocarbon normally used for SID experiments , based on SIMION simulation (data not shown). It has previously been shown that stainless steel surfaces can successfully generate SID products , and they have been used for SID of protonated peptides , proteins , and protein complexes . Optimization of the SID design for high mass ions in FTICR will require additional studies.
The SID spectra shown in this study were acquired with the acceleration voltage (i.e., the potential difference between the quadrupole and the offset on the shortened collision cell) set to 57 V. The front top and middle bottom deflectors of the SID device were set to –40 V and –50 V, respectively. Other SID lenses were set to ground. Within this collision cell, the protein ions presumably hit and generate SID products after being steered off axis by the voltages in the SID device. For collecting mass spectra, all lenses in the SID device were set to near ground, and the collision voltage was set to 4 V for transmission with minimal activation. Because the quadrupole in the system cannot isolate ions with m/z above 6000, the m/z in the quadrupole tune method was set to 1000 without isolation and used as a high pass filter to remove unwanted low mass species. The formulas of the three subunits of Mnx protein are obtained from the reported sequences in UniProt (Supplementary Table S1). The sequences of MnxE and MnxF have both been validated with top-down analysis . It is noted that MnxF has only been observed with the sequence starting after the second methionine in the sequence reported in UniProt. In addition, the top-down data suggested there is a disulfide bond in MnxE that is responsible for a –2Da mass shift from the expected sequence. The MnxG sequence was confirmed by ~70% coverage with bottom-up analysis (data not shown). However, we have not yet been able to detect the intact MnxG under denaturing conditions, possibly due to low ionization efficiency and/or likelihood of precipitation in organic solvent. The theoretical isotopic distributions of the assigned species were generated using Bruker Data Analysis software using the calculated molecular formula, with peak width set to 0.02. We defined a background noise level to be 2 × 106 (arbitrary unit) by taking the average of the signal in the 16500 < m/z < 18500 where no protein species were detected. All the species assigned in the spectra have intensities significantly above the noise level (S/N > 5).
For bottom-up LC-MS, the higher-energy collisional dissociation (HCD) spectra of Mnx peptides were acquired on a Thermo Orbitrap Elite (data-dependent MS/MS of top six peaks; HCD normalized collision energy 28) equipped with a Waters NanoAcquity liquid chromatography system (70 cm, 75 μm i.d. C18 reversed phase column; mobile phase 0.1% formic acid in H2O/acetonitrile at 0.3 μL/min; acetonitrile gradient from 5% to 35% over 2 h). Mnx protein was denatured in urea, reduced by dithiothreitol, alkylated with iodoacetimide, and digested with trypsin to obtain the Mnx peptides. The LC-MS data were first analyzed with MassMatrix [39, 40] with a database containing Mnx protein sequences and the E coli. proteome. Carbamidomethyl of cysteine was set as a fixed modification. Several custom variable modifications were included: oxidation on methionine, gluconoylation on the protein N-terminus, phosphogluconoylation on the protein N-terminus, and (2-aminoethyl) benzenesulfonyl fluoride hydrochloride on tyrosine. Peptide mass tolerance was set to 10 ppm and fragments mass tolerance was set to 0.01 Da. Spectra associated with modifications of interest were selected and manually annotated.
Results and Discussion
SID Dissects Mnx into Smaller Building Blocks for Determining the Quaternary Structure
Several of the MnxE/F multimers at m/z 5000–7500, especially the MnxE/F tetramers and pentamers, were only present at relatively low abundance and masked by the other more intense signals (Figure 1). The ion mobility separation afforded by the IM-TOF instrument provided an additional dimension of separation and significantly improved the resolving power for adequately interpreting the highly convoluted multi-subunit SID spectrum . However, with the high resolution offered by the FTICR, most of these underlying species can be isotopically resolved and distinguished from overlapping species. The data acquired with the two instruments are highly complementary: the two-dimensional IM-MS provides an effective survey of all the species in the spectra, and the high resolution FTICR provides a means to confidently assign specific peaks unassignable in the IM-MS data and reveal unexpected modifications as discussed below.
Ultra-High Resolution Helps to Decipher the Heterogeneity of Mnx by Identifying Multiple Metal Binding Events and Modifications
The unexpected covalent modifications are further confirmed after the removal of the majority of bound copper. By treating Mnx with ethylenediaminetetraacetic acid (EDTA) prior to MS, we were able to obtain highly simplified SID spectra of released MnxE and MnxF monomers (Supplementary Figure S5). Although all of the unexpected modifications were only detected at less than ~10% of the “native”, unmodified proteoforms, these modifications were the source of additional heterogeneity precluding us from confidently defining metal binding stoichiometry of Mnx, especially when these different proteoforms were assembled into multi-subunit complexes. Gluconoylation is one of the known, undesired post-translational modifications for heterologously expressed proteins . AEBSF has also been reported to covalently modify proteins . Optimization of protein expression and purification conditions to minimize such unwanted modifications would be beneficial, in particular for production of proteins for pharmaceutical and medical applications. It is noted that both the gluconoylation (Figure 4b) and AEBSF (Figure 4c) have a similar unit mass to that of multiple Cu bound MnxF (Figure 4d). Hence, high resolution is essential for characterization of highly heterogeneous systems because some components (including unexpected modifications) could easily become indistinguishable at lower resolution. As a proof of principle, the high resolution spectrum was smoothed and the resolution was reduced by a factor of 10 from about 100 k to 10 k, as shown by the original spectrum (green line) and the smoothed spectrum (dotted line) in Figure 4a. The MnxF+4Cu species became buried and invisible, whereas the gluconoylation and AEBSF species coalesced into a mixed isotope distribution that is difficult to interpret. For larger MnxE/F multimers (Supplementary Figure S6), overlapping isotope distributions (consistent with multiple bound Cu and modifications) may not be sufficiently resolved and distinguished from each other even with the high resolution of FTICR. Such sample heterogeneity arising from undesirable artifacts introduced during sample handling remains a significant challenge for achieving the ultimate resolution for large proteins and protein complexes despite the ever increasing resolving power of the mass analyzer. Nonetheless, the ability to confidently identify unexpected modifications is critical for improving the homogeneity of protein samples for other techniques such as X-ray crystallography, which benefit from obtaining highly homogeneous material .
Sample Heterogeneity and Incomplete Desolvation are Major Obstacles for High Resolution Analysis of Native Proteins
The theoretical isotope distribution of completely desolvated MnxE3F3 hexamer carrying 7–9 Cu (Figure 6c) can be matched to the three major peaks in the 7050 < m/z < 7070 range. It is interesting to note that the baseline of the peak is increased starting from m/z 7050 to 7150. Apart from the modifications, the elevated baseline likely originates from extra adducts (solvent, salt, etc.) which adds to the heterogeneity that cannot be resolved even with high resolution. The ability to resolve different Cu bound species in Figure 6 is attributed to the sufficient desolvation that occurred upon ionization, SID, and measurement in the FTICR, yielding several relatively well-defined, homogenous species at high abundance above the baseline. Instrument conditions and salts in sample buffer tend to affect desolvation and may hamper differentiation of species bound to varying number of Cu. Fluctuations in such minor experimental details are usually overlooked because the small buffer adducts typically cannot be resolved at low resolution. But they can affect spectral quality to a remarkable extent in high resolution measurements, affecting the ability to resolve metal binding on large protein complexes.
A heterogeneous protein complex Mnx, previously characterized only by native MS on Q-IM-TOF and refractive to characterization by other structural biology tools, has been interrogated by SID coupled with high resolution MS. Dissecting the intact complex into smaller subunits by SID reduces the complexity and allowed the heterogeneity of each subunit to be readily determined. The noncovalently bound metals were maintained in the released subunits for further characterization because of the minimal unfolding introduced by SID in the activation process. Two of the protein subunits, MnxE and MnxF, released from the intact complex were shown to bind Cu at varying ratios (average Cu load 1.2 and 1.4 per monomer, respectively), and were partially modified to varying degrees. The Cu load on the MnxE3F3 hexamer was directly measured to be 7–9 with the isotopically resolved hexamer peaks despite the heterogeneity of the protein, and is qualitatively consistent with an average of about 1.3 Cu per monomer unit. The metal binding stoichiometry derived from the most abundant charge states of the same species was similar. Previously, the Cu load in Mnx was estimated to be at least 8 by electron paramagnetic resonance (EPR)  and a variable value of 10–15 by ICP-OES depending on the dialysis buffer . Apart from the four canonical Cu in MnxG that are essential for catalytic activity for MCOs, the majority of the remaining 6–11 Cu are most likely located in MnxE and MnxF as suggested by the SID spectra. In addition, Cu can be partially removed from Mnx with metal chelators, resulting in change of catalytic activity [26, 53]. Recent SID experiments also showed that the Cu bound to MnxF are labile and could be the primary cause for the varying Cu load of Mnx under different buffer conditions . Although our goal of this manuscript is to demonstrate the value of high resolution MS for studying metalloprotein complexes, future work will utilize SID to characterize Mnx under different conditions to quantitatively monitor the dynamics of metal binding within individual subunits.
Combining SID and FTICR allowed “high resolution” dissection of the Mnx complex at each substructure level, from MnxE/F monomers up to MnxE3F3 hexamer. Furthermore, unexpected modifications, gluconoylation, and AEBSF, were identified based on the accurate mass. These modified species can easily be misinterpreted as iron-containing species at lower resolution. High resolution was critical for eliminating the ambiguity of the peak assignment for species with very similar masses. Information regarding metal binding and modification on individual subunits within a protein complex cannot be easily obtained using techniques that only examine large ensembles. The native MS and SID method described here offers a unique opportunity for structural analysis of individual building blocks through dissecting intact protein complexes in the gas phase.
There are still significant challenges in resolving isotopic distributions of very large proteins and protein complexes due to heterogeneity, incomplete desolvation, and decreased sensitivity for detection of high mass ions. Future instrument developments are expected to increase ion transmission in the high m/z range and reduce the acquisition time (i.e., number of averages) needed for high resolution native MS, thus making it more amenable to coupling with online separations for high throughput analysis. A combination of different methods, in particular effective ion activation methods such as SID, is indispensable for thorough characterization of heterogeneous protein complexes. While both CID and SID primarily induce cleavage of noncovalent interactions in protein complexes, other activation methods, including ultraviolet photodissociation (UVPD) , electron capture dissociation (ECD) [56, 57], electron transfer dissociation , and electron ionization dissociation (EID)  have been applied for native proteins. These methods generate protein backbone fragments for structural elucidation at the residue level. Ideally, the folded subunits released by SID can be examined by these methods in order to probe metal/ligand binding sites and potentially further break down the complexity of large proteins. Development of “top-down” workflows for intact proteins [60, 61, 62] and native protein complexes is anticipated to provide an alternative structural biology tool for in-depth and rapid characterization of proteins.
The authors thank Yang Song and Arpad Somogyi at The Ohio State University for helping with the SID experiments; Jeremy Wolff and Michael Easterling at Bruker Corporation, Randy Pedder at Ardara Technologies for helping with the instrument modification. This work was funded by the National Science Foundation (NSF DBI 1455654; SID development and installation) and National Institute of Health (NIH 1S10OD018507; FTICR purchase) to V.H.W., NSF CHE-1410688 to B.M.T, and an NSF Postdoctoral Research Fellowship in Biology Award ID: DBI-1202859 to C.A.R. A portion of the research was supported by the Environmental and Molecular Sciences Laboratory (EMSL), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory.
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