METHODS AND APPLICATIONS Electron-capture dissociation and ion mobility mass spectrometry for characterization of the hemoglobin protein assembly
Weidong Cui,1 Hao Zhang,1 Robert E. Blankenship,1,2 and Michael L. Gross1* 1
Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130
2
Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130
Received 12 December 2014; Accepted 19 May 2015 DOI: 10.1002/pro.2712 Published online 1 June 2015 proteinscience.org
Abstract: Native spray has the potential to probe biophysical properties of protein assemblies. Here we report an investigation using both ECD top-down sequencing with an FTICR mass spectrometer and ion mobility (IM) measurements on a Q-TOF to investigate the collisionally induced unfolding of a native-like heterogeneous tetrameric assembly, human hemoglobin (hHb), in the gas phase. To our knowledge, this is the first report combining ECD and ion-mobility data on the same target protein assembly to delineate the effects of collisional activation on both assembly size and the extent and location of fragmentation. Although the collision-induced unfolding of the hemoglobin assembly is clearly seen by both IMMS and ECD, the latter delineates the regions that increasingly unfold as the collision energy is increased. The results are consistent with previous outcomes for homogeneous protein assemblies and reinforce our interpretation that activation opens the structure of the protein assembly from the flexible regions to make available ECD fragmentation, without dissociating the component proteins. Keywords: electron capture dissociation; ion mobility; protein assembly; mass spectrometry; hemoglobin; B factor
Introduction Additional Supporting Information may be found in the online version of this article. Grant sponsor: Photosynthetic Antenna Research Center, an Energy Frontier Research Center funded by the U.S. DOE, Office of Basic Energy Sciences; Grant numbers: DE-SC 0001035; Grant sponsor: National Institute of General Medical Science; Grant numbers: 8 P41 GM103422; Grant sponsors: DOE, NIH. *Correspondence to: Michael L. Gross, Department of Chemistry, Washington University in St Louis, One Brookings Drive, Box 1134, St Louis, MO 63130. E-mail: [email protected]
C 2015 The Protein Society Published by Wiley-Blackwell. V
Determining the higher order structure of proteins is key to understanding protein function. Although many structural-biology methods are available, solving protein high order structure remains a large challenge. The functional units in most biological systems are multi-subunit protein assemblies. Such assemblies have molecular weights (MW) ranging from kilo- to mega-daltons and contain many different proteins and ligands.1 The subunits are held together by noncovalent interactions that are
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affected when the assembly experiences different environments other than their native one. Because topology, stoichiometry, and protein flexibility are missed by studying the subunits alone, methods to characterize intact protein assemblies by top-down methods are needed in structural biology.2 X-ray crystallography and NMR spectroscopy are capable of providing high resolution models of protein assemblies.3,4 The requirements of crystallization (X-ray) and relatively small protein size and high concentration (NMR) limit their applications, however. In the low resolution end of the spectrum of structural methods are CD, fluorescence, calorimetry, and ultra-centrifugation. Information from those methods complement and guide other high resolution approaches. We are motivated by the notion that mass spectrometry (MS) can occupy a middle position in structural biology owing to its sensitivity, speed, and ability to afford more structural information than the low resolution approaches.5 Currently there are two major approaches to characterizing intact protein assemblies by using modern MS and analytical proteomics.6 One includes protein footprinting and crosslinking, which have in common the need for proteomic analysis to complete the experiments. The usual bottom-up MS analysis provides detailed peptide and even amino-acid residue information.7–11 The other approach is native electrospray ionization (ESI or native MS) and topdown analysis, which give a global view of the system.12–14 The advantages of both approaches are small sample consumption, high upper mass, and capacity to probe the native or near-native states of protein assemblies. Whereas footprinting is tolerant of highly complex solution media, even those containing MS-unfriendly small molecules, native ESI and top-down analysis can add specificity and high throughput to systems with sample heterogeneity. Native ESI and top-down approaches are attractive because intact protein assemblies and their constituents can be directly observed and interrogated in near native states15 even after the assemblies are transferred to the gas phase. The advantages of such an approach were recognized decades ago.12 As high-MW mass spectrometers and new methods of ion activation emerged, a new field of MS-based studies of protein assemblies opened16 to afford stoichiometry, topology, and subunit interactions of both soluble and membrane-embedded protein assemblies.17 Measurements of ion mobility (IM) can inform on conformational changes and intermediates formed upon activation of protein assemblies.18,19 Activation methods now include electron-transfer dissociation (ETD) and electron-capture dissociation (ECD); the use of these approaches can locate fragile modifications (e.g., phosphorylation and glycosylation).20 Fragmentation of small noncovalent protein-
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ligand complexes by ECD without breaking the noncovalent interactions can provide new structural information as to locate the ligand-binding pocket.21 We discovered in 2010 that native spray coupled with top-down ECD can generate sequence information from certain subunits in modest sized protein assemblies.22 Mapping the region that fragments upon ECD onto the X-ray crystal structure demonstrates that the fragmentation occurs in those regions possessing high B-factors (i.e., those highly flexible regions that undergo diffuse X-ray scattering23). Thus far, we have studied only homogeneous protein assemblies by using this approach. Given that 25% of the protein assemblies in the PDB are heterogeneous,24 we address the first of these assemblies, namely human adult hemoglobin (hHb) tetramer, which is composed of one pair of a and one pair of b-chains. Owing to the clinical importance of hemoglobin, identification of the chain variants in the primary sequence by MS was addressed in previous studies using top-down.25–28 In these analytical studies, the Hb assemblies were denatured to release the constituent chains. For example, Costello and coworkers27 sequenced the individual chains by direct infusion of a whole blood sample dissolved in 50/50 water/acetonitrile with 0.1% formic acid. For our biophysical characterization of intact hHb assembly by MS; however, we cannot use denaturing ESI to maintain intact the tetrameric form. Instead we used native spray to investigate the effect of collisional activation on hHb by both IM and ECD top-down sequencing.
Materials and Methods Materials Human hemoglobin (hHb), ammonium acetate and HPLC-grade water were purchased from Sigma Aldrich (St. Louis, MO).
Top-down MS ECD top-down experiments were performed on a 12 T FTICR mass spectrometer (Bruker Daltonics, Billerica, MA). hHb was dissolved in 100 mM aqueous ammonium acetate solution and introduced by nanospray into the 12 T FTICR mass spectrometer. Polymicro silicon tubing (O.D. 360 mm and I.D. 150 mm) was custom pulled in a microcapillary puller (Sutter Instrument, Novato, CA) to make spray tips. A PHD/Ultra syringe pump (Harvard Apparatus, Holliston, MA) was used to infuse the hHb tetramer solution at 2 lM. By adjusting the front-end ionoptics parameters, the complete narrow charge state envelop was subjected to ECD. Data analysis of the spectra and fragment assignments were performed with Bruker software and ProsightPTM (https:// prosightptm.northwestern.edu/).
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outcome is significantly different than the high charge and wide charge-state distribution of a single protein component by normal ESI. In addition to the tetrameric-assembly ions, monomer, dimer, and lowabundant hexamer species were also produced (Supporting Information, Fig. S1). The production of monomer and dimer species from commercially available hemoglobin is not surprising.39–41 Hexamers likely form through nonspecific interactions of smaller oligomers. Our focus here, however, is on the intact hemoglobin assembly, and only results from hHb tetramer are presented and discussed.
Ion mobility (IM) measurements Figure 1. Drift time measurement of the 171 charge state at various collisional activation voltages.
Ion mobility MS A 5 lL sample was loaded into an offline electrospray capillary (GlassTip 2 lm ID, New Objective, Woburn, MA). The sample solution was injected into a hybrid ion-mobility quadrupole time-of-flight mass spectrometer (Q-IM-TOF, SYNAPT G2 HDMS, Waters, Milford, MA). The instrument was operated under gentle ESI conditions (capillary voltage 1.5– 1.8 kV, sampling cone 20–100 V, extraction cone 2–3 V, source temperature 308C). The collision energy at the trap and transfer region was optimized for dissociating the complex. The pressure of the vacuum/ backing region was 5.1–5.6 mbar. For the ionmobility measurements, the helium gas flow to the collision cell was 180 mL/min, the IMS gas flow was 90 mL/min, the IMS wave velocity was 650 m/s, and the IMS wave height was 40 V. Nitrogen was used as the mobility carrier gas. Each spectrum was acquired over the range m/z 1500–7500 every 1 s. The instrument was externally calibrated up to 8000 m/z with the clusters produced by ESI of a 100 mg/mL NaI solution. The peak picking and data processing were achieved by using Masslynx (v 4.1) and DriftScope software (Water, Milford, MA). The collisional-cross sections for protein ions were converted by following previous published calibration protocols and databases.29,30
Results and Disscussion Native ESI of hHB The adult human hemoglobin assembly contains two pairs of polypeptide chains, a and b, and four heme groups containing iron ions. The assemblies have been investigated by MS-based approaches for some time.31–39 Native spray of the intact assemblies generated charge states spanning 141 to 181 with 161 the most abundant on both the QToF and the FTICR instruments that we used in this research (Fig. 1 inset, see also Supporting Information S1). This
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IM depends on the collisional cross-section (CCS) of an ion drifting through an inert buffer gas in a low electric field. The ion experiences many collisions with inert buffer-gas molecules such that its drift time is related to a two-dimensional projection (or CCS) of the three-dimensional shape of the ion in free rotation. This drift time [or arrival-time distribution (ATD)] is long for ions with a large cross section in a manner similar to that of a native-gel experiment. Thus, proteins and protein assemblies can be distinguished based on differences in their 3D structures.42,43 The cross-sections of human hemoglobin ions have been measured by several groups.34,37,44 Here we use IM to provide evidence that a heterogeneous native-protein assembly, when submitted to collisional activation in a tandem mass spectrometer,45 undergoes conformational changes to increase its cross-section, an idea tested earlier by Robinson and coworkers.46,47 Upon introduction of the hemoglobin assembly into a quadrupole time-of-flight tandem mass spectrometer (Waters Synapt G2) and an increase in the collision energy, the drift time profile of the hemoglobin assembly (171 charge state) shifted continuously to longer times, as expected (Fig. 1). Each of the drift-time profiles prior to smoothing is comprised of a set of small cross sectional distributions. At 18 V collisional activation, the first CCS of 171 hemoglobin tetramer at 20% ˚ 2, and the last is maximum peak amplitude is 4380 A 2 ˚ 4950 A . This range agrees with the value of about ˚ 2 measured by Scrivens and coworkers37 4300 A using a Waters Synapt HDMS system. To assess more quantitatively the consequences of increasing collisional activation, we determined the range of CCSs at 88 V activation, the highest in our experi˚ 2. Clearly, collisional activament, to be 6270–6810 A tion opens up the Hb complex ions and increases their CCS by 40% for the largest activation used here. Furthermore, Figure 1 shows that the abundance of the 171 charge state decreased at higher activation voltages. This was caused in part by scattering and dissociation of ions in the regions where collision voltages were applied. Although the
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should unfold by breaking non-covalent interactions that hold together the protein-assembly structure, and expose more the flexible constituent polypeptide chains. In the case of the hemoglobin assembly, increasing numbers of high m/z fragment ions are produced when the collision voltage for activation is increased from 10 to 125 V (Fig. 3). This trend strongly suggests that, upon collisional activation, the whole assembly starts to unfold along the N terminus, allowing ECD to extend more deeply along the sequence starting from the flexible regions.
ECD sequence coverage on both a- and bchains
Figure 2. ECD spectra of the complete, mass-selected charge state envelop (inset) of hHb tetramer after activation in the nozzle-skimmer region by collisions at 10, 50, 75, 100, and 125 V. IS stands for in-source.
increase in the drift time at higher activation reveals that a fraction of the tetramer is unfolding while remaining assembled, IM cannot locate the regions that are unfolding. For this, we rely on ECD and our hypothesis that ECD generates fragments starting in flexible regions of the assembly.
ECD of hHB tetramer ECD of the whole charge state envelop of hHb (from 141 to 181, Fig. 2) produced a similar fragmentation pattern to that of the homogeneous complexes published by us before and recently by the Loo group,48,49 and to that of formed by ETD as reported by the Sabott group.50 In the low m/z region, usually below m/z 2000, are the sequence ions of the constituent subunits; in the high m/z region, beginning at the m/z of the intact assembly ions, are the charge-reduced intact tetramers. The sequence ions generated upon native MS introduction and ECD top-down sequencing not only permit identification of subunits of the hemoglobin assembly but also afford information on regions we have hypothesized to be flexible in the assembly. The question we address now is the effect of collisional activation on the assembly. Specifically, can we learn those regions that open to increase the collision cross-section, as determined by IM? To answer the question, we submitted the assembly to ECD. Previous studies showed that activated-ion, electroncapture dissociation (AI-ECD) generates higher sequence coverage in top-down sequencing of denatured protein ions.51,52 This principle can be applied to the hHb protein assembly introduced by native spray. By activation in the source or in the collisioncell region, the flexible region of protein assembly
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The high mass resolving power spectra from the FTICR instrument provide confident identification of sequence ions from both chains. We plotted the assigned ion signals with normalized intensities as a function of the activation voltages (Fig. 3). The sequence coverage at 100 V activation is highlighted on the amino-acid sequence (Fig. 3, insert). Most of the assigned ions are c-type from N termini (highlighted in red). Only a small number of z ions were observed, as highlighted in purple in the sequence. It is clear that N-terminal fragmentation can reach deeper along both a- and b-chains when the whole assembly is activated. The approximate minimum voltage setting that allows ions to traverse the entire path is 10 V in the nozzle-skimmer region (in-source activation). The fragment ions, formed with this minimum collisional pre-excitation contain c ions that contain up to 23 residues from both chains. At 100 and 125 V activation (laboratory frame), fragmentation occurs at peptide bonds for over 60 residues of the C terminus, demonstrating a considerable amount of unfolding can occur upon collisional activation without rupturing the assembly.
Correlation of B-factors and ECD We previously observed that ECD of homogenous protein assemblies involves flexible regions of an assembly, as revealed by correlating sites of fragmentation and B-factors from the X-ray crystal structure.23 hHb is an example of a heterogeneous tetramer. Because ECD fragments were produced from both component chains, their flexibility must be similar. We chose the crystal structure of the deoxy-Hb (PDB code 4hhb) to assist our interpretation of the MS results. From the crystal structure of this tetramer, we see that the N-termini of the two a- and b-chains are both exposed and have the highest B-factors (Fig. 4). The observed ECD fragments map faithfully onto those regions of the structure, supporting that both collisional activation and electron capture cause flexible regions of a heterogeneous assembly to
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Figure 3. Assignment of N-terminal ECD cleavage sites on both a- (top) and b- (bottom) chains of hemoglobin tetramer, and comparison with the average B-factor values of the two a- and b-chains, respectively.
fragment similarly to homogeneous assemblies. This is seen most clearly by the trends in fragmentation and in average B-factor values of the two a- and bchains (Fig. 3), respectively. For both a- and bchains, the B-factor goes from a high value at the termini to a low value around the 30–35th residues and then returns to intermediate values around the 50th residue. Without prior collisional activation, ECD stops before the low B-factor region. Ramping the activation energy presumably unfolds more of this flexible region, making more regions along the polypeptide chain more vulnerable for fragmentation. The C-terminus is buried beneath this region and has low B-factors for both chains, prohibiting formation of z-ions (Fig. 3, 100 V in-source
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activation). This was likely caused by unfolding of buried regions to expose them and allow a small number of fragments to be generated. Hydrogen deuterium exchange mass spectrometry (HDX MS) can reflect protein dynamics and conformational changes and report on flexible regions. The Konermann group reported the HDX behavior of oxy-Hb, deoxy-Hb, and aquomet-Hb.32,53 They identified several regions that showed deuterium saturation (90%) in a short time frame across the three forms of hemoglobin, even the ligand binding states. For the deoxy-Hb, those regions are the amino acids of (1) the a-chain 1–23, 67–80, 135–141 and (2) the b-chain 1–8, 41–47. Because vertebrate hemoglobins share almost identical 3D structures,54
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different charge states to be the salt bridges of its high-resolution structure. For protein assemblies, it is highly desirable to obtain more structural information, for example, salt bridge sites between subunits and/or among a subunit, from the types and the abundance changes of the ECD fragments. We are in the process of analyzing such data in protein systems we have investigated, seeking rules regarding protein– protein interaction interfaces. Although we cannot compare the exact collision energies on both the FTICR and Synapt G2 instruments, the data trends are consistent. IM reports conformational changes of protein assemblies upon collisional activation and shows that the cross section of the protein is increasing with collision energy increments. AI-ECD not only reveals that whole assembly becomes reactive as collisional energy increases, but also provides the location of the change/unfolding. An instrument with integrated capabilities of both IM and ETD/ECD would be ideal for such purposes, as we pointed out earlier.56 Approaches of this nature should be invaluable in guiding theoretical modeling of the behavior of protein complexes in vacuo. For example, an electrostatic model was developed to elucidate the charge-partitioning mechanism during CID of hemoglobin complexes.34 If it had been known that unfolding started from the N-terminus, the model could be made more detailed and accurate. Moreover, our results could provide insights into establishing a molecular dynamics (MD) model of protein structural folding, unfolding and dynamics, by which, for example, Robinson57 obtained structural information complementary to IM to understand the charge-state dependent compaction and dissociation of protein complexes. ECD provides an experimental approach to this problem. Figure 4. X-ray structure of hHb tetramer shown in B-factor scale (PDB code 4hhb). Top, two a-chains in color; bottom, two b-chains in color.
the HDX results support our interpretation of the ECD top-down data to indicate flexibility at the Nterminal regions. The changes in relative abundances of specific cions as a function of activation locates the structural unfolding of the assembly upon activation, as was determined by IM. For example, c23 at 10 V in-source activation, increased in abundance at 50 and 75 V activation then decreased at 100 and 125 V activation. Most of the c-ions larger than c34, seen at 50 V activation, show increased abundances as the activation voltage increases. The abundance variation of fragment ions observed upon activation-voltage ramping may contain 3D structural information of the hemoglobin assembly. Breuker and coworkers55 followed the abundance changes of ECD fragments of a 10 kDa protein, KIX, and attributed those that change at
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Conclusions Protein unfolding induced by collisional activation of protein assemblies can be seen by IM and located by ECD. The unfolding increases the protein cross sections measured by IM, and ECD reveals where unfolding occurs. The unfolded regions are predictable from the B factors of the protein assembly. Use of various fragmentation methods (i.e., CAD, SORICAD, ETD, ECD, IRMPD) available on the FTICR platform, and combinations of these methods augmented by ultraviolet photodissociation58 and trapped-IM spectrometry (TIMS),59 promise to generate even more structural information about protein assemblies. This work is a needed stepping stone for the development of a top-down approach generally applicable for the biophysical characterization of intact biomolecular assemblies.
ACKNOWLEDGMENTS HZ thanks the Am. Soc. for Mass Spectrom for a postdoctoral award.
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ECD & IM for Hemoglobin
Weidong Cui,1 Hao Zhang,1 Robert E. Blankenship,1,2 and Michael L. Gross1* 1
Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130
2
Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130
Received 12 December 2014; Accepted 19 May 2015 DOI: 10.1002/pro.2712 Published online 1 June 2015 proteinscience.org
Abstract: Native spray has the potential to probe biophysical properties of protein assemblies. Here we report an investigation using both ECD top-down sequencing with an FTICR mass spectrometer and ion mobility (IM) measurements on a Q-TOF to investigate the collisionally induced unfolding of a native-like heterogeneous tetrameric assembly, human hemoglobin (hHb), in the gas phase. To our knowledge, this is the first report combining ECD and ion-mobility data on the same target protein assembly to delineate the effects of collisional activation on both assembly size and the extent and location of fragmentation. Although the collision-induced unfolding of the hemoglobin assembly is clearly seen by both IMMS and ECD, the latter delineates the regions that increasingly unfold as the collision energy is increased. The results are consistent with previous outcomes for homogeneous protein assemblies and reinforce our interpretation that activation opens the structure of the protein assembly from the flexible regions to make available ECD fragmentation, without dissociating the component proteins. Keywords: electron capture dissociation; ion mobility; protein assembly; mass spectrometry; hemoglobin; B factor
Introduction Additional Supporting Information may be found in the online version of this article. Grant sponsor: Photosynthetic Antenna Research Center, an Energy Frontier Research Center funded by the U.S. DOE, Office of Basic Energy Sciences; Grant numbers: DE-SC 0001035; Grant sponsor: National Institute of General Medical Science; Grant numbers: 8 P41 GM103422; Grant sponsors: DOE, NIH. *Correspondence to: Michael L. Gross, Department of Chemistry, Washington University in St Louis, One Brookings Drive, Box 1134, St Louis, MO 63130. E-mail: [email protected]
C 2015 The Protein Society Published by Wiley-Blackwell. V
Determining the higher order structure of proteins is key to understanding protein function. Although many structural-biology methods are available, solving protein high order structure remains a large challenge. The functional units in most biological systems are multi-subunit protein assemblies. Such assemblies have molecular weights (MW) ranging from kilo- to mega-daltons and contain many different proteins and ligands.1 The subunits are held together by noncovalent interactions that are
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affected when the assembly experiences different environments other than their native one. Because topology, stoichiometry, and protein flexibility are missed by studying the subunits alone, methods to characterize intact protein assemblies by top-down methods are needed in structural biology.2 X-ray crystallography and NMR spectroscopy are capable of providing high resolution models of protein assemblies.3,4 The requirements of crystallization (X-ray) and relatively small protein size and high concentration (NMR) limit their applications, however. In the low resolution end of the spectrum of structural methods are CD, fluorescence, calorimetry, and ultra-centrifugation. Information from those methods complement and guide other high resolution approaches. We are motivated by the notion that mass spectrometry (MS) can occupy a middle position in structural biology owing to its sensitivity, speed, and ability to afford more structural information than the low resolution approaches.5 Currently there are two major approaches to characterizing intact protein assemblies by using modern MS and analytical proteomics.6 One includes protein footprinting and crosslinking, which have in common the need for proteomic analysis to complete the experiments. The usual bottom-up MS analysis provides detailed peptide and even amino-acid residue information.7–11 The other approach is native electrospray ionization (ESI or native MS) and topdown analysis, which give a global view of the system.12–14 The advantages of both approaches are small sample consumption, high upper mass, and capacity to probe the native or near-native states of protein assemblies. Whereas footprinting is tolerant of highly complex solution media, even those containing MS-unfriendly small molecules, native ESI and top-down analysis can add specificity and high throughput to systems with sample heterogeneity. Native ESI and top-down approaches are attractive because intact protein assemblies and their constituents can be directly observed and interrogated in near native states15 even after the assemblies are transferred to the gas phase. The advantages of such an approach were recognized decades ago.12 As high-MW mass spectrometers and new methods of ion activation emerged, a new field of MS-based studies of protein assemblies opened16 to afford stoichiometry, topology, and subunit interactions of both soluble and membrane-embedded protein assemblies.17 Measurements of ion mobility (IM) can inform on conformational changes and intermediates formed upon activation of protein assemblies.18,19 Activation methods now include electron-transfer dissociation (ETD) and electron-capture dissociation (ECD); the use of these approaches can locate fragile modifications (e.g., phosphorylation and glycosylation).20 Fragmentation of small noncovalent protein-
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ligand complexes by ECD without breaking the noncovalent interactions can provide new structural information as to locate the ligand-binding pocket.21 We discovered in 2010 that native spray coupled with top-down ECD can generate sequence information from certain subunits in modest sized protein assemblies.22 Mapping the region that fragments upon ECD onto the X-ray crystal structure demonstrates that the fragmentation occurs in those regions possessing high B-factors (i.e., those highly flexible regions that undergo diffuse X-ray scattering23). Thus far, we have studied only homogeneous protein assemblies by using this approach. Given that 25% of the protein assemblies in the PDB are heterogeneous,24 we address the first of these assemblies, namely human adult hemoglobin (hHb) tetramer, which is composed of one pair of a and one pair of b-chains. Owing to the clinical importance of hemoglobin, identification of the chain variants in the primary sequence by MS was addressed in previous studies using top-down.25–28 In these analytical studies, the Hb assemblies were denatured to release the constituent chains. For example, Costello and coworkers27 sequenced the individual chains by direct infusion of a whole blood sample dissolved in 50/50 water/acetonitrile with 0.1% formic acid. For our biophysical characterization of intact hHb assembly by MS; however, we cannot use denaturing ESI to maintain intact the tetrameric form. Instead we used native spray to investigate the effect of collisional activation on hHb by both IM and ECD top-down sequencing.
Materials and Methods Materials Human hemoglobin (hHb), ammonium acetate and HPLC-grade water were purchased from Sigma Aldrich (St. Louis, MO).
Top-down MS ECD top-down experiments were performed on a 12 T FTICR mass spectrometer (Bruker Daltonics, Billerica, MA). hHb was dissolved in 100 mM aqueous ammonium acetate solution and introduced by nanospray into the 12 T FTICR mass spectrometer. Polymicro silicon tubing (O.D. 360 mm and I.D. 150 mm) was custom pulled in a microcapillary puller (Sutter Instrument, Novato, CA) to make spray tips. A PHD/Ultra syringe pump (Harvard Apparatus, Holliston, MA) was used to infuse the hHb tetramer solution at 2 lM. By adjusting the front-end ionoptics parameters, the complete narrow charge state envelop was subjected to ECD. Data analysis of the spectra and fragment assignments were performed with Bruker software and ProsightPTM (https:// prosightptm.northwestern.edu/).
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outcome is significantly different than the high charge and wide charge-state distribution of a single protein component by normal ESI. In addition to the tetrameric-assembly ions, monomer, dimer, and lowabundant hexamer species were also produced (Supporting Information, Fig. S1). The production of monomer and dimer species from commercially available hemoglobin is not surprising.39–41 Hexamers likely form through nonspecific interactions of smaller oligomers. Our focus here, however, is on the intact hemoglobin assembly, and only results from hHb tetramer are presented and discussed.
Ion mobility (IM) measurements Figure 1. Drift time measurement of the 171 charge state at various collisional activation voltages.
Ion mobility MS A 5 lL sample was loaded into an offline electrospray capillary (GlassTip 2 lm ID, New Objective, Woburn, MA). The sample solution was injected into a hybrid ion-mobility quadrupole time-of-flight mass spectrometer (Q-IM-TOF, SYNAPT G2 HDMS, Waters, Milford, MA). The instrument was operated under gentle ESI conditions (capillary voltage 1.5– 1.8 kV, sampling cone 20–100 V, extraction cone 2–3 V, source temperature 308C). The collision energy at the trap and transfer region was optimized for dissociating the complex. The pressure of the vacuum/ backing region was 5.1–5.6 mbar. For the ionmobility measurements, the helium gas flow to the collision cell was 180 mL/min, the IMS gas flow was 90 mL/min, the IMS wave velocity was 650 m/s, and the IMS wave height was 40 V. Nitrogen was used as the mobility carrier gas. Each spectrum was acquired over the range m/z 1500–7500 every 1 s. The instrument was externally calibrated up to 8000 m/z with the clusters produced by ESI of a 100 mg/mL NaI solution. The peak picking and data processing were achieved by using Masslynx (v 4.1) and DriftScope software (Water, Milford, MA). The collisional-cross sections for protein ions were converted by following previous published calibration protocols and databases.29,30
Results and Disscussion Native ESI of hHB The adult human hemoglobin assembly contains two pairs of polypeptide chains, a and b, and four heme groups containing iron ions. The assemblies have been investigated by MS-based approaches for some time.31–39 Native spray of the intact assemblies generated charge states spanning 141 to 181 with 161 the most abundant on both the QToF and the FTICR instruments that we used in this research (Fig. 1 inset, see also Supporting Information S1). This
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IM depends on the collisional cross-section (CCS) of an ion drifting through an inert buffer gas in a low electric field. The ion experiences many collisions with inert buffer-gas molecules such that its drift time is related to a two-dimensional projection (or CCS) of the three-dimensional shape of the ion in free rotation. This drift time [or arrival-time distribution (ATD)] is long for ions with a large cross section in a manner similar to that of a native-gel experiment. Thus, proteins and protein assemblies can be distinguished based on differences in their 3D structures.42,43 The cross-sections of human hemoglobin ions have been measured by several groups.34,37,44 Here we use IM to provide evidence that a heterogeneous native-protein assembly, when submitted to collisional activation in a tandem mass spectrometer,45 undergoes conformational changes to increase its cross-section, an idea tested earlier by Robinson and coworkers.46,47 Upon introduction of the hemoglobin assembly into a quadrupole time-of-flight tandem mass spectrometer (Waters Synapt G2) and an increase in the collision energy, the drift time profile of the hemoglobin assembly (171 charge state) shifted continuously to longer times, as expected (Fig. 1). Each of the drift-time profiles prior to smoothing is comprised of a set of small cross sectional distributions. At 18 V collisional activation, the first CCS of 171 hemoglobin tetramer at 20% ˚ 2, and the last is maximum peak amplitude is 4380 A 2 ˚ 4950 A . This range agrees with the value of about ˚ 2 measured by Scrivens and coworkers37 4300 A using a Waters Synapt HDMS system. To assess more quantitatively the consequences of increasing collisional activation, we determined the range of CCSs at 88 V activation, the highest in our experi˚ 2. Clearly, collisional activament, to be 6270–6810 A tion opens up the Hb complex ions and increases their CCS by 40% for the largest activation used here. Furthermore, Figure 1 shows that the abundance of the 171 charge state decreased at higher activation voltages. This was caused in part by scattering and dissociation of ions in the regions where collision voltages were applied. Although the
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should unfold by breaking non-covalent interactions that hold together the protein-assembly structure, and expose more the flexible constituent polypeptide chains. In the case of the hemoglobin assembly, increasing numbers of high m/z fragment ions are produced when the collision voltage for activation is increased from 10 to 125 V (Fig. 3). This trend strongly suggests that, upon collisional activation, the whole assembly starts to unfold along the N terminus, allowing ECD to extend more deeply along the sequence starting from the flexible regions.
ECD sequence coverage on both a- and bchains
Figure 2. ECD spectra of the complete, mass-selected charge state envelop (inset) of hHb tetramer after activation in the nozzle-skimmer region by collisions at 10, 50, 75, 100, and 125 V. IS stands for in-source.
increase in the drift time at higher activation reveals that a fraction of the tetramer is unfolding while remaining assembled, IM cannot locate the regions that are unfolding. For this, we rely on ECD and our hypothesis that ECD generates fragments starting in flexible regions of the assembly.
ECD of hHB tetramer ECD of the whole charge state envelop of hHb (from 141 to 181, Fig. 2) produced a similar fragmentation pattern to that of the homogeneous complexes published by us before and recently by the Loo group,48,49 and to that of formed by ETD as reported by the Sabott group.50 In the low m/z region, usually below m/z 2000, are the sequence ions of the constituent subunits; in the high m/z region, beginning at the m/z of the intact assembly ions, are the charge-reduced intact tetramers. The sequence ions generated upon native MS introduction and ECD top-down sequencing not only permit identification of subunits of the hemoglobin assembly but also afford information on regions we have hypothesized to be flexible in the assembly. The question we address now is the effect of collisional activation on the assembly. Specifically, can we learn those regions that open to increase the collision cross-section, as determined by IM? To answer the question, we submitted the assembly to ECD. Previous studies showed that activated-ion, electroncapture dissociation (AI-ECD) generates higher sequence coverage in top-down sequencing of denatured protein ions.51,52 This principle can be applied to the hHb protein assembly introduced by native spray. By activation in the source or in the collisioncell region, the flexible region of protein assembly
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The high mass resolving power spectra from the FTICR instrument provide confident identification of sequence ions from both chains. We plotted the assigned ion signals with normalized intensities as a function of the activation voltages (Fig. 3). The sequence coverage at 100 V activation is highlighted on the amino-acid sequence (Fig. 3, insert). Most of the assigned ions are c-type from N termini (highlighted in red). Only a small number of z ions were observed, as highlighted in purple in the sequence. It is clear that N-terminal fragmentation can reach deeper along both a- and b-chains when the whole assembly is activated. The approximate minimum voltage setting that allows ions to traverse the entire path is 10 V in the nozzle-skimmer region (in-source activation). The fragment ions, formed with this minimum collisional pre-excitation contain c ions that contain up to 23 residues from both chains. At 100 and 125 V activation (laboratory frame), fragmentation occurs at peptide bonds for over 60 residues of the C terminus, demonstrating a considerable amount of unfolding can occur upon collisional activation without rupturing the assembly.
Correlation of B-factors and ECD We previously observed that ECD of homogenous protein assemblies involves flexible regions of an assembly, as revealed by correlating sites of fragmentation and B-factors from the X-ray crystal structure.23 hHb is an example of a heterogeneous tetramer. Because ECD fragments were produced from both component chains, their flexibility must be similar. We chose the crystal structure of the deoxy-Hb (PDB code 4hhb) to assist our interpretation of the MS results. From the crystal structure of this tetramer, we see that the N-termini of the two a- and b-chains are both exposed and have the highest B-factors (Fig. 4). The observed ECD fragments map faithfully onto those regions of the structure, supporting that both collisional activation and electron capture cause flexible regions of a heterogeneous assembly to
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Figure 3. Assignment of N-terminal ECD cleavage sites on both a- (top) and b- (bottom) chains of hemoglobin tetramer, and comparison with the average B-factor values of the two a- and b-chains, respectively.
fragment similarly to homogeneous assemblies. This is seen most clearly by the trends in fragmentation and in average B-factor values of the two a- and bchains (Fig. 3), respectively. For both a- and bchains, the B-factor goes from a high value at the termini to a low value around the 30–35th residues and then returns to intermediate values around the 50th residue. Without prior collisional activation, ECD stops before the low B-factor region. Ramping the activation energy presumably unfolds more of this flexible region, making more regions along the polypeptide chain more vulnerable for fragmentation. The C-terminus is buried beneath this region and has low B-factors for both chains, prohibiting formation of z-ions (Fig. 3, 100 V in-source
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activation). This was likely caused by unfolding of buried regions to expose them and allow a small number of fragments to be generated. Hydrogen deuterium exchange mass spectrometry (HDX MS) can reflect protein dynamics and conformational changes and report on flexible regions. The Konermann group reported the HDX behavior of oxy-Hb, deoxy-Hb, and aquomet-Hb.32,53 They identified several regions that showed deuterium saturation (90%) in a short time frame across the three forms of hemoglobin, even the ligand binding states. For the deoxy-Hb, those regions are the amino acids of (1) the a-chain 1–23, 67–80, 135–141 and (2) the b-chain 1–8, 41–47. Because vertebrate hemoglobins share almost identical 3D structures,54
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different charge states to be the salt bridges of its high-resolution structure. For protein assemblies, it is highly desirable to obtain more structural information, for example, salt bridge sites between subunits and/or among a subunit, from the types and the abundance changes of the ECD fragments. We are in the process of analyzing such data in protein systems we have investigated, seeking rules regarding protein– protein interaction interfaces. Although we cannot compare the exact collision energies on both the FTICR and Synapt G2 instruments, the data trends are consistent. IM reports conformational changes of protein assemblies upon collisional activation and shows that the cross section of the protein is increasing with collision energy increments. AI-ECD not only reveals that whole assembly becomes reactive as collisional energy increases, but also provides the location of the change/unfolding. An instrument with integrated capabilities of both IM and ETD/ECD would be ideal for such purposes, as we pointed out earlier.56 Approaches of this nature should be invaluable in guiding theoretical modeling of the behavior of protein complexes in vacuo. For example, an electrostatic model was developed to elucidate the charge-partitioning mechanism during CID of hemoglobin complexes.34 If it had been known that unfolding started from the N-terminus, the model could be made more detailed and accurate. Moreover, our results could provide insights into establishing a molecular dynamics (MD) model of protein structural folding, unfolding and dynamics, by which, for example, Robinson57 obtained structural information complementary to IM to understand the charge-state dependent compaction and dissociation of protein complexes. ECD provides an experimental approach to this problem. Figure 4. X-ray structure of hHb tetramer shown in B-factor scale (PDB code 4hhb). Top, two a-chains in color; bottom, two b-chains in color.
the HDX results support our interpretation of the ECD top-down data to indicate flexibility at the Nterminal regions. The changes in relative abundances of specific cions as a function of activation locates the structural unfolding of the assembly upon activation, as was determined by IM. For example, c23 at 10 V in-source activation, increased in abundance at 50 and 75 V activation then decreased at 100 and 125 V activation. Most of the c-ions larger than c34, seen at 50 V activation, show increased abundances as the activation voltage increases. The abundance variation of fragment ions observed upon activation-voltage ramping may contain 3D structural information of the hemoglobin assembly. Breuker and coworkers55 followed the abundance changes of ECD fragments of a 10 kDa protein, KIX, and attributed those that change at
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Conclusions Protein unfolding induced by collisional activation of protein assemblies can be seen by IM and located by ECD. The unfolding increases the protein cross sections measured by IM, and ECD reveals where unfolding occurs. The unfolded regions are predictable from the B factors of the protein assembly. Use of various fragmentation methods (i.e., CAD, SORICAD, ETD, ECD, IRMPD) available on the FTICR platform, and combinations of these methods augmented by ultraviolet photodissociation58 and trapped-IM spectrometry (TIMS),59 promise to generate even more structural information about protein assemblies. This work is a needed stepping stone for the development of a top-down approach generally applicable for the biophysical characterization of intact biomolecular assemblies.
ACKNOWLEDGMENTS HZ thanks the Am. Soc. for Mass Spectrom for a postdoctoral award.
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