Proteomics 2015, 15, 2813–2822
2813
DOI 10.1002/pmic.201400516
RESEARCH ARTICLE
Electron transfer dissociation provides higher-order structural information of native and partially unfolded protein complexes Frederik Lermyte1,2 and Frank Sobott1,2 1 2
UA-VITO Center for Proteomics, University of Antwerp, Antwerp, Belgium Biomolecular & Analytical Mass Spectrometry group, Department of Chemistry, University of Antwerp, Antwerp, Belgium
Top–down sequencing approaches are becoming ever more popular for protein characterization, due to the ability to distinguish and characterize different protein isoforms. Under nondenaturing conditions, electron transfer dissociation (ETD) can furthermore provide important information on the exposed surface of proteins or complexes, thereby contributing to the characterization of their higher-order structure. Here, we investigate this approach using top–down ETD of tetrameric hemoglobin, concanavalin A, and alcohol dehydrogenase combined with ion mobility (IM) on a commercially available quadrupole/ion mobility/time-of-flight instrument (Waters Synapt G2). By applying supplemental activation in the transfer cell (post-IM), we release ETD fragments and attain good sequence coverage in the exposed terminal regions of the protein. We investigate the correlation between observed sites of fragmentation with regions of solvent accessibility, as derived from the crystal structure. Ion acceleration prior to ETD is also used to cause collision-induced unfolding (CIU) of the complexes without monomer ejection, as evidenced by the IM profiles. These partially unfolded tetramers show efficient fragmentation in some regions which are not sequenced under more gentle MS conditions. We show that by increasing CIU in small increments and monitoring the changes in the fragmentation pattern, it is possible to follow the initial steps of gas-phase protein unfolding. Fragments from partially unfolded protein complexes are released immediately after electron transfer, prior to IM (they do not share the drift time of their precursor), and observed without the need for supplemental activation. This is further evidence that the higher-order structure in these protein regions has been disrupted.
Received: November 2, 2014 Revised: March 13, 2015 Accepted: June 15, 2015
Keywords: Electron transfer dissociation / Ion mobility / Noncovalent complex / Surface mapping / Technology / Top–down sequencing
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Correspondence: Professor Frank Sobott, Biomolecular & Analytical Mass Spectrometry group, Department of Chemistry, University of Antwerp, Antwerpen, Belgium E-mail: [email protected] Abbreviations: ADH, alcohol dehydrogenase; CIU, collisioninduced unfolding; Con A, concanavalin A; ETD, electron transfer dissociation; ETnoD, non-dissociative electron transfer; FAIMS, high-field asymmetric waveform ion mobility spectrometry; Hb, hemoglobin; IM, ion mobility; PDB, Protein data bank; SASA, solvent-accessible surface area
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Introduction
High-throughput genome sequencing efforts such as the Human Genome Project have shown that the number of genes in organisms is insufficient to explain the observed biological complexity [1, 2]. Part of this discrepancy can be accounted for by the appearance of protein variants, collectively known as proteoforms [3], resulting from, e.g. alternate
Colour Online: See the article online to view Fig. 1 in colour.
www.proteomics-journal.com
2814
F. Lermyte and F. Sobott
RNA splicing, site-specific mutations, truncations, and posttranslational modifications. For some time, MS has been a key technology in proteomics. In recent years, the use of high-resolution, high-mass accuracy instruments, allowing accurate mass and charge state assignment, has become the norm. At the same time, top–down proteomics – superior to conventional bottom–up proteomics in detecting many of these variants – is becoming ever more important [4–7]. For example, in a recent effort, 74 proteoforms of the human histone H4 were identified using top–down methods [8]. However, it is evident that the biological complexity of organisms is not only determined by the number of proteoforms, but much more so by the even greater number of specific interactions which these proteins participate in [9, 10]. There is, hence, a need to study the conformation and interactions present within protein complexes. This is often done using X-ray diffraction or NMR spectroscopy. While these methods yield high-resolution information, they are also timeconsuming, require a relatively large amount of protein, and have their limitations with regards to what types of complex are amenable to analysis. On the other hand, MS-based methods which rely on non-native analysis such as chemical crosslinking, hydrogen–deuterium exchange, and footprinting techniques, e.g. covalent or hydroxyl radical labeling, can provide high-resolution information about the interactions in a protein complex [11, 12], next to native MS and ion mobility (IM) approaches [13–19]. A key challenge in structural proteomics is to obtain sequence-specific information from a defined folding state of the protein or complex. Fragmentation by conventional collision-induced dissociation (CID) causes a loss of tertiary and quaternary structure before covalent bonds are broken, and therefore does not provide information about the noncovalent interactions present in the complex. Alternative fragmentation techniques such as surface-induced dissociation can provide data about the subunit connectivity in a protein complex [20], and noncovalent contacts are also found to be preserved during ultraviolet photodissociation [21]. Ultraviolet photodissociation has recently also been combined with IM, showing that different conformations of ubiquitin lead to different fragmentation patterns [22, 23]. Electron-based methods such as electron capture dissociation (ECD) [24] and electron transfer dissociation (ETD) [25] cleave the backbone of a protein or peptide while leaving labile PTMs and noncovalent interactions largely intact [26–39]. It should be noted that in these experiments, the gain of information about the higher-order structure of the native protein typically comes at the cost of a reduced fragmentation efficiency and sequence coverage, compared to ECD or ETD of denatured proteins [26, 40, 41]. Two factors contribute to this: first, if a native-like structure is retained, certain parts of the protein sequence will be protected against dissociation [26, 38, 42]. Second, as near-native proteins typically exhibit lower charge states than denatured proteins in electrospray ionization, their reaction cross-section for ETD is much lower, as it scales with the charge state [43]. The combination of ETD with IM, specifi C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2015, 15, 2813–2822
cally, high-field asymmetric waveform ion mobility spectrometry (FAIMS) [44, 45] has also proven to be a powerful one, as this has been shown to allow separation of isobaric phosphopeptides prior to fragmentation on a hybrid linear trap quadrupole-Orbitrap instrument [46]. However, one downside of this approach is the difficulty of obtaining structural information from FAIMS spectra, as opposed to drift-time or travelling-wave IM, where measurement of the arrival time allows direct inference of the collision cross-section of an ion. Recently, we showed that top–down ETD of the native alcohol dehydrogenase (ADH) tetramer on a quadrupole/TOF instrument results in selective cleavage at residues that are exposed on the surface of the complex, and can in this way be used as a footprinting technique [38]. As the ETD process leaves noncovalent interactions intact, the resulting fragments did not readily separate from each other, and significant supplemental activation of so-called non-dissociative electron transfer (ETnoD) products was required to promote fragment release. In the current work, we further study the behavior of noncovalent complexes in native ETD by expanding these experiments to two other complexes with important structural differences compared to ADH: concanavalin A (Con A), where both the N- and C-terminus are exposed on the surface, and hemoglobin (Hb), where the N-terminus is comprised of an exposed alpha helix not significantly bound to the bulk of the complex by strong noncovalent interactions such as hydrogen bonds. Further, we also study gas-phase unfolded states of these protein complexes by monitoring the changes in accessible surface area, for the first time using top–down ETD fragmentation in combination with IM. Our hypothesis is that ETD cleavage sites are predominantly located on the exposed surface of the protein, while the ease of fragment release may depend on the degree of noncovalent interactions which hold the fragments in place. Fragments resulting from cleavage at the surface, but which are bound to the rest of the complex by strong noncovalent interactions will require supplemental activation in order to be released and become visible in the spectra. An interesting question in this context pertains to potential differences between ECD and ETD, particularly regarding the accessibility of cleavage sites to the reagent. Temperature-resolved monitoring of gas-phase unfolding and time-resolved refolding of small proteins, after annihilation of the tertiary structure by an IR laser pulse, using ECD have previously been performed by McLafferty and colleagues [26, 47–49]. In earlier work, this group had demonstrated increased ECD fragmentation efficiency and sequence coverage if ions are subjected to collisional activation during the ECD process [50]. ECD with a variable delay after IR irradiation has also been used to study the gas-phase folding kinetics of small peptides [51]. In these ECD studies however, fragmentation was assumed to have occurred throughout the sequence, with higher-order structure only affecting whether or not fragments were subsequently released. In contrast, there have been relatively few reports in the literature of ECD www.proteomics-journal.com
2815
Proteomics 2015, 15, 2813–2822
followed by supplemental activation (most commonly applied by using an IR laser) in order to destroy the higher-order structure and release noncovalently bound fragments. Furthermore, most of these studies were conducted using peptides or small proteins such as ubiquitin, which, due to their small size can be expected not to have any significant ‘buried’ (i.e. solvent-inaccessible) regions [52–54]. Calculation of the solvent-accessible surface area (SASA) of the crystal structure of ubiquitin (PDB accession code 1UBQ) indicates no buried regions, although it is believed that this protein undergoes significant structural changes in the gas phase, refolding into a three-helix structure at low charge states [52, 55]. We therefore also calculated the solvent accessibility of residues for the solution structure of the slightly larger (10.7 kDa) KIX protein, which also forms a three-helix bundle [56] (PDB accession code 2AGH), finding that no buried regions are present here, either. Our hypothesis is therefore that most likely the entire sequence of ubiquitin is surface-exposed and available for ECD or ETD fragmentation. An interesting result was recently reported by Ganisl and Breuker [57], in which the 180-residue (20.0 kDa) soybean trypsin inhibitor, which also has two disulfide bonds, was subjected to ECD and the resulting non-dissociative electron capture product exposed to IR laser radiation. This resulted in extensive dissociation in non-cyclic regions (i.e. parts of the sequence not located between C(39)-C(86) or C(136)C(145)), including a beta strand between residues T(158) and V(163), which according to the crystal structure (PDB accession code 1AVU) should not be exposed in the native structure. As the protein was however analyzed from 50:50:1 water:methanol:acetic acid (pH 2.5), it is likely that the native structure was not fully retained in the gas phase. This is further supported by the observation of fragments from this same beta strand already under the gentlest conditions reported in the study (5 V pre-ECD collisional activation, no supplemental activation), implying that this strand might have been surface-exposed in the gas phase after all (as this is necessary for fragment release). We therefore conclude that there is insufficient information available in the literature to ascertain whether ECD and ETD differ in terms of surfaceselectivity. Recently, Vachet and colleagues reported the use of ETD followed by supplemental activation on a quadrupole ion trap to map salt bridges in small, monomeric proteins [58]. The authors of this study also used collisional activation to induce unfolding of ubiquitin prior to ETD, although interestingly, this only resulted in a change in fragmentation pattern when supplemental activation was also applied. Denatured states of protein complexes can also be prepared in solution by changing solvent composition [59]; these nonnative states will however typically appear at higher charge states compared to the native complex [60–64], increasing ETD reactivity and thus complicating a direct comparison [40, 43]. Alternatively, collision-induced unfolding (CIU) of protein ions in the gas phase has been shown to produce conformational states which can continue to exist on the timescale of an MS experiment [34, 65]. As it is generally C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
accepted that the charge state of an ion is determined during the final stages of the electrospray ionization (ESI) process, rather than in the gas phase [60–64, 66], the charge state distribution of the protein complex is not affected by this type of unfolding, thus enabling separation of the effect of protein conformation from that of the charge state on the observed fragmentation behavior. The drawback of this approach is that the appearance of these partially unfolded states cannot be verified by MS alone. The Synapt G2 instrument used in this study is however equipped with IM capabilities, which can be used to confirm the existence of extended protein conformers. Here, we test the hypothesis that the changing fragmentation pattern observed with an increasing extent of CIU illustrates the initial unfolding steps of the proteins. Furthermore, we show that disruption of the higher-order structure allows fragment release from partially unfolded regions without the use of supplemental activation. As the IM cell of the instrument is located between the trap cell where the electron transfer reaction occurs, and the transfer cell where supplemental activation can be applied, the arrival time of the ETD fragments reveals whether they were released from the protein complex immediately after electron transfer, or only upon application of supplemental activation.
2
Materials and methods
Tetrameric Hb (Bos taurus, Sigma H2500, 64 kDa), Con A (Canavalia ensiformis, Sigma C2010, 103 kDa), and ADH (Saccharomyces cerevisiae, Sigma A3263, 148 kDa) were used as model proteins. The sequences of these proteins, including observed ETD cleavage sites, can be found in the Supporting Information (Fig. S1). These proteins were dissolved at a concentration of 1 mg/mL in 100 mM aqueous ammonium acetate (pH = 6.8) and desalted twice using Bio-Rad Micro Bio-Spin 6 columns, yielding an estimated final concentration of protein tetramer between 5 and 11 M. Approximately 5 L of protein solution was transferred to in-house prepared gold-coated capillaries and infused into the mass spectrometer using the nanoflow version of the Z-spray ion source, with a capillary voltage of 1.0 – 1.3 kV, minimal (
2813
DOI 10.1002/pmic.201400516
RESEARCH ARTICLE
Electron transfer dissociation provides higher-order structural information of native and partially unfolded protein complexes Frederik Lermyte1,2 and Frank Sobott1,2 1 2
UA-VITO Center for Proteomics, University of Antwerp, Antwerp, Belgium Biomolecular & Analytical Mass Spectrometry group, Department of Chemistry, University of Antwerp, Antwerp, Belgium
Top–down sequencing approaches are becoming ever more popular for protein characterization, due to the ability to distinguish and characterize different protein isoforms. Under nondenaturing conditions, electron transfer dissociation (ETD) can furthermore provide important information on the exposed surface of proteins or complexes, thereby contributing to the characterization of their higher-order structure. Here, we investigate this approach using top–down ETD of tetrameric hemoglobin, concanavalin A, and alcohol dehydrogenase combined with ion mobility (IM) on a commercially available quadrupole/ion mobility/time-of-flight instrument (Waters Synapt G2). By applying supplemental activation in the transfer cell (post-IM), we release ETD fragments and attain good sequence coverage in the exposed terminal regions of the protein. We investigate the correlation between observed sites of fragmentation with regions of solvent accessibility, as derived from the crystal structure. Ion acceleration prior to ETD is also used to cause collision-induced unfolding (CIU) of the complexes without monomer ejection, as evidenced by the IM profiles. These partially unfolded tetramers show efficient fragmentation in some regions which are not sequenced under more gentle MS conditions. We show that by increasing CIU in small increments and monitoring the changes in the fragmentation pattern, it is possible to follow the initial steps of gas-phase protein unfolding. Fragments from partially unfolded protein complexes are released immediately after electron transfer, prior to IM (they do not share the drift time of their precursor), and observed without the need for supplemental activation. This is further evidence that the higher-order structure in these protein regions has been disrupted.
Received: November 2, 2014 Revised: March 13, 2015 Accepted: June 15, 2015
Keywords: Electron transfer dissociation / Ion mobility / Noncovalent complex / Surface mapping / Technology / Top–down sequencing
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Correspondence: Professor Frank Sobott, Biomolecular & Analytical Mass Spectrometry group, Department of Chemistry, University of Antwerp, Antwerpen, Belgium E-mail: [email protected] Abbreviations: ADH, alcohol dehydrogenase; CIU, collisioninduced unfolding; Con A, concanavalin A; ETD, electron transfer dissociation; ETnoD, non-dissociative electron transfer; FAIMS, high-field asymmetric waveform ion mobility spectrometry; Hb, hemoglobin; IM, ion mobility; PDB, Protein data bank; SASA, solvent-accessible surface area
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Introduction
High-throughput genome sequencing efforts such as the Human Genome Project have shown that the number of genes in organisms is insufficient to explain the observed biological complexity [1, 2]. Part of this discrepancy can be accounted for by the appearance of protein variants, collectively known as proteoforms [3], resulting from, e.g. alternate
Colour Online: See the article online to view Fig. 1 in colour.
www.proteomics-journal.com
2814
F. Lermyte and F. Sobott
RNA splicing, site-specific mutations, truncations, and posttranslational modifications. For some time, MS has been a key technology in proteomics. In recent years, the use of high-resolution, high-mass accuracy instruments, allowing accurate mass and charge state assignment, has become the norm. At the same time, top–down proteomics – superior to conventional bottom–up proteomics in detecting many of these variants – is becoming ever more important [4–7]. For example, in a recent effort, 74 proteoforms of the human histone H4 were identified using top–down methods [8]. However, it is evident that the biological complexity of organisms is not only determined by the number of proteoforms, but much more so by the even greater number of specific interactions which these proteins participate in [9, 10]. There is, hence, a need to study the conformation and interactions present within protein complexes. This is often done using X-ray diffraction or NMR spectroscopy. While these methods yield high-resolution information, they are also timeconsuming, require a relatively large amount of protein, and have their limitations with regards to what types of complex are amenable to analysis. On the other hand, MS-based methods which rely on non-native analysis such as chemical crosslinking, hydrogen–deuterium exchange, and footprinting techniques, e.g. covalent or hydroxyl radical labeling, can provide high-resolution information about the interactions in a protein complex [11, 12], next to native MS and ion mobility (IM) approaches [13–19]. A key challenge in structural proteomics is to obtain sequence-specific information from a defined folding state of the protein or complex. Fragmentation by conventional collision-induced dissociation (CID) causes a loss of tertiary and quaternary structure before covalent bonds are broken, and therefore does not provide information about the noncovalent interactions present in the complex. Alternative fragmentation techniques such as surface-induced dissociation can provide data about the subunit connectivity in a protein complex [20], and noncovalent contacts are also found to be preserved during ultraviolet photodissociation [21]. Ultraviolet photodissociation has recently also been combined with IM, showing that different conformations of ubiquitin lead to different fragmentation patterns [22, 23]. Electron-based methods such as electron capture dissociation (ECD) [24] and electron transfer dissociation (ETD) [25] cleave the backbone of a protein or peptide while leaving labile PTMs and noncovalent interactions largely intact [26–39]. It should be noted that in these experiments, the gain of information about the higher-order structure of the native protein typically comes at the cost of a reduced fragmentation efficiency and sequence coverage, compared to ECD or ETD of denatured proteins [26, 40, 41]. Two factors contribute to this: first, if a native-like structure is retained, certain parts of the protein sequence will be protected against dissociation [26, 38, 42]. Second, as near-native proteins typically exhibit lower charge states than denatured proteins in electrospray ionization, their reaction cross-section for ETD is much lower, as it scales with the charge state [43]. The combination of ETD with IM, specifi C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2015, 15, 2813–2822
cally, high-field asymmetric waveform ion mobility spectrometry (FAIMS) [44, 45] has also proven to be a powerful one, as this has been shown to allow separation of isobaric phosphopeptides prior to fragmentation on a hybrid linear trap quadrupole-Orbitrap instrument [46]. However, one downside of this approach is the difficulty of obtaining structural information from FAIMS spectra, as opposed to drift-time or travelling-wave IM, where measurement of the arrival time allows direct inference of the collision cross-section of an ion. Recently, we showed that top–down ETD of the native alcohol dehydrogenase (ADH) tetramer on a quadrupole/TOF instrument results in selective cleavage at residues that are exposed on the surface of the complex, and can in this way be used as a footprinting technique [38]. As the ETD process leaves noncovalent interactions intact, the resulting fragments did not readily separate from each other, and significant supplemental activation of so-called non-dissociative electron transfer (ETnoD) products was required to promote fragment release. In the current work, we further study the behavior of noncovalent complexes in native ETD by expanding these experiments to two other complexes with important structural differences compared to ADH: concanavalin A (Con A), where both the N- and C-terminus are exposed on the surface, and hemoglobin (Hb), where the N-terminus is comprised of an exposed alpha helix not significantly bound to the bulk of the complex by strong noncovalent interactions such as hydrogen bonds. Further, we also study gas-phase unfolded states of these protein complexes by monitoring the changes in accessible surface area, for the first time using top–down ETD fragmentation in combination with IM. Our hypothesis is that ETD cleavage sites are predominantly located on the exposed surface of the protein, while the ease of fragment release may depend on the degree of noncovalent interactions which hold the fragments in place. Fragments resulting from cleavage at the surface, but which are bound to the rest of the complex by strong noncovalent interactions will require supplemental activation in order to be released and become visible in the spectra. An interesting question in this context pertains to potential differences between ECD and ETD, particularly regarding the accessibility of cleavage sites to the reagent. Temperature-resolved monitoring of gas-phase unfolding and time-resolved refolding of small proteins, after annihilation of the tertiary structure by an IR laser pulse, using ECD have previously been performed by McLafferty and colleagues [26, 47–49]. In earlier work, this group had demonstrated increased ECD fragmentation efficiency and sequence coverage if ions are subjected to collisional activation during the ECD process [50]. ECD with a variable delay after IR irradiation has also been used to study the gas-phase folding kinetics of small peptides [51]. In these ECD studies however, fragmentation was assumed to have occurred throughout the sequence, with higher-order structure only affecting whether or not fragments were subsequently released. In contrast, there have been relatively few reports in the literature of ECD www.proteomics-journal.com
2815
Proteomics 2015, 15, 2813–2822
followed by supplemental activation (most commonly applied by using an IR laser) in order to destroy the higher-order structure and release noncovalently bound fragments. Furthermore, most of these studies were conducted using peptides or small proteins such as ubiquitin, which, due to their small size can be expected not to have any significant ‘buried’ (i.e. solvent-inaccessible) regions [52–54]. Calculation of the solvent-accessible surface area (SASA) of the crystal structure of ubiquitin (PDB accession code 1UBQ) indicates no buried regions, although it is believed that this protein undergoes significant structural changes in the gas phase, refolding into a three-helix structure at low charge states [52, 55]. We therefore also calculated the solvent accessibility of residues for the solution structure of the slightly larger (10.7 kDa) KIX protein, which also forms a three-helix bundle [56] (PDB accession code 2AGH), finding that no buried regions are present here, either. Our hypothesis is therefore that most likely the entire sequence of ubiquitin is surface-exposed and available for ECD or ETD fragmentation. An interesting result was recently reported by Ganisl and Breuker [57], in which the 180-residue (20.0 kDa) soybean trypsin inhibitor, which also has two disulfide bonds, was subjected to ECD and the resulting non-dissociative electron capture product exposed to IR laser radiation. This resulted in extensive dissociation in non-cyclic regions (i.e. parts of the sequence not located between C(39)-C(86) or C(136)C(145)), including a beta strand between residues T(158) and V(163), which according to the crystal structure (PDB accession code 1AVU) should not be exposed in the native structure. As the protein was however analyzed from 50:50:1 water:methanol:acetic acid (pH 2.5), it is likely that the native structure was not fully retained in the gas phase. This is further supported by the observation of fragments from this same beta strand already under the gentlest conditions reported in the study (5 V pre-ECD collisional activation, no supplemental activation), implying that this strand might have been surface-exposed in the gas phase after all (as this is necessary for fragment release). We therefore conclude that there is insufficient information available in the literature to ascertain whether ECD and ETD differ in terms of surfaceselectivity. Recently, Vachet and colleagues reported the use of ETD followed by supplemental activation on a quadrupole ion trap to map salt bridges in small, monomeric proteins [58]. The authors of this study also used collisional activation to induce unfolding of ubiquitin prior to ETD, although interestingly, this only resulted in a change in fragmentation pattern when supplemental activation was also applied. Denatured states of protein complexes can also be prepared in solution by changing solvent composition [59]; these nonnative states will however typically appear at higher charge states compared to the native complex [60–64], increasing ETD reactivity and thus complicating a direct comparison [40, 43]. Alternatively, collision-induced unfolding (CIU) of protein ions in the gas phase has been shown to produce conformational states which can continue to exist on the timescale of an MS experiment [34, 65]. As it is generally C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
accepted that the charge state of an ion is determined during the final stages of the electrospray ionization (ESI) process, rather than in the gas phase [60–64, 66], the charge state distribution of the protein complex is not affected by this type of unfolding, thus enabling separation of the effect of protein conformation from that of the charge state on the observed fragmentation behavior. The drawback of this approach is that the appearance of these partially unfolded states cannot be verified by MS alone. The Synapt G2 instrument used in this study is however equipped with IM capabilities, which can be used to confirm the existence of extended protein conformers. Here, we test the hypothesis that the changing fragmentation pattern observed with an increasing extent of CIU illustrates the initial unfolding steps of the proteins. Furthermore, we show that disruption of the higher-order structure allows fragment release from partially unfolded regions without the use of supplemental activation. As the IM cell of the instrument is located between the trap cell where the electron transfer reaction occurs, and the transfer cell where supplemental activation can be applied, the arrival time of the ETD fragments reveals whether they were released from the protein complex immediately after electron transfer, or only upon application of supplemental activation.
2
Materials and methods
Tetrameric Hb (Bos taurus, Sigma H2500, 64 kDa), Con A (Canavalia ensiformis, Sigma C2010, 103 kDa), and ADH (Saccharomyces cerevisiae, Sigma A3263, 148 kDa) were used as model proteins. The sequences of these proteins, including observed ETD cleavage sites, can be found in the Supporting Information (Fig. S1). These proteins were dissolved at a concentration of 1 mg/mL in 100 mM aqueous ammonium acetate (pH = 6.8) and desalted twice using Bio-Rad Micro Bio-Spin 6 columns, yielding an estimated final concentration of protein tetramer between 5 and 11 M. Approximately 5 L of protein solution was transferred to in-house prepared gold-coated capillaries and infused into the mass spectrometer using the nanoflow version of the Z-spray ion source, with a capillary voltage of 1.0 – 1.3 kV, minimal (
