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H/D exchange (HDX) mass spectrometry (MS) is a widely used technique for interrogating protein structure and dynamics. Backbone HDX is mediated by opening/closing (unfolding/refolding) fluctuations. In traditional HDX-MS, proteins are incubated in D2O as a function of time at constant temperature (T). There is an urgent need to complement this traditional approach with experiments that probe proteins in a T-dependent fashion, e.g., for assessing the stability of therapeutic antibodies. A key problem with such studies is the absence of strategies for interpreting HDX-MS data in the context of T-dependent protein dynamics. Specifically, it has not been possible thus far to separate T-induced changes of the chemical labeling step (kch) from thermally enhanced protein fluctuations. Focusing on myoglobin, the current work solves this problem by dissecting T-dependent HDX-MS profiles into contributions from kch(T), as well as local and global protein dynamics. Experimental profiles started off with surprisingly shallow slopes that seemed to defy the quasi-exponential kch(T) dependence. Just below the melting temperature (Tm) the profiles showed a sharp increase. Our analysis revealed that local dynamics dominate at low T, while global events become prevalent closer to Tm. About half of the backbone NH sites exhibited a canonical scenario, where local opening/closing was associated with positive ΔH and ΔS. Many of the remaining sites had negative ΔH and ΔS, thereby accounting for the shallowness of the experimental HDX-MS profiles at low T. In summary, this work provides practitioners with the tools to analyze proteins over a wide temperature range, paving the way toward T-dependent high-throughput screening applications by HDX-MS.

Covalent modifications by reactive oxygen species can modulate the function and stability of proteins. Thermal unfolding experiments in solution are a standard tool for probing oxidation-induced stability changes. Complementary to such solution investigations, the stability of electrosprayed protein ions can be assessed in the gas phase by collision-induced unfolding (CIU) and ion-mobility spectrometry. A question that remains to be explored is whether oxidation-induced stability alterations in solution are mirrored by the CIU behavior of gaseous protein ions. Here, we address this question using chloramine-T-oxidized cytochrome c (CT-cyt c) as a model system. CT-cyt c comprises various proteoforms that have undergone MetO formation (+16 Da) and Lys carbonylation (LysCH2-NH2 → LysCHO, -1 Da). We found that CT-cyt c in solution was destabilized, with a ∼5 °C reduced melting temperature compared to unmodified controls. Surprisingly, CIU experiments revealed the opposite trend, i.e., a stabilization of CT-cyt c in the gas phase. To pinpoint the source of this effect, we performed proteoform-resolved CIU on CT-cyt c fractions that had been separated by cation exchange chromatography. In this way, it was possible to identify MetO formation at residue 80 as the key modification responsible for stabilization in the gas phase. Possibly, this effect is caused by newly formed contacts of the sulfoxide with aromatic residues in the protein core. Overall, our results demonstrate that oxidative modifications can affect protein stability in solution and in the gas phase very differently.

The structure and reactivity of electrosprayed protein ions is governed by their net charge. Native proteins in non-denaturing aqueous solutions produce low charge states. More highly charged ions are formed when electrospraying proteins that are unfolded and/or exposed to organic supercharging agents. Numerous studies have explored the electrospray process under these various conditions. One phenomenon that has received surprisingly little attention is the charge enhancement caused by multivalent metal ions such as La3+ when electrospraying proteins out of non-denaturing solutions. Here, we conducted mass spectrometry and ion mobility spectrometry experiments, in combination with molecular dynamics (MD) simulations, to uncover the mechanistic basis of this charge enhancement. MD simulations of aqueous ESI droplets reproduced the experimental observation that La3+ boosts protein charge states relative to monovalent metals (e.g., Na+). The simulations showed that gaseous proteins were released by solvent evaporation to dryness, consistent with the charged residue model. Metal ion ejection kept the shrinking droplets close to the Rayleigh limit until ∼99% of the solvent had left. For droplets charged with Na+, metal adduction during the final stage of solvent evaporation produced low protein charge states. Droplets containing La3+ showed a very different behavior. The trivalent nature of La3+ favored adduction to the protein at a very early stage, when most of the solvent had not evaporated yet. This irreversible binding via multidentate contacts suppressed La3+ ejection from the vanishing droplets, such that the resulting gaseous proteins carried significantly more charge. Our results illustrate that MD simulations are suitable for uncovering intricate aspects of electrospray mechanisms, paving the way toward an atomistic understanding of mass spectrometry based analytical workflows.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription regulator that plays a pivotal role in coordinating the cellular response to oxidative stress. Through interactions with other proteins, such as Kelch-like ECH-associated protein 1 (Keap1), CREB-binding protein (CBP), and retinoid X receptor alpha (RXRα), Nrf2 mediates the transcription of cytoprotective genes critical for removing toxicants and preventing DNA damage, thereby playing a significant role in chemoprevention. Dysregulation of Nrf2 is linked to tumorigenesis and chemoresistance, making Nrf2 a promising target for anticancer therapeutics. However, despite the physiological importance of Nrf2, the molecular details of this protein and its interactions with most of its targets remain unknown, hindering the rational design of Nrf2-targeted therapeutics. With this in mind, we used a combined bioinformatics and experimental approach to characterize the structure of full-length Nrf2 and its interaction with Keap1. Our results show that Nrf2 is partially disordered, with transiently structured elements in its Neh2, Neh7, and Neh1 domains. Moreover, interaction with the Kelch domain of Keap1 leads to protection of the binding motifs in the Neh2 domain of Nrf2, while the rest of the protein remains highly dynamic. This work represents the first detailed structural characterization of full-length Nrf2 and provides valuable insights into the molecular basis of Nrf2 activity modulation in oxidative stress response.

Proteins that are unfolded in solution produce higher charge states during electrospray ionization (ESI) than their natively folded counterparts. Protein charge states can be further increased by the addition of supercharging agents (SCAs) such as sulfolane. The mechanism whereby these supercharged [M + zH] z+ ions are formed under unfolded conditions remains unclear. Here we employed a combination of mass spectrometry (MS), ion mobility spectrometry (IMS), and molecular dynamics (MD) simulations for probing the ESI mechanism under denatured supercharging conditions. ESI of acid-unfolded apo-myoglobin (aMb) in the presence of sulfolane produced charge states around 27+, all the way to fully protonated (33+) aMb. MD simulations of aMb 27+ to 33+ in Rayleigh-charged water/sulfolane droplets culminated in electrostatically driven protein expulsion, consistent with the chain ejection model (CEM). The electrostatically stretched conformations predicted by these simulations were in agreement with IMS experiments. The CEM involves partitioning of mobile H+ between the droplet and the departing protein. Our results imply that supercharging of unfolded proteins is caused by residual sulfolane that stabilizes protonated sites on the protruding chains, thereby promoting H+ retention on the protein. The stabilization of charged sites is due to charge-dipole interactions mediated by the large dipole moment and the low volatility of sulfolane. Support for this mechanism comes from the experimental observation of sulfolane adducts on the most highly charged ions, a phenomenon previously noted by Venter ( J. Am. Soc. Mass Spectrom. 2012, 23, 489-497). The "CEM supercharging model" proposed here for unfolded proteins is distinct from the charge trapping mechanism believed to be operative during native ESI supercharging.

Electrospray ionization (ESI) is an essential technique for transferring proteins from solution into the gas phase for mass spectrometry and ion mobility spectrometry. The mechanisms whereby [M + zH]z+ protein ions are released from charged nanodroplets during ESI have been controversial for many years. Here we discuss recent computational and experimental studies that have shed light on many of the mysteries in this area. Four types of protein ESI experiments can be distinguished, each of which appears to be associated with a specific mechanism. (i) Native ESI proceeds according to the charged residue model (CRM) that entails droplet evaporation to dryness, generating compact protein ions in low charge states. (ii) Native ESI supercharging is also a CRM process, but the dried-out proteins accumulate additional charge because supercharging agents such as sulfolane interfere with the ejection of small ions (Na+, NH4+, etc.) from the shrinking droplets. (iii) Denaturing ESI follows the chain ejection model (CEM), where protein ions are gradually expelled from the droplet surface. H+ equilibration between the droplets and the protruding chains culminates in highly charged gaseous proteins, analogous to the collision-induced dissociation of multi-protein complexes. (iv) Denatured ESI supercharging also generates protein ions via the CEM. Supercharging agents stabilize protonated sites on the protein tail via charge-dipole interactions, causing the chain to acquire additional charge. There will likely be scenarios that fall outside of these four models, but it appears that the framework outlined here covers most of the experimentally relevant conditions.

Previous structural studies of osteoprotegerin (OPG), a crucial negative regulator of bone remodeling and osteoclastogenesis, were mostly limited to the N-terminal ligand-binding domains. It is now known that the three C-terminal domains of OPG also play essential roles in its function by mediating OPG dimerization, OPG–heparan sulfate (HS) interactions, and formation of the OPG–HS–receptor activator of nuclear factor κB ligand (RANKL) ternary complex. Employing hydrogen–deuterium exchange MS methods, here we investigated the structure of full-length OPG in complex with HS or RANKL in solution. Our data revealed two noteworthy aspects of the OPG structure. First, we found that the interconnection between the N- and C-terminal domains is much more rigid than previously thought, possibly because of hydrophobic interactions between the fourth cysteine-rich domain and the first death domain. Second, we observed that two hydrophobic clusters located in two separate C-terminal domains directly contribute to OPG dimerization, likely by forming a hydrophobic dimerization interface. Aided by site-directed mutagenesis, we further demonstrated that an intact dimerization interface is essential for the biological activity of OPG. Our study represents an important step toward deciphering the structure–function relationship of the full-length OPG protein.

Oxidation of key residues in cytochrome

Recent advances in molecular dynamics (MD) simulations have made it possible to examine the behavior of large charged droplets that contain analytes such as proteins or polymers, thereby providing insights into electrospray ionization (ESI) mechanisms. In the present study, we use this approach to investigate the release of polylactide (PLA) ions from water/acetonitrile ESI droplets. We found that cationized gaseous PLA ions can be formed via various competing pathways. Some MD runs showed extrusion and subsequent separation of polymer chains from the droplet, as envisioned by the chain ejection model (CEM). On other occasions the PLA chains remained inside the droplets and were released after solvent evaporation to dryness, consistent with the charge residue model (CRM). Following their release from ESI droplets, the nascent gaseous PLA ions were subjected to structural relaxation for several μs in vacuo. The MD conformations generated in this way for various PLA charge states compared favorably to experimental results obtained by ion mobility spectrometry-mass spectrometry (IMS-MS). The structures of all PLA ions evolved during relaxation in the gas phase. However, some macroion species retained features that resembled their nascent structures. For this subset of ions, the IMS-MS response appears to be strongly correlated with the ESI release mechanism (CEM vs. CRM). The former favored extended structures, whereas the latter preferentially generated compact conformers.

The ion evaporation model (IEM) and the charged residue model (CRM) represent cornerstones of any discussion related to the mechanism of electrospray ionization (ESI). Molecular dynamics (MD) simulations have confirmed that small ions such as Na+ are ejected from the surface of aqueous ESI droplets (IEM), while folded proteins in native ESI are released by water evaporation to dryness (CRM). ESI of unfolded proteins yields [M + zH] z+ ions that are much more highly charged than their folded counterparts. A chain ejection model (CEM) has been proposed to account for the protein ESI behavior under such non-native conditions (Konermann, L., et al. Anal. Chem. 2013, 85, 2-9). The CEM envisions that unfolded proteins are driven to the droplet surface by hydrophobic and electrostatic factors, followed by gradual ejection via intermediates where droplets carry extended protein tails. Thus far, it has not been possible to support the CEM through MD simulations using realistic protein models and atomistic force fields. Such endeavors require much larger droplets than in previous MD studies. Also, the incorporation of CEM-related H+ migration is difficult. This work overcomes these challenges in MD simulations on unfolded apo-myoglobin (aMb) in droplets with a 5.5 nm radius (∼22500 water molecules). We focused on solutions at pH ∼4 where the aMb solution charge coincides with the charge on some of the electrosprayed ions (22+ to 27+), such that H+ migration could be neglected. Na+ ions were added to ensure a droplet charge close to the Rayleigh limit. We found that 16 of 17 MD runs on various protonation patterns produced [M + zH] z+ ions via chain ejection. The predicted stretched-out aMb conformations were consistent with experimental collision cross sections. These results support the view that unfolded proteins follow the CEM. Overall, the IEM/CRM/CEM triad can account for a wide range of ESI scenarios involving various types of analytes.

Electrospray ionization (ESI) allows the production of intact gas-phase ions from proteins in solution. Nondenaturing solvent conditions usually culminate in low ESI charge states. However, many mass spectrometric applications benefit from protein ions that are more highly charged. One way to boost protein charge is the addition of supercharging agents (SCAs) such as sulfolane or m-nitrobenzyl alcohol (m-NBA) to the aqueous solution. The supercharging mechanism remains controversial. We use molecular dynamics (MD) simulations to examine how SCAs affect the behavior of ESI nanodroplets. Simulations were conducted on myoglobin in water, water/sulfolane, and water/m-NBA. Na(+) ions served as surrogate charge carriers instead of H(+). We focus on conditions where the protein initially adopts its native conformation. MD-generated charge states show remarkable agreement with experimental data. Droplet shrinkage is accompanied by Na(+) ejection, consistent with the ion evaporation model (IEM). The droplets segregate into an outer SCA shell and an aqueous core. This core harbors protein and Na(+). Unfavorable SCA solvation restricts Na(+) access to the droplet surface, thereby impeding IEM ejection. Rapid water loss causes SCA enrichment, ultimately forcing all remaining Na(+) to bind the protein. IEM ejection is no longer feasible after this point, such that the protein becomes supercharged by Na(+) trapping. SCA-free droplets produce lower charge states because the aqueous environment ensures a higher IEM efficiency. For all scenarios examined here, proteins are released via solvent evaporation to dryness, as envisioned by the charged residue model. Our data provide the first atomistic view of the supercharging mechanism.

F

The ability to transfer intact proteins and protein complexes into the gas phase by electrospray ionization (ESI) has opened up numerous mass spectrometry (MS)-based avenues for exploring biomolecular structure and function. However, many details regarding the ESI process and the properties of gaseous analyte ions are difficult to decipher when relying solely on experimental data. Molecular dynamics (MD) simulations can provide additional insights into the behavior of ESI droplets and protein ions. This review is geared primarily towards experimentalists who wish to adopt MD simulations as a complementary research tool. We touch on basic points such as force fields, the choice of a proper water model, GPU-acceleration, possible artifacts, as well as shortcomings of current MD models. Following this technical overview, we highlight selected applications. Simulations on aqueous droplets confirm that "native" ESI culminates in protein ion release via the charged residue model. MD-generated charge states and collision cross sections match experimental data. Gaseous protein ions produced by native ESI retain much of their solution structure. Moving beyond classical fixed-charge algorithms, we discuss a simple strategy that captures the mobile nature of H+ within gaseous biomolecules. These mobile proton simulations confirm the high propensity of gaseous proteins to form salt bridges, as well as the occurrence of charge migration during collision-induced unfolding and dissociation. It is hoped that this review will promote the use of MD simulations in ESI-related research. We also hope to encourage the development of improved algorithms for charged droplets and gaseous biomolecular ions.

It is believed that enzyme catalysis is facilitated by conformational dynamics of the protein scaffold that surrounds the active site, yet the exact nature of catalytically relevant protein motions remains largely unknown. Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) reports on backbone H-bond fluctuations. HDX/MS thus represents a promising avenue for probing the relationship between enzyme dynamics and catalysis. A seemingly straightforward strategy for such studies involves comparative measurements during substrate turnover and in the resting state. We examined the feasibility of this approach using rabbit muscle pyruvate kinase (rM1-PK) which catalyzes the conversion of phosphoenolpyruvate and Mg-ADP to pyruvate and Mg-ATP. HDX/MS revealed that catalytically active rM1-PK undergoes significant rigidification in the active site. This finding is counterintuitive, considering the purported correlation between dynamics and catalysis. Interestingly, virtually the same rigidification was seen upon exposing rM1-PK to substrates or products in the absence of turnover. These data imply that the active site dynamics during turnover are dominated by protein-ligand binding interactions. These interactions stabilize H-bonds in the vicinity of the active site, thereby masking subtle dynamic features that might be uniquely associated with catalysis. Our data uncover an inherent problem with side-by-side turnover/resting state measurements, i.e., the difficulty to design a suitable reference state against which the working enzyme can be compared. Comparative HDX/MS experiments on enzyme dynamics should therefore be interpreted with caution.

Gas/water interfaces (such as air bubbles or foam) are detrimental to the stability of proteins, often causing aggregation. This represents a potential problem for industrial processes, for example, the production and handling of protein drugs. Proteins possess surfactant-like properties, resulting in a high affinity for gas/water interfaces. The tendency of previously buried nonpolar residues to maximize contact with the gas phase can cause significant structural distortion. Most earlier studies in this area employed spectroscopic tools that could only provide limited information. Here we use hydrogen/deuterium exchange (HDX) mass spectrometry (MS) for probing the conformational dynamics of the model protein myoglobin (Mb) in the presence of N2 bubbles. HDX/MS relies on the principle that unfolded and/or highly dynamic regions undergo faster deuteration than tightly folded segments. In bubble-free solution Mb displays EX2 behavior, reflecting the occurrence of short-lived excursions to partially unfolded conformers. A dramatically different behavior is seen in the presence of N2 bubbles; EX2 dynamics still take place, but in addition the protein shows EX1 behavior. The latter results from interconversion of the native state with conformers that are globally unfolded and long-lived. These unfolded species likely correspond to Mb that is adsorbed to the surface of gas bubbles. N2 sparging also induces aggregation. To explain the observed behavior we propose a simple model, that is, “semi-unfolded” ↔ “native” ↔ “globally unfolded” → “aggregated”. This model quantitatively reproduces the experimentally observed kinetics. To the best of our knowledge, the current study marks the first exploration of surface denaturation phenomena by HDX/MS.

Royal jelly (RJ) triggers the development of female honeybee larvae into queens. This effect has been attributed to the presence of major royal jelly protein 1 (MRJP1) in RJ. MRJP1 isolated from royal jelly is tightly associated with apisimin, a 54-residue α-helical peptide that promotes the noncovalent assembly of MRJP1 into multimers. No high-resolution structural data are available for these complexes, and their binding stoichiometry remains uncertain. We examined MRJP1/apisimin using a range of biophysical techniques. We also investigated the behavior of deglycosylated samples, as well as samples with reduced apisimin content. Our mass spectrometry (MS) data demonstrate that the native complexes predominantly exist in a (MRJP14 apisimin4) stoichiometry. Hydrogen/deuterium exchange MS reveals that MRJP1 within these complexes is extensively disordered in the range of residues 20-265. Marginally stable secondary structure (likely antiparallel β-sheet) exists around residues 266-432. These weakly structured regions interchange with conformers that are extensively unfolded, giving rise to bimodal (EX1) isotope distributions. We propose that the native complexes have a "dimer of dimers" quaternary structure in which MRJP1 chains are bridged by apisimin. Specifically, our data suggest that apisimin acts as a linker that forms hydrophobic contacts involving the MRJP1 segment 316VLFFGLV322. Deglycosylation produces large soluble aggregates, highlighting the role of glycans as aggregation inhibitors. Samples with reduced apisimin content form dimeric complexes with a (MRJP12 apisimin1) stoichiometry. The information uncovered in this work will help pave the way toward a better understanding of the unique physiological role played by MRJP1 during queen differentiation.

F0F1 ATP synthase harnesses a transmembrane electrochemical gradient for the production of ATP. When operated in reverse, this multiprotein complex catalyzes ATP hydrolysis. In bacteria, the ε subunit is involved in regulating this ATPase activity. Also, ε is essential for coupling ATP hydrolysis (or synthesis) to proton translocation. The ε subunit consists of a β sandwich and two C-terminal helices, α1 and α2. The protein can switch from a compact fold to an alternate conformation where α1 and α2 are separated, resulting in an extended structure. ε from the thermophile Bacillus PS3 (Tε) binds ATP with high affinity such that this protein may function as an intracellular ATP level sensor. ATP binding to isolated Tε triggers a major conformational transition. Earlier data were interpreted in terms of an ATP + Tεextended → ATP·Tεcompact transition that may mimic aspects of the regulatory switching within F0F1 (Yagi et al. (2007) Proc. Natl. Acad. Sci. U.S.A., 104, 11233–11238). In this work, we employ complementary biophysical techniques for examining the ATP-induced conformational switching of isolated Tε. CD spectroscopy confirmed the occurrence of a large-scale conformational transition upon ATP binding, consistent with the formation of stable helical structure. Hydrogen/deuterium exchange (HDX) mass spectrometry revealed that this transition is accompanied by a pronounced stabilization in the vicinity of the ATP-binding pocket. Surprisingly, dramatic stabilization is also seen in the β8−β9 region, which is remote from the site of ATP interaction. Analytical ultracentrifugation uncovered a previously unrecognized feature of Tε: a high propensity to undergo dimerization in the presence of ATP. Comparison with existing crystallography data strongly suggests that the unexpected β8−β9 HDX protection is due to newly formed protein–protein contacts. Hence, ATP binding to isolated Tε proceeds according to 2ATP + 2Tεextended → (ATP·Tεcompact)2. Implications of this dimerization propensity for the possible role of Tε as an antibiotic target are discussed.

The mechanisms whereby protein ions are liberated from charged droplets during electrospray ionization (ESI) remain under investigation. Compact conformers electrosprayed from aqueous solution in positive ion mode likely follow the charged residue model (CRM), which envisions analyte release after solvent evaporation to dryness. The concentration of nonvolatile salts such as NaCl increases sharply within vanishing CRM droplets, promoting nonspecific pairing of Cl(-) and Na(+) with charged groups on the protein surface. For unfolded proteins, it has been proposed that ion formation occurs via the chain ejection model (CEM). During the CEM proteins are expelled from the droplet long before complete solvent evaporation has taken place. Here we examine whether salt adduction levels support the view that folded and unfolded proteins follow different ESI mechanisms. Solvent evaporation during the CEM is expected to be less extensive and, hence, the salt concentration at the point of protein release should be substantially lower than for the CRM. CEM ions should therefore exhibit lower adduction levels than CRM species. We explore the adduction behavior of several proteins that were chosen to allow comparative studies on folded and unfolded structures in the same solution. In-source activation eliminates chloride adducts via HCl release, generating protein ions that are heterogeneously charged because of sodiation and protonation. Sodiation levels measured under such conditions provide estimates of the salt adduction behavior experienced by the "nascent" analyte ions. Sodiation levels are significantly reduced for unfolded proteins, supporting the view that these species are indeed formed via the CEM.

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) is a widely used technique for probing protein structure and dynamics. Exposure to D2O induces the deuteration of backbone N-H groups via a process that involves transient excursions to partially unfolded protein conformers. The resulting mass shifts can be probed by MS, usually in combination with proteolytic digestion and/or electron-based fragmentation. Studies on protein-ligand complexes represent a particularly important HDX/MS application. The prevailing view is that ligand binding should reduce deuteration rates, and it is often expected that this reduction will be most pronounced in the vicinity of the interaction site. Many protein-ligand systems do indeed behave in a fashion that is consistent with this paradigm. In this review we point out that the opposite effect may be encountered as well. Also, mixed scenarios are possible where ligand binding induces elevated HDX rates in some protein regions, whereas rates in other segments are reduced. We present a framework that links ligand-induced changes in HDX kinetics to alterations in the occupancy of excited protein conformers. Spontaneous ligand binding will always lower the free energy of the ground state. In contrast, the corresponding free energy shifts of excited states are largely unpredictable, giving rise to a range of possible HDX responses. "Type 1" scenarios, characterized by a reduction of HDX rates are just as feasible as "Type 2" behavior where deuteration is accelerated. Even "Type 0" phenomena may be encountered, where HDX rates are unaffected by the presence of ligand. Type 0/1/2 scenarios can coexist in the same protein (these terms are not to be confused with the EX1/EX2 expressions which refer to a different aspect of protein HDX). Allosteric effects and ligand-induced protein-protein contacts can affect the outcome of protein-ligand binding studies as well. In summary, comparative HDX measurements conducted in the presence and in the absence ligand provide a detailed fingerprint of biomolecular interactions. However, protein-ligand interactions can elicit a wide range of responses, and the interpretation of binding site mapping experiments may not always be straightforward.

Many protein investigations by electrospray ionization (ESI) mass spectrometry (MS) strive to ensure a "native" solvent environment, i.e., nondenaturing conditions up to the point of gas-phase ion formation. Ideally, these studies would employ a volatile pH buffer to mitigate changes in H(+) concentration that can occur during ESI. Ammonium acetate is a commonly used additive, despite its low buffering capacity at pH 7. Ammonium bicarbonate provides greatly improved pH stabilization, thus offering an interesting alternative. Surprisingly, protein analyses in bicarbonate at pH 7 tend to result in the formation of very high charge states, similar to those obtained when electrospraying unfolded proteins in a denaturing solvent. This effect has been reported previously (Sterling, H. J.; Cassou, C. A.; Susa, A. C.; Williams, E. R. Anal. Chem. 2012, 84, 3795), but its exact mechanistic origin remains unclear. ESI-mediated unfolding does not take place in acetate under otherwise identical conditions. We demonstrate that heating of protein-containing bicarbonate solutions results in extensive foaming, caused by CO2 outgassing. In contrast, acetate solutions do not generate foam. Protein denaturation caused by gas bubbles is a well-known phenomenon. Adsorption to the gas/liquid interface is accompanied by major conformational changes that allow the protein to act as a surfactant. The foaming of beer is a manifestation of this effect. Bubble formation in bicarbonate during ESI is facilitated by collisional and blackbody droplet heating. Our data imply that heat and bubbles act synergistically to cause unfolding during the electrospray process, while proteins reside in ESI droplets. Because of this effect we advise against the use of ammonium bicarbonate for native ESI-MS. Ammonium acetate represents a gentler droplet environment, despite its low buffering capacity.

The function of hemoglobin (Hb) as oxygen transporter is mediated by reversible O2 binding to Fe(2+) heme in each of the α and β subunits. X-ray crystallography revealed different subunit arrangements in oxy-Hb and deoxy-Hb. The deoxy state is stabilized by additional contacts, causing a rigidification that results in strong protection against hydrogen/deuterium exchange (HDX). Aquomet-Hb is a dysfunctional degradation product with four water-bound Fe(3+) centers. Heme release from aquomet-Hb is relatively facile, triggering oxidative damage of membrane lipids. Aquomet-Hb crystallizes in virtually the same conformation as oxy-Hb. Hence, it is commonly implied that the solution-phase properties of aquomet-Hb should resemble those of the oxy state. This work compares the structural dynamics of oxy-Hb and aquomet-Hb by HDX mass spectrometry (MS). It is found that the aquomet state exhibits a solution-phase structure that is significantly more dynamic, as manifested by elevated HDX levels. These enhanced dynamics affect the aquomet α and β subunits in a different fashion. The latter undergoes global destabilization, whereas the former shows elevated HDX levels only in the heme binding region. It is proposed that these enhanced dynamics play a role in facilitating heme release from aquomet-Hb. Our findings should be of particular interest to the MS community because oxy-Hb and aquomet-Hb serve as widely used test analytes for probing the relationship between biomolecular structure in solution and in the gas phase. We are not aware of any prior comparative HDX/MS experiments on oxy-Hb and aquomet-Hb.

Kinetic folding experiments by pulsed hydrogen/deuterium exchange (HDX) mass spectrometry (MS) are a well-established tool for water-soluble proteins. To the best of our knowledge, the current study is the first that applies this approach to an integral membrane protein. The native state of bacteriorhodopsin (BR) comprises seven transmembrane helices and a covalently bound retinal cofactor. BR exposure to sodium dodecyl sulfate (SDS) induces partial unfolding and retinal loss. We employ a custom-built three-stage mixing device for pulsed-HDX/MS investigations of BR refolding. The reaction is triggered by mixing SDS-denatured protein with bicelles. After a variable folding time (10 ms to 24 h), the protein is exposed to excess D2O buffer under rapid exchange conditions. The HDX pulse is terminated by acid quenching after 24 ms. Subsequent off-line analysis is performed by size exclusion chromatography and electrospray MS. These measurements yield the number of protected backbone N–H sites as a function of folding time, reflecting the recovery of secondary structure. Our results indicate that much of the BR secondary structure is formed quite late during the reaction, on a time scale of 10 s and beyond. It is hoped that in the future it will be possible to extend the pulsed-HDX/MS approach employed here to membrane proteins other than BR. Copyright © 2012 John Wiley & Sons, Ltd.

Hydrogen/deuterium exchange mass spectrometry reveals that the antiapoptotic protein MCL-1 is inhibited by a covalent modification far from its functional site. This finding opens new avenues for cancer therapy, but it also highlights that much remains to be learned about the fundamental basis of allosteric regulation.

Electrospray ionization mass spectrometry (ESI-MS) has become an indispensable technique for examining noncovalent protein complexes. Collision-induced dissociation (CID) of these multiply protonated gaseous ions usually culminates in ejection of a single subunit with a disproportionately large amount of charge. Experiments suggest that this process involves subunit unfolding prior to separation from the residual complex, as well as H(+) migration onto the unravelling chain. Molecular dynamics (MD) simulations are a promising avenue for gaining detailed insights into these CID events. Unfortunately, typical MD algorithms do not allow for mobile protons. Here we address this limitation by implementing a strategy that combines atomistic force fields (such as OPLS/AA and CHARMM36) with a proton hopping algorithm, focusing on the tetrameric complexes transthyretin and streptavidin. Protons are redistributed over all acidic and basic sites in 20 ps intervals, subject to an energy function that reflects electrostatic interactions and proton affinities. Our simulations predict that nativelike conformers at the onset of collisional heating contain multiple salt bridges. Collisional heating initially causes subtle structural changes that lead to a gradual decline of these zwitterionic patterns. Many of the MD runs show gradual unfolding of a single subunit in conjunction with H(+) migration, culminating in subunit separation from the complex. However, there are also instances where two or more chains start to unfold simultaneously, giving rise to charge competition. The scission point where the "winning" subunit separates from the complex can be attained for different degrees of unfolding, giving rise to product ions in various charge states. The simulated product ion distributions are in close agreement with experimental CID data. Proton enrichment in the departing subunit is driven by charge-charge repulsion, but the combination of salt bridge depletion, charge migration, and proton affinity causes surprising compensation effects among the various energy terms. It appears that this work provides the most detailed account to date of the mechanism whereby noncovalent protein complexes disassemble during CID.

FoF1 is a membrane-bound molecular motor that uses proton-motive force (PMF) to drive the synthesis of ATP from ADP and Pi. Reverse operation generates PMF via ATP hydrolysis. Catalysis in either direction involves rotation of the γe shaft that connects the α3β3 head and the membrane-anchored cn ring. X-ray crystallography and other techniques have provided insights into the structure and function of FoF1 subcomplexes. However, interrogating the conformational dynamics of intact membrane-bound FoF1 during rotational catalysis has proven to be difficult. Here, we use hydrogen/deuterium exchange mass spectrometry to probe the inner workings of FoF1 in its natural membrane-bound state. A pronounced destabilization of the γ C-terminal helix during hydrolysis-driven rotation was observed. This behavior is attributed to torsional stress in γ, arising from γ⋅⋅⋅α3β3 interactions that cause resistance during γ rotation within the apical bearing. Intriguingly, we find that destabilization of γ occurs only when FoF1 operates against a PMF-induced torque; the effect disappears when PMF is eliminated by an uncoupler. This behavior resembles the properties of automotive engines, where bearings inflict greater forces on the crankshaft when operated under load than during idling.

The protein Chibby (Cby) is an antagonist of the Wnt signaling pathway, where it inhibits the binding between the transcriptional coactivator β‐catenin and the Tcf/Lef transcription factors. The 126 residue Cby is partially disordered; its N‐terminal half is unstructured while its C‐terminal half comprises a coiled‐coil domain. Previous structural analyses of Cby using NMR spectroscopy suffered from severe line broadening for residues within the protein's C‐terminal half, hindering detailed characterization of the coiled‐coil domain. Here, we use hydrogen/deuterium exchange‐mass spectrometry (HDX‐MS) to examine Cby's C‐terminal half. Results reveal that Cby is divided into three structural elements: a disordered N‐terminal half, a coiled‐coil domain, and a C‐terminal unstructured extension consisting of the last ∼ 25 residues (which we term C‐terminal extension). A series of truncation constructs were designed to assess the roles of individual structural elements in protein stability and Cby binding to TC‐1, a positive regulator of the Wnt signaling pathway. CD and NMR data show that Cby maintains coiled‐coil structure upon deletion of either disordered region. NMR and ITC binding experiments between Cby and TC‐1 illustrate that the interaction is retained upon deletion of either Cby's N‐terminal half or its C‐terminal extension. Intriguingly, Cby's C‐terminal half alone binds to TC‐1 with significantly greater affinity compared to full‐length Cby, implying that target binding of the coiled‐coil domain is affected by the flanking disordered regions.

Mass spectrometry (MS)-based protein conformational studies are a rapidly growing field. The characterization of partially disordered conformers is of particular interest because these species are not amenable to classical high-resolution techniques. Such equilibrium intermediates can often be populated by exposure to mildly acidic pH. Hydroxyl radical (·OH) introduces oxidative modifications at solvent-accessible side chains, while buried sites are protected. ·OH can be generated by laser photolysis of H(2)O(2) (fast photochemical oxidation of proteins-FPOP). The resulting labeling pattern can be analyzed by MS. The characterization of partially disordered intermediates usually involves comparative measurements under different solvent conditions. It can be challenging to separate structurally induced labeling changes from pH-mediated "secondary" effects. The issue of secondary effects in FPOP has received little prior attention. We demonstrate that with a proper choice of conditions (e.g., in the absence of pH-dependent ·OH scavengers) such undesired phenomena can be almost completely eliminated. Using apomyoglobin as a model system, we map the structure of an intermediate that is formed at pH 4. This species retains a highly protected helix G that is surrounded by partially protected helices A, B, and H. Our results demonstrate the utility of FPOP for the structural characterization of equilibrium intermediates. The near absence of an intrinsic pH dependence represents an advantage compared to hydrogen/deuterium exchange MS.

Computer-assisted topology predictions are widely used to build low-resolution structural models of integral membrane proteins (IMPs). Experimental validation of these models by traditional methods is labor intensive and requires modifications that might alter the IMP native conformation. This work employs oxidative labeling coupled with mass spectrometry (MS) as a validation tool for computer-generated topology models. ·OH exposure introduces oxidative modifications in solvent-accessible regions, whereas buried segments (e.g., transmembrane helices) are non-oxidizable. The Escherichia coli protein WaaL (O-antigen ligase) is predicted to have 12 transmembrane helices and a large extramembrane domain (Pérez et al., Mol. Microbiol. 2008, 70, 1424). Tryptic digestion and LC-MS/MS were used to map the oxidative labeling behavior of WaaL. Met and Cys exhibit high intrinsic reactivities with ·OH, making them sensitive probes for solvent accessibility assays. Overall, the oxidation pattern of these residues is consistent with the originally proposed WaaL topology. One residue (M151), however, undergoes partial oxidation despite being predicted to reside within a transmembrane helix. Using an improved computer algorithm, a slightly modified topology model was generated that places M151 closer to the membrane interface. On the basis of the labeling data, it is concluded that the refined model more accurately reflects the actual topology of WaaL. We propose that the combination of oxidative labeling and MS represents a useful strategy for assessing the accuracy of IMP topology predictions, supplementing data obtained in traditional biochemical assays. In the future, it might be possible to incorporate oxidative labeling data directly as constraints in topology prediction algorithms.

The structural characterization of gaseous biomolecular ions remains a challenging task. Here, we employ a combination of gas-phase hydrogen-deuterium exchange (HDX) and electron capture dissociation (ECD) mass spectrometry for gaining insights into the properties of two electrosprayed peptides: RA(9)K and RG(9)K. Mass analysis of ECD fragments provides spatially resolved labeling information. ND(3)-mediated HDX at peptide termini and amino acid side chains goes to completion within 1 s. Backbone amide labeling occurs more slowly, and proceeds in a structurally sensitive fashion. HDX is more extensive for RG(9)K than for RA(9)K, suggesting a more "open" conformation for the former. Residues 7-10 in RA(9)K are strongly protected, which indicates the presence of stable backbone hydrogen bonds at these sites. Our findings are consistent with the results of previous ion mobility measurements and computational investigations. Overall, it appears that the combination of gas-phase HDX and ECD represents a viable approach for uncovering structural features of biomolecular ions in the gas phase.

Many proteins act as molecular machines that are fuelled by a nonthermal energy source. Examples include transmembrane pumps and stator-rotor complexes. These systems undergo cyclic motions (CMs) that are being driven along a well-defined conformational trajectory. Superimposed on these CMs are thermal fluctuations (TFs) that are coupled to stochastic motions of the solvent. Here we explore whether the TFs of a molecular machine are affected by the occurrence of CMs. Bacteriorhodopsin (BR) is a light-driven proton pump that serves as a model system in this study. The function of BR is based on a photocycle that involves trans/cis isomerization of a retinal chromophore, as well as motions of transmembrane helices. Hydrogen/deuterium exchange (HDX) mass spectrometry was used to monitor the TFs of BR, focusing on the monomeric form of the protein. Comparative HDX studies were conducted under illumination and in the dark. The HDX kinetics of BR are dramatically accelerated in the presence of light. The isotope exchange rates and the number of backbone amides involved in EX2 opening transitions increase roughly 2-fold upon illumination. In contrast, light/dark control experiments on retinal-free protein produced no discernible differences. It can be concluded that the extent of TFs in BR strongly depends on photon-driven CMs. The light-induced differences in HDX behavior are ascribed to protein destabilization. Specifically, the thermodynamic stability of the dark-adapted protein is estimated to be 5.5 kJ mol(-1) under the conditions of our work. This value represents the free energy difference between the folded state F and a significantly unfolded conformer U. Illumination reduces the stability of F by 2.2 kJ mol(-1). Mechanical agitation caused by isomerization of the chromophore is transferred to the surrounding protein scaffold, and subsequently, the energy dissipates into the solvent. Light-induced retinal motions therefore act analogously to an internal heat source that promotes the occurrence of TFs. Overall, our data highlight the potential of HDX methods for probing the structural dynamics of molecular machines under "engine on" and "engine off" conditions.

Deciphering the mechanisms of protein folding remains a considerable challenge. In this review we discuss the application of pulsed oxidative labeling for tracking protein structural changes in a time-resolved fashion. Exposure to a microsecond OH pulse at selected time points during folding induces the oxidation of solvent-accessible side chains, whereas buried residues are protected. Oxidative modifications can be detected by mass spectrometry. Folding is associated with dramatic accessibility changes, and therefore this method can provide detailed mechanistic insights. Solvent accessibility patterns are complementary to H/D exchange investigations, which report on the extent of hydrogen bonding. This review highlights the application of pulsed OH labeling to soluble proteins as well as membrane proteins.

Protein structural studies are particularly challenging under conditions in which several conformational species (e.g., monomers and aggregated forms) coexist in solution. Most spectroscopic techniques provide population-averaged data. Hence, it is usually not possible to obtain detailed structural information on individual protein species in heterogeneous samples. The current work employs an experimental strategy that addresses this issue. Solution-phase hydrogen exchange (HX) is used in combination with tandem mass spectrometry. Electrosprayed intact ions exhibiting specific HX mass shifts are selected in the gas phase, followed by electron capture dissociation. The resulting fragment ion deuteration pattern provides amide hydrogen bonding information in a conformer-specific and spatially resolved fashion. The feasibility of this approach is demonstrated by applying it to neurotoxic Aβ(1-42) oligomers that coexist with disordered monomers in solution. The findings of this study point to similarities between oligomers and mature amyloid fibrils with regard to the Aβ(1-42) backbone organization. Specifically, fibrils and oligomers appear to share a β-loop-β secondary structure motif. The spatial resolution obtained with the "top-down" approach used here exceeds that of earlier proteolysis-based HX data on Aβ.

Electrospray ionization (ESI) allows the transfer of multi-protein complexes into the gas phase, thereby providing a simple approach for monitoring the stoichiometry of these noncovalent assemblies by mass spectrometry (MS). It remains unclear, however, whether the measured ion abundance ratios of free and bound species are suitable for determining solution-phase binding affinities (K d values). Many types of mass spectrometers employ rf-only quadrupoles as ion guides. This work demonstrates that the settings used for these devices are a key factor for ensuring uniform transmission behavior, which is a prerequisite for meaningful affinity measurements. Using bovine β-lactoglobulin and hemoglobin as model systems, it is demonstrated that under carefully adjusted conditions the “direct” ESI-MS approach is capable of providing K d values that are in good agreement with previously published solution-phase data. Of the several ion sources tested, a regular ESI emitter operated with pressure-driven flow at 1 μL min–1 provided the most favorable results. Potential problems in these experiments include conformationally-induced differences in ionization efficiencies, inadvertent collision-induced dissociation, and ESI-induced clustering artifacts. A number of simple tests can be conducted to assess whether or not these factors are prevalent under the conditions used. In addition, the fidelity of the method can be scrutinized by performing measurements over a wide concentration range. Overall, this work supports the viability of the direct ESI-MS approach for determining binding affinities of protein–protein complexes in solution.

Laser-induced oxidative labeling of proteins provides insights into biomolecular structures and interactions. In these experiments, the hydroxyl radical ((*)OH) formed by photolysis of H(2)O(2) generates covalent modifications that are detectable by mass spectrometry. Under conditions where individual protein molecules are irradiated only once, the short (*)OH lifetime ( approximately 1 mus) ensures that covalent modifications are formed before any oxidation-induced conformational changes take place. This feature implies that the method should be free of structural artifacts. It has been proposed that single-exposure conditions can be achieved by passing the solution through a capillary where successive laser pulses generate a string of irradiated flow segments that are well separated from one another. The current work explores the convection phenomena within the labeling capillary in more detail. The experiments are conducted at Reynolds numbers <

Protein-metal complexes may be transferred from solution into the gas phase by electrospray ionization (ESI), such that they can be directly analyzed by mass spectrometry (MS). In principle, therefore, ESI-MS represents a simple and elegant approach for gaining insights into the binding stoichiometry and affinity of these assemblies. Unfortunately, the formation of nonspecific metal adducts during ESI can be a severe problem, often leading to binding levels that are dramatically higher than those in bulk solution. Focusing on several calcium binding proteins as test systems, this work explores the suitability of different salts to serve as metal source. Despite their widespread use in previous ESI-MS studies, calcium chloride and acetate induce extensive nonspecific adduction. In contrast, much lower levels of artifactual metal binding are observed in the presence of calcium tartrate. In the case of high and intermediate affinity proteins, the resulting ESI-MS data are in excellent agreement with the calcium binding behavior in bulk solution. The situation is more challenging when studying proteins with very low affinities, but in the presence of tartrate qualitative information on protein-metal interactions can still be obtained. The beneficial effects of tartrate also extend to zinc binding experiments. This work does not directly explore the mechanism by which tartrate suppresses nonspecific metalation. However, it seems likely that weak chelators such as tartrate sequester metal ions within rapidly shrinking droplets during the final stages of ESI, thereby reducing nonspecific metal adduction to protein carboxylates. The use of tartrate and possibly other weak chelators will greatly enhance the reliability of future ESI-MS studies on the interactions of proteins with divalent metal ions.

The exposure of solution-phase proteins to reactive oxygen species (ROS) causes oxidative modifications, giving rise to the formation of covalent +16 Da adducts. Electrospray ionization (ESI) mass spectrometry (MS) is the most widely used method for monitoring the extent of these modifications. Unfortunately, protein oxidation can also take place as an experimental artifact during ESI, such that it may be difficult to assess the actual level of oxidation in bulk solution. Previous work has demonstrated that ESI-induced oxidation is highly prevalent when operating at strongly elevated capillary voltage V(0) (e.g., +8 kV) and with oxygen nebulizer gas in the presence of a clearly visible corona discharge. Protein oxidation under these conditions is commonly attributed to OH radicals generated in the plasma of the discharge. On the other hand, charge balancing oxidation reactions are known to take place at the metal/liquid interface of the emitter. Previous studies have not systematically explored whether such electrochemical processes could be responsible for the formation of oxidative +16 Da adducts instead of (or in combination with) plasma-generated ROS. Using hemoglobin as a model system, this work illustrates the occurrence of extensive protein oxidation even under typical operating conditions (e.g., V(0) = 3.5 kV, N(2) nebulizer gas). Surprisingly, measurements of the current flowing in the ESI circuit demonstrate that a weak corona discharge persists for these relatively gentle settings. On the basis of comparative experiments with nebulizer gases of different dielectric strength, it is concluded that ROS generated under discharge conditions are solely responsible for ESI-induced protein oxidation. This result is corroborated through off-line electrolysis experiments designed to mimic the electrochemical processes taking place during ESI. Our findings highlight the necessity of using easily oxidizable internal standards in biophysical or biomedical ESI-MS studies where knowledge of protein oxidation in bulk solution is desired. Strategies for eliminating ESI-induced oxidation artifacts are discussed.

Exposure of proteins to hydroxyl radicals induces the incorporation of oxygen atoms into solvent-exposed side chains. Earlier studies have employed this approach for mapping protein-protein interactions in mass spectrometry-based footprinting experiments. This work explores whether the overall level of gamma-ray mediated oxidative labeling can be used for monitoring large-scale conformational changes. According to a recently developed kinetic model (Tong, X.; Wren, J. C.; Konermann, L. Anal. Chem. 2007, 79, 6376-6382), the apparent first-order rate constant for oxidative labeling can be approximated as k(app) = k(RAD)/([P](tot) + C/k(u)), where k(RAD) is the primary rate of *OH formation, [P](tot) is the protein concentration, C reflects the presence of competing radical deactivation channels, and ku is the rate constant at which hydroxyl radicals react with the protein. The current study introduces conformational effects into this model by proposing that k(u) = [see text for formula] , where N is the number of amino acids, alphai is a measure for the solvent exposure of residue i, and k(ch)(i) is the oxidation rate constant that would apply for a completely solvent-exposed side chain. Using myoglobin and cytochrome c as model systems, it is demonstrated that unfolding by addition of H(3)PO(4) increases k(app) by up to 30% and 70%, respectively. Unfolding by other commonly used denaturants such as organic acids or urea results in dramatically lower oxidation levels than for the native state, a behavior that is due to the radical scavenging activity of these substances (corresponding to an increased value of C). Control experiments on model peptides are suitable for identifying such "secondary" effects, i.e., factors that modify oxidation levels without being related to conformational changes. In conclusion, the overall *OH labeling level represents a viable probe of large-scale protein conformational changes only under conditions where secondary effects are known to be minimal and where [P](tot) is constant.

Electrospray ionization mass spectrometry (ESI-MS) is a commonly used tool for characterizing conformational changes of proteins in solution. Different conformations can be distinguished on the basis of their ESI charge state distributions. ESI-MS studies carried out under semidenaturing conditions result in bi- or multimodal distributions that reflect the presence of coexisting conformers. This study explores whether the concentration ratios of these species in solution are reflected in the measured ion intensities. Experiments on two model proteins, lysozyme and myoglobin, reveal that non-native polypeptide chains tend to result in a much stronger signal response than natively folded species. The measured ion intensity ratios can differ from the actual concentration ratios by as much as 2 orders of magnitude. It is proposed that the higher ionization efficiency of unfolded proteins is due to their partially hydrophobic character, which results in a larger surface activity and facilitates protein transfer into ion-producing progeny droplets. Conversely, natively folded proteins have a lower affinity for the air/liquid interface, such that ionization of these conformers is suppressed. The extent of ion suppression is strongly dependent on the experimental conditions such as flow rate and protein concentration, which determine if ESI occurs in a charge deficient or a charge surplus regime. These aspects should be taken into account for the design of ESI-MS-based protein folding experiments and for studies that use ion intensity ratios for the determination of protein-ligand binding affinities.

Electrospray ionization (ESI)-mass spectrometry (MS) can provide information on protein-ligand interactions via detection of the corresponding complexes as gaseous ions. Unfortunately, some systems are prone to dissociation upon transfer into the gas phase. The reversible oxygen binding to hemoglobin (Hb) has been extensively studied in solution using a wide range of biophysical techniques. In addition to O2, ferrous (Fe(II)) Hb can bind CO. High affinity interactions with CN(-) are limited to the ferric (Fe(III)) state. In analogous fashion, CN(-), CO, and O2 bind to myoglobin (Mb). It remains unclear whether any of these ligand-bound forms can be observed by ESI-MS. In this work we demonstrate the successful detection of MbCN, while MbCO and MbO2 do not survive under ESI-MS conditions. Control experiments suggest that an older report of "MbO2" detection by ESI-MS may involve the misassignment of oxidation artifacts formed under corona discharge conditions. The situation is more favorable for ESI-MS studies on Hb. The most intense signal in the HbCN mass distribution corresponds to the expected complex with four cyanide moieties bound. Ligand loss during ESI-MS is around 20%. HbCO is detectable as well, albeit with a more noticeable level of ligand dissociation (∼50%) which produces the 2CO-bound state as the highest intensity ion in the spectrum. In addition, our data suggest that low levels of HbO2 can survive the transition into the gas phase, evident from +64 Da and +128 Da signals that can be assigned to Hb carrying two and four oxygen molecules, respectively. The application of collisional activation induces neutral ligand loss for all three Hb derivatives. It appears that this is the first report on the detection of MbCN, HbCO, and HbO2 in the gas phase. We hope that this work will pave the way towards future spectroscopic investigations of desolvated Mb and Hb, complementing the extensive literature on CN(-), CO, and O2 bound globins in the condensed phase.

Protein analyses by electrospray ionization (ESI) mass spectrometry can suffer from interferences caused by nonvolatile salts. The mechanistic basis of this effect remains to be fully investigated. In the current work we explore the behavior of proteins under native and denaturing conditions in the presence of NaCl, CsCl, and tetrabutyl ammonium chloride (NBu4Cl). All three salts interfere with the formation of "clean" [M + zH](z+) protein ions by progressively deteriorating spectral S/N ratios. We propose that salt interferences can be dissected into two independent aspects, i.e., (i) peak splitting by adduct formation and (ii) protein ion suppression. NaCl degrades the spectral quality by forming heterogeneous [M + zH + n(Na - H) + m(Cl + H)](z+) ions, while the integrated protein ion intensity remains surprisingly robust. Conversely, NBu4Cl does not cause any adduction, while dramatically reducing the protein ion yield. These findings demonstrate that adduct formation and protein ion suppression are indeed unrelated effects that may occur independently of one another. Other salts, such as CsCl, can give rise to a combination of the two scenarios. Molecular dynamics simulations of water droplets charged with either Na(+) or NBu4(+) provide insights into the mechanism underlying the observed effects. Na(+) containing droplets evolve relatively close to the Rayleigh limit (z/z(R) ≈ 0.74), whereas the z/z(R) values of NBu4(+) charged droplets are considerably lower (∼0.59). This difference is due to the high surface affinity of NBu4(+), which facilitates charge ejection from the droplet. We propose that the low z/z(R) values encountered in the presence of NBu4(+) suppress the Rayleigh fission of parent droplets in the ESI plume, thereby reducing the yield of progeny droplets that represent the precursors of gaseous protein ions. In addition, the rate of solvent evaporation is reduced in the presence of NBu4(+). Both of these factors lower the protein signal intensity. NaCl does not interfere with droplet fission, such that protein ions continue to form with high yield—albeit in heavily adducted form. Our findings expand on earlier proposals of charge competition as a key factor during the ESI process for salt-contaminated solutions.

Mass spectrometry (MS) owes its remarkable rise as a bioanalytical tool to the development of two “soft” ionization techniques: matrix-assisted laser desorption/ ionization (MALDI) (1,2) and electrospray ionization (ESI) (3). The salient feature of soft ionization is that a wide range of analytes, from small metabolites to very large biomolecular systems, can be transferred intact into the gas phase, thus making them amenable to mass spectrometric analysis. For MALDI-MS, the sample is embedded into a crystalline matrix prior to analysis. For ESI, in contrast, ionization is initiated directly from the liquid phase by spraying analyte solution from the tip of an electrically charged capillary. The small droplets formed by the ESI emitter undergo rapid evaporation and fi ssion, ultimately releasing the analyte as multiply protonated (or deprotonated) species (4).

Electrospray ionization (ESI) mass spectrometry (MS) has become a popular tool for monitoring ligand–protein and protein–protein interactions. Due to the "gentle" nature of the ionization process, it is often possible to transfer weakly bound complexes into the gas phase, thus making them amenable to MS detection. One problem with this technique is the potential occurrence of fragmentation events during ESI. Also, some analytes tend to cluster together during ionization, thus forming nonspecific gas-phase assemblies that do not represent solution-phase complexes. In this work, we implemented a hydrogen–deuterium exchange (HDX) approach that can reveal whether or not the free and (or) bound constituents of a complex observed in ESI-MS reflect the binding situation in solution. Proteins are subjected to ESI immediately following an isotopic labeling pulse; only ligand-free and ligand-bound protein ions that were formed directly from the corresponding solution-phase species showed different HDX levels. Using myoglobin as a model system, it is demonstrated that this approach can readily distinguish scenarios where the heme–protein interactions were disrupted in solution from those where dissociation of the complex occurred in the gas phase. Experiments on cytochrome c strongly suggest that dimeric protein ions observed in ESI-MS reflect aggregates that were formed in solution.Key words: electrospray mass spectrometry, ligand–protein interaction, noncovalent complex, hydrogen–deuterium exchange, protein folding.

The possible involvement of "hidden" kinetic intermediates in the apparent two-state folding of some proteins is currently a matter of debate. This study uses time-resolved electrospray ionization (ESI) mass spectrometry with on-line pulsed hydrogen-deuterium exchange (HDX) for monitoring the refolding of acid/methanol-denatured ubiquitin. It is demonstrated that the ESI charge-state distribution (CSD) and the extent of HDX represent nonredundant probes of the protein structure in solution. When considered in isolation, the data provided by both of these probes are consistent with a two-state behavior, involving only denatured ubiquitin D and refolded protein F. However, a careful comparison of the CSD and HDX kinetics reveals the presence of an additional species, exhibiting a CSD like the folded protein but showing non-native HDX characteristics. This kinetic intermediate, D*, is in rapid equilibrium with D, such that the overall reaction is consistent with the mechanism D--D* --F. The results of this work suggest that the occurrence of transient intermediates may be more widespread than commonly thought, especially in cases where a cursory analysis indicates two-state behavior.

The application of a novel method for the identification of low-molecular-weight noncovalent ligands to a macromolecular target is reported. This technique is based on the measurement of analyte diffusion coefficients by electrospray mass spectrometry (ESI-MS) (Clark et al., Rapid Commun. Mass Spectrom. 2002, 16, 1454-1462). Potential ligands have large diffusion coefficients as long as they are free in solution. Binding to a macromolecular target, however, drastically reduces the diffusional mobility of any ligand species. Mixtures containing six different saccharides [ribose, rhamnose, glucose, maltose, maltotriose, and N,N',N''-triacetylchitotriose (NAG(3))] were screened for noncovalent binding to lysozyme. Of these six compounds, only NAG(3) is known to bind to the protein. In "direct" binding tests, NAG(3) shows a significantly reduced diffusion coefficient in the presence of the protein. No changes were observed for any of the other saccharides. In a second set of experiments, the use of a "competition" screening method was explored in which mixtures of candidate saccharides were tested for their ability to displace a reference ligand from the target. The addition of NAG(3)-containing mixtures significantly increased the diffusion coefficient of the reference ligand NAG(4) (N,N',N'',N'''-tetraacetylchitotetrose), whereas mixtures that did not contain NAG(3) had no effect. These data clearly indicate the potential of ESI-MS-based diffusion measurements as a novel tool to screen compound libraries for binding to proteins and other macromolecular targets. In contrast to conventional ESI-MS-based ligand-receptor binding studies, this method does not rely on the preservation of noncovalent interactions in the gas phase.

The "protein-folding problem" refers to the question of how and why a denatured polypeptide chain can spontaneously fold into a compact and highly ordered conformation. The classical description of this process in terms of reaction pathways has been complemented by models that describe folding as a biased conformational diffusion on a multidimensional energy landscape. The identification and characterization of short-lived intermediates provide important insights into the mechanism of folding. Pulsed hydrogen/deuterium exchange (HDX) methods are among the most powerful tools for studying the properties of kinetic intermediates. Analysis of pulse-labeled proteins by mass spectrometry (MS) provides information that is complementary to that obtained in nuclear magnetic resonance (NMR) studies; NMR data represent an average of entire protein ensembles, whereas MS can detect co-existing protein species. MS-based pulse-labeling experiments can distinguish between folding scenarios that involve parallel pathways, and those where folding is channeled through obligatory intermediates. The proteolytic digestion/MS technique provides spatially resolved information on the HDX pattern of folding intermediates. This method is especially important for proteins that are too large to be studied by NMR. Although traditional pulsed HDX protocols are based on quench-flow techniques, it is also possible to use electrospray (ESI) MS to analyze the reaction mixture on-line and "quasi-instantaneously" after labeling. This approach allows short-lived protein conformations to be studied by their HDX level, their ESI charge-state distribution, and their ligand-binding state. Covalent labeling of free cysteinyl residues provides an alternative approach to pulsed HDX experiments. Another promising development is the use of synchrotron X-rays to induce oxidation at specific sites within a protein for studying their solvent accessibility during folding.

A novel technique for studying protein folding kinetics is presented. It is based on a continuous-flow setup that is coupled to an electrospray (ESI) mass spectrometer and allows initiation of a folding reaction, followed by isotopic pulse labeling. The protein is electrosprayed "quasi-instantaneously" after exposure to the deuterated solvent. This approach yields structural information from the ESI charge state distribution and from the H/D exchange levels of individual protein states, while at the same time noncovalent interactions can be monitored. This technique is used to study the reconstitution of holomyoglobin (hMb) from unfolded apomyoglobin (aMb) and free heme. MS/MS is used to establish that a short-lived folding intermediate with two heme groups attached represents a protein-bound heme dimer. This state appears to have a compactness close to that of native hMb; however, isotopic labeling indicates a significantly perturbed structure. Another intermediate is bound to a single heme group and shows a charge state distribution similar to that of unfolded aMb. Exchange levels exhibited by this state are lower than for unfolded aMb, indicating that fewer hydrogens are exposed to the solvent and/or that more of them are involved in hydrogen bonding. Native hMb leads to the formation of low charge state ions (hMb(9+), hMb(8+)) and shows low exchange levels. However, early during reconstitution, a slightly unfolded form of the heme-protein complex contributes to the observed hMb(9+) ions. A peak width analysis reveals that the structural heterogeneity of some of the observed protein species decreases as reconstitution proceeds.

Hydrogen/deuterium exchange (HDX) methods are widely used for monitoring protein-ligand interactions. This approach relies on the fact that ligand binding can modulate the extent of protein structural fluctuations that transiently disrupt hydrogen bonds and expose backbone amides to the solvent. It is commonly observed that ligand binding causes a reduction of HDX rates. This reduction can be restricted to elements adjacent to the binding site, but other regions can be affected as well. Qualitatively, ligand-induced HDX protection can be rationalized on the basis of two-state models that equate structural dynamics with global unfolding/refolding. Unfortunately, such models tend to be unrealistic because the dynamics of native proteins are dominated by subglobal transitions and local fluctuations. Ligand binding lowers the ground-state free energy. It is not obvious why this should necessarily be accompanied by a depletion of excited-state occupancies, which would be required for a reduction of HDX rates. Here, we propose a framework that implies that ligand binding can either slow or accelerate amide deuteration throughout the protein. These scenarios are referred to as "type 1" and "type 2", respectively. Evidence for type 1 binding is abundant in the literature, whereas the viability of type 2 interactions is less clear. Using HDX mass spectrometry (MS), we demonstrate that the oxygenation of hemoglobin (Hb) provides a dramatic example of a type 2 scenario. The observed behavior is consistent with cooperative T → R switching, where part of the intrinsic O2 binding energy is reinvested for destabilization of the ground state. This destabilization increases the Boltzmann occupancy of unfolded conformers, thereby enhancing HDX rates. Surprisingly, O2 binding to myoglobin (Mb) also induces elevated HDX rates. These Mb data reveal that type 2 behavior is not limited to cooperative multisubunit systems. Although enhanced protection from deuteration is widely considered to be a hallmark of protein-ligand interactions, this work establishes that an overall deuteration increase also represents a viable outcome. HDX-based ligand screening assays, therefore, have to allow for canonical as well as noncanonical effects.

The operation of an electrospray ion source in the positive ion mode involves charge-balancing oxidation reactions at the liquid/metal interface of the sprayer capillary. One of these reactions is the electrolytic oxidation of water. The protons generated in this process acidify the analyte solution within the electrospray capillary. This work explores the effects of this acidification on the electrospray ionization (ESI) mass spectrum of the protein cytochrome c (cyt c). In aqueous solution containing 40% propanol, cyt c unfolds around pH 5.6. Mass spectra recorded under these conditions, using a simple ESI series circuit, display a bimodal charge-state distribution that reflects an equilibrium mixture of folded and unfolded protein in solution. These spectra are not strongly affected by electrochemical acidification. An "external loop" is added to the ESI circuit when the metal needle of the sample injection syringe is connected to ground. The resulting circuit represents two coupled electrolytic cells that share the ESI capillary as a common anode. Under these conditions, the rate of charge-balancing oxidation reactions is dramatically increased because the ion source has to supply electrons for both, the external circuit and the ESI circuit. The analytical implications of this effect are briefly discussed. Mass spectra of cyt c recorded with the syringe needle grounded are shifted to higher charge states, indicating that electrochemical acidification has caused the protein to unfold in the ion source. The acidification can be suppressed by increasing the flow rate and lowering the electrolyte concentration of the solution and by using an electrolyte that acts as redox buffer. The observed acidification is similar for sprayer capillaries made of platinum and stainless steel. Removal of the protective oxide layer on the stainless steel surface results in effective redox buffering for a few minutes.