Gas-Phase Collisions with Trimethylamine-N-Oxide Enable Activation-Controlled Protein Ion Charge Reduction
Modulating protein ion charge is a useful tool for the study of protein folding and interactions by electrospray ionization mass spectrometry. Here, we investigate activation-dependent charge reduction of protein ions with the chemical chaperone trimethylamine-N-oxide (TMAO). Based on experiments carried out on proteins ranging from 4.5 to 35 kDa, we find that when combined with collisional activation, TMAO removes approximately 60% of the charges acquired under native conditions. Ion mobility measurements furthermore show that TMAO-mediated charge reduction produces the same end charge state and arrival time distributions for native-like and denatured protein ions. Our results suggest that gas-phase collisions between the protein ions and TMAO result in proton transfer, in line with previous findings for dimethyl- and trimethylamine. By adjusting the energy of the collisions experienced by the ions, it is possible to control the degree of charge reduction, making TMAO a highly dynamic charge reducer that opens new avenues for manipulating protein charge states in ESI-MS and for investigating the relationship between protein charge and conformation.
KeywordsCharge reduction Protein structure Native mass spectrometry Gas-phase basicity
The analysis of intact proteins by electrospray ionization (ESI)-mass spectrometry (MS) relies on the generation of multiply charged ions from solution, where protein solution structure and ion charge are intricately related. The prevailing hypothesis is that the number of charges on a protein ion is largely determined by the protein’s conformation in solution [1, 2, 3]. Here, globular proteins give rise to narrow charge state distributions, whereas denatured proteins acquire a higher number of charges and a broad charge state distribution. The gas-phase conformations of the ionized proteins are thus closely correlated with charge: ions with a relatively low number of charges generally exhibit compact conformations, while highly charged ions preferentially populate extended conformations.
The number of charges on a protein ion can be manipulated through the use of supercharging or charge-reducing agents. The technique of charge reduction is often used to preserve protein conformations and interactions for MS analysis, and obtain better resolution of ions with similar masses [4, 5]. Charge reduction in ESI is most commonly carried out by adding compounds with high gas-phase basicities (GB) directly to the ESI solution [4, 5, 6], where the degree of charge reduction is believed to largely correlate with the GB of the additive and the protein [5, 7]. Alternatively, charge reduction can be achieved through gas-phase proton transfer reactions. It has been proposed that gas-phase collisions between protein ions and ammonia, methyl-, or ethylamines effectively reduce ion charges, and found that intact disulfide bonds, but not initial ion conformation, affects the proton transfer reactivity of desolvated proteins [8, 9, 10].
We have recently demonstrated that addition of the chemical chaperone trimethylamine-N-oxide (TMAO) in combination with collisional activation in the ion source of the mass spectrometer substantially reduces the charges of protein ions . Here, we find that activation-dependent charge reduction by TMAO produces the same end charge states and arrival time distributions for denatured and native-like protein ions. Taken together, our findings indicate that charge reduction with TMAO is determined by the number and energy of the collisions experienced by the protein ions, and thus likely scales with the protein ion’s collision cross-sections (CCSs). The ability to tune the energy of the collisions and thus the degree of charge reduction by TMAO offers new opportunities to study the relationship between ion charge and gas-phase conformation.
TMAO-Mediated Charge Reduction Is pH-Independent
TMAO Produces Similar Charge States and Arrival Time Distributions for Native-Like and Denatured Proteins
Removing Residues with High Gas-Phase Basicity Increases TMAO-Mediated Charge Reduction
In the present study, we show that TMAO in combination with collisional activation produces the same end charge state for folded and denatured proteins independent of solution pH. Strikingly, we find that the weak base TMAO (GB 953 kJ/mol) in combination with collisional activation is as effective for charge reduction as highly basic proton sponges with significantly higher GB values (1006–1022 kJ/mol) . In the absence of collisional activation, on the other hand, insignificant charge reduction by TMAO is observed, despite its GB being higher than that of common charge-reducing agents like dimethyl sulfoxide (853 kJ/mol) or imidazole (909 kJ/mol). Although we and others previously showed that TMAO, trimethylamine, and triethylamine adducts can dissociate as charged species [11, 16], our observations cannot be explained based on proton affinities alone.
In a series of studies, Smith and co-workers demonstrated that gas-phase collisions of desolvated proteins with ammonia, methylamines, and ethylamines resulted in effective charge reduction that roughly correlated with the number of arginines available for protonation [8, 9, 10, 17]. They were able to show that protein ions generated from denaturing and native-like ESI solutions , but not under reducing and non-reducing conditions [8, 10], yielded the same end charge states. These findings are in good agreement with the observations made for TMAO. Here, the high TMAO concentration present in the ESI solution likely facilitates collisions with protein ions in the source and trap regions of the mass spectrometer. The fact that we find a strict dependence on collisional activation suggests that we are able to tune the energy and intervals of these collisions to determine the degree of charge reduction at a given TMAO concentration. Similar, but not as pronounced, effects were previously reported for trimethylamine and triethylamine .
In the original studies by Smith and co-workers, it was concluded that denatured and native-like protein ions display very similar proton transfer reactivity thus likely have similar gas-phase conformations . This led us to analyze the conformations of charge-reduced myoglobin by ion mobility. We found that the 3+ ions of both denatured apo- and native-like holo-myoglobin have identical drift times. In line with results from post-ionization charge reduction studies , this suggests that with progressive charge reduction, denatured and native-like ions adopt gas-phase conformations with similar CCS. As their CCSs converge, the ions likely experience a similar number of collisions, and thus reach a similar final charge state regardless of their starting conformation. It is then tempting to speculate that intact disulfide bonds restrict the number of gas-phase conformers, and thus, ions produced under reducing and non-reducing conditions may have different CCSs and thus reach different end charge states.
In summary, activation-dependent charge reduction of protein ions by TMAO as demonstrated here can provide a simple means to manipulate the charge state and gas-phase conformations of protein ions independent of solution conditions, opening new avenues for the study of their structures in the gas-phase.
NÖ is supported by a doctoral scholarship from the Sven and Lilly Lawski Foundation. E.G.M. holds a Marie Skłodowska Curie International Career Grant from the European Commission and the Swedish Research Council (2015-00559). ML is supported by an Ingvar Carlsson Award from the Swedish Foundation for Strategic Research, a KI faculty-funded Career Position, and a KI-StratNeuro starting grant. Part of this work was facilitated by the Protein Science Facility at Karolinska Institutet and SciLifeLab (http://psf.ki.se). Special thanks to Prof. Sir David P. Lane, Karolinska Institutet, for support through Swedish Research Council grant 2013_08807.
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