Lars Konermann

The association of proteins into amorphous aggregates is linked to various detrimental phenomena, ranging from disease mechanisms to the degradation of biopharmaceuticals. The development of aggregation suppression strategies is of considerable interest. Experiments on aggregation (and aggregation suppression) are commonly performed under accelerated conditions, where proteins are exposed to elevated temperatures. Focusing on the thermal aggregation of myoglobin (Mb, a paradigmatic model protein), our group previously reported that intermolecular Lys+/Glu– and Lys+/Asp– salt bridges are a key driver of aggregate formation. Additives such as free arginine and guanidinium salts suppress aggregation by “blocking” Glu– and Asp– side chains, thereby preventing them from participating in salt bridges (Ng & Konermann (2024) JACS 146, 8394). However, the use of such additives, e.g., as excipients in biopharmaceutical formulations, can be problematic. The current work highlights a complementary approach. Using spectroscopic experiments and molecular dynamics simulations, we show that acetylation of Lys side chains is highly effective for suppressing the thermal aggregation of Mb. Acetylation (R–NH3+ → R–NH–CO–CH3) eliminates the positive charge of Lys, thereby rendering the corresponding side chains incapable of salt bridge formation. It is demonstrated that acetylation only has relatively minor effects on the native protein structure under physiological conditions (room temperature, pH 7). In summary, Lys acetylation is a simple yet effective aggregation suppression approach that may also be applicable to other proteins, including biopharmaceuticals such as monoclonal antibodies.

Many operating principles of electrospray ionization (ESI) emitters have been uncovered, but some aspects are yet to be fully elucidated. One of these is the electrophoretic migration of dissolved ions in the solution near the emitter outlet. Also, it remains unclear to what extent the electric field generated by the ESI voltage penetrates the solution (keeping in mind that dielectric effects tend to suppress external fields). We performed molecular dynamics (MD) simulations of ESI emitters containing aqueous NaCl to tackle these topics. ESI in positive ion mode requires the accumulation of excess positive charge in the solution at the emitter outlet. The electric field between the emitter and counter electrode acts on this charge, producing a Taylor cone. We show that this excess charge has two contributions: (i) a polarization charge resulting from H₂O dipole alignment and (ii) cation accumulation resulting from electrophoretic charge separation. Factors (i) and (ii) weaken the electric field in the solution, but a residual component remains that drives cation migration in the direction of the counter electrode. The residual field is strongest in the jet that forms at the Taylor cone apex. Electrophoretic movement of solvated cations in the jet promotes elongation and subsequent rupture of the jet, thereby producing ESI droplets. This work marks the first time that basic operating principles of an ESI emitter have been captured in MD simulations, using a model that employs mass spectrometry-relevant dimensions and realistic electrostatic properties.

MD simulations of droplets in an unprecedented size range capture key steps of native ESI, including droplet fission and evaporation to dryness, culminating in the formation of gaseous protein ions via the charged residue mechanism.

Most enzyme reactions occur in aqueous solution, but catalytic activity is often retained in nonaqueous solvents. It is an intriguing question whether enzymes can also remain active in the gas phase, without any solvent. Here, we address this question by examining electrosprayed [L·NAG₅ + 8H]⁸⁺ complexes (L = lysozyme, NAG_n = N-acetyl glucosamine oligomer, a lysozyme substrate in solution). Collisional activation triggered cleavage of NAG₅ into NAG₄ and a monomeric NAG unit. The reaction proceeded via a [L·NAG₄ + 8H]⁸⁺ enzyme·product complex, confirming that cleavage took place while NAG₅ was bound to lysozyme. We demonstrate that the reaction exhibits hallmarks of enzyme catalysis, including the participation of specific active site residues (E35 and D52), substrate specificity, and a lowered activation energy. The gas-phase products differ from those in solution, as dictated by the absence of water. Our experimental findings are bolstered by mobile-proton molecular dynamics simulations of gaseous lysozyme complexes with substrates and products. In conclusion, this work for the first time demonstrates that an enzyme can be catalytically active in vacuo. While it is yet to be determined if this finding can be generalized to other enzymes, our study may provide a stepping stone to novel enzyme-catalyzed (bio)molecular transformations in the absence of a solvent.

Thermal unfolding ("melting") experiments are widely used for protein stability measurements. These assays probe spectroscopic properties of the protein while gradually increasing the solution temperature until unfolding is complete. Thermodynamic parameters are obtained from fits of the resulting profiles. Differential scanning calorimetry relies on similar concepts. Meaningful stability measurements require reversible conditions, where the protein fluctuates between its native and unfolded states (N ⇌ U), as governed by the temperature-dependent equilibrium constant. A simple reversibility test is to ensure that heating and cooling (unfolding and refolding) profiles are superimposable. Unfortunately, such tests are not commonly performed. Here, we focused on cytochrome c, one of the most widely used model proteins for folding studies. Surprisingly, thermal unfolding/refolding was found to be irreversible, even in the absence of aggregation. Using mass spectrometry (MS), we traced the origin of this irreversibility to oxidative side chain modifications that accumulate during thermal assays. By gradually altering the covalent composition of the protein, oxidation creates a scenario far from ideal N ⇌ U conditions, rendering the validity of fitted thermodynamic parameters questionable. Oxidation at Tyr, Trp, and Met residues was promoted by dissolved O₂. It appears that the role of oxidation as an impediment for protein stability assays has been overlooked in the past. While the use of deoxygenated solutions represents a partial remedy, it is hoped that better oxidation suppression strategies will be developed in the future. In any case, it is advisable to perform MS measurements alongside thermal protein stability experiments to ensure that problems related to oxidation-mediated irreversibility are properly recognized.

The electric field emanating from an electrospray ionization (ESI) capillary triggers the formation of a Taylor cone and the release of charged droplets. Uncovering the properties of this electric field is essential for developing a comprehensive understanding of the ESI process. The field is the negative gradient of the electric potential. The potential can be obtained by numerically solving the Poisson or Laplace equations, e.g., by using commercial software packages. However, such software tools do not necessarily provide mass spectrometry practitioners (many of whom were trained as biological or analytical chemists) with an intuitive grasp of the underlying concepts. The current work proposes an equivalent but much simpler method for uncovering the electrostatic properties of an ESI source. We focus on a charged capillary with an adjacent counter electrode (the latter represents the mass spectrometer). Instead of solving differential equations, the algorithm developed here adjusts the charge distribution on the ESI capillary until the potential anywhere on the capillary surface is constant. By definition, this scenario defines electrostatic equilibrium. The counter electrode can be included via simple image charge arguments. Our algorithm correctly predicts charge accumulation at the narrow capillary outlet, giving rise to a strong and highly divergent electric field in this area. Application of the method to various capillary geometries provides insights into the effects of experimental parameters. The code developed here is not intended to replace existing software packages, but it may provide practitioners with a more intuitive understanding of basic electrostatic principles. In addition, the method should be helpful for designing improved molecular dynamics simulations of ESI emitters.

Numerous analytical workflows involve electrospray ionization (ESI), a process that converts solution species into gaseous ions for detection by mass spectrometry (MS). Upon exposure of a solution-filled emitter capillary to an electric field, ESI proceeds via a liquid jet that forms at the apex of a Taylor cone. Breakup of this jet generates charged droplets that ultimately release analyte ions into the gas phase. Many facets of these events remain incompletely understood. Molecular dynamics (MD) simulations of ESI droplets have become an important tool for ESI mechanistic investigations. Here, we extend these modeling efforts to entire ESI emitter capillaries. Such simulations face significant challenges. Chief among these is the fact that experimental ESI emitters operate in steady-state mode, where ejected solution is constantly replenished from an upstream reservoir. Previous MD simulations employed capillaries with a finite amount of solution, resulting in only short bursts of sprayer operation. The current work develops an MD algorithm that combines trajectory stitching with "solution recycling". This algorithm periodically removes ejected droplets, while simultaneously replenishing the emitter inlet with fresh solution. For the first time, this approach provides continuous MD-based ESI operation, while keeping the instantaneous number of atoms (and hence computational cost) manageable. For a 50 ns simulation, we illustrate a reduction of wall clock time from an estimated 5000 days to 20 days. The ESI emitter MD simulations performed here are the most detailed to date, providing insights into the properties of the Taylor cone and the behavior of nascent ESI droplets. Focusing on aqueous NaCl solution, this work provides the first comprehensive perspective of the entire ESI process, from analyte solution in the emitter to MS-detectable gaseous ions.

Reversed-phase liquid chromatography (RPLC) is an essential tool for separating complex mixtures such as proteolytic digests in bottom-up proteomics. There is growing interest in methods that can predict the RPLC retention behavior of peptides and other analytes. Already, existing algorithms provide excellent performance based on empirical rules or large sets of RPLC training data. Here we explored a new type of retention prediction strategy that relies on first-principles modeling of peptide interactions with a C18 stationary phase. We recently demonstrated that molecular dynamics (MD) simulations can provide atomistic insights into the behavior of peptides under RPLC conditions (Anal. Chem. 2023, 95, 3892). However, the current work found that it is problematic to use conventional MD data for retention prediction, evident from a poor correlation between experimental retention times and MD-generated "fraction bound" values. We thus turned to umbrella sampling MD, a complementary technique that has previously been applied to probe noncovalent contacts in other types of systems. By restraining the peptide dynamic motions at various positions inside a C18-lined pore, we determined the free energy of the system as a function of peptide-stationary phase distance. ΔG_binding values determined in this way under various mobile phase conditions were linearly correlated with experimental retention times of tryptic test peptides. This work opens retention prediction avenues for novel types of stationary and mobile phases, and for peptides (or other analytes) having arbitrary chemical properties, without the need for RPLC reference data. Umbrella sampling can be used as a stand-alone tool, or it may serve to enhance existing retention prediction algorithms.

Electrospray ionization (ESI) of serine (Ser) solution generates Ser₈H⁺ as an abundant magic number cluster. ESI clustering of most other solutes yields nonspecific stoichiometries. It is unclear why Ser₈H⁺ dominates in the case of Ser, and how Ser₈H⁺ forms during ESI. Even the location of Ser₈H⁺ formation is contentious (in solution, in ESI droplets, or elsewhere). Here we unravel key aspects of the l-Ser₈H⁺ formation pathway. Harsh ion sampling conditions promote the collision-induced dissociation (CID) of regular ESI analytes. Unexpectedly, Ser₈H⁺ was seemingly resistant against CID during ion sampling, despite its extremely low tandem mass spectrometry (MS/MS) stability. This unusual behavior reveals that Ser₈H⁺ forms during ion sampling. We propose the following pathway: (1) Nonspecific Ser clusters are released when ESI droplets evaporate to dryness. These initial clusters cover a wide size range, from a few Ser to hundreds or thousands of monomers. (2) The clusters undergo dissociation during ion sampling, mostly via successive loss of neutral monomers. For any source activation voltage, there is a subpopulation of clusters for which this CID cascade tends to terminate at the octamer level, culminating in Ser₈H⁺-dominated product distributions. Mobile proton molecular dynamics simulations were used to model the entire pathway. Ser₈H⁺ structures formed in these simulations were consistent with ion mobility experiments. The most compact structures resembled the model of [Scutelnic, V. J. Am. Chem. Soc. 2018, 140, 7554-7560], with numerous intermolecular salt bridges and H-bonds. Our findings illustrate how the interplay of association and dissociation reactions across phase boundaries can culminate in magic number clusters.