Native Electrospray Ionization Mass Spectrometry Research Articles

Machine Learning Models Identify Inhibitors of New Delhi Metallo-β-lactamase.

The worldwide spread of the metallo-β-lactamases (MBL), especially New Delhi metallo-β-lactamase-1 (NDM-1), is threatening the efficacy of β-lactams, which are the most potent and prescribed class of antibiotics in the clinic. Currently, FDA-approved MBL inhibitors are lacking in the clinic even though many strategies have been used in inhibitor development, including quantitative high-throughput screening (qHTS), fragment-based drug discovery (FBDD), and molecular docking. Herein, a machine learning-based prediction tool is described, which was generated using results from HTS of a large chemical library and previously published inhibition data. The prediction tool was then used for virtual screening of the NIH Genesis library, which was subsequently screened using qHTS. A novel MBL inhibitor was identified and shown to lower minimum inhibitory concentrations (MICs) of Meropenem for a panel of E. coli and K. pneumoniae clinical isolates expressing NDM-1. The mechanism of inhibition of this novel scaffold was probed utilizing equilibrium dialyses with metal analyses, native state electrospray ionization mass spectrometry, UV-vis spectrophotometry, and molecular docking. The uncovered inhibitor, compound 72922413, was shown to be 9-hydroxy-3-[(5-hydroxy-1-oxa-9-azaspiro[5.5]undec-9-yl)carbonyl]-4H-pyrido[1,2-a]pyrimidin-4-one.

On the Chemistry of Aqueous Ammonium Acetate Droplets during Native Electrospray Ionization Mass Spectrometry.

Ammonium acetate (NH4Ac) is a widely used solvent additive in native electrospray ionization (ESI) mass spectrometry. NH4Ac can undergo proton transfer to form ammonia and acetic acid (NH4+ + Ac- → NH3 + HAc). The volatility of these products ensures that electrosprayed ions are free of undesired adducts. NH4Ac dissolution in water yields pH 7, providing "physiological" conditions. However, NH4Ac is not a buffer at pH 7 because NH4+ and Ac- are not a conjugate acid/base pair (Konermann, L. J. Am. Soc. Mass Spectrom. 2017, 28, 1827-1835.). In native ESI, it is desirable that analytes experience physiological conditions not only in bulk solution but also while they reside in ESI droplets. Little is known about the internal milieu of NH4Ac-containing ESI droplets. The current work explored the acid/base chemistry of such droplets, starting from a pH 7 analyte solution. We used a two-pronged approach involving evaporation experiments on bulk solutions under ESI-mimicking conditions, as well as molecular dynamics simulations using a newly developed algorithm that allows for proton transfer. Our results reveal that during droplet formation at the tip of the Taylor cone, electrolytically generated protons get neutralized by Ac-, making NH4+ the net charge carriers in the weakly acidic nascent droplets. During the subsequent evaporation, the droplets lose water as well as NH3 and HAc that were generated by proton transfer. NH3 departs more quickly because of its greater volatility, causing the accumulation of HAc. Together with residual Ac-, these HAc molecules form an acetate buffer that stabilizes the average droplet pH at 5.4 ± 0.1, as governed by the Henderson-Hasselbalch equation. The remarkable success of native ESI investigations in the literature implies that this pH drop by ∼1.6 units relative to the initially neutral analyte solution can be tolerated by most biomolecular analytes on the short time scale of the ESI process.

Stabilization of Labile Lysozyme-Ligand Interactions in Native Electrospray Ionization Mass Spectrometry.

Flavonoids are polyphenolic secondary metabolites with extensive biological activities and pharmacological effects. Exploring the interactions of flavonoids with proteins may be helpful for understanding their biological processes. Electrospray ionization mass spectrometry (ESI-MS) is a powerful tool to characterize the noncovalent protein-ligand (PL) complexes. However, some protein-flavonoid complexes are labile during electrospray ionization. Here, the labile lysozyme-flavonoid (rutin, icariin, and naringin) complexes were determined by direct ESI-MS without derivation. It has been found that low amounts of N-methylpyrrolidinone and dimethylformamide can protect labile lysozyme-flavonoid complexes away from dissociation during electrospray ionization process. The intact lysozyme-flavonoid complexes were specifically observed in mass spectra, and the measured binding affinities by ESI-MS were matched with the fluorescence data. The effects of additives on the analysis of lysozyme-flavonoid complexes were investigated by ESI-MS, combined with the molecular docking and fluorescence. This strategy was helpful to investigate the labile PL interactions by direct ESI-MS.

Proton Transfers to DNA in Native Electrospray Ionization Mass Spectrometry: A Quantum Mechanics/Molecular Mechanics Study.

Native electrospray ionization-ion mobility mass spectrometry (N-ESI/IM-MS) is a powerful approach for low-resolution structural studies of DNAs in the free state and in complex with ligands. Solvent vaporization is coupled with proton transfers from ammonium ions to the DNA, resulting in a reduction of the DNA charge. Here we provide insight into these processes by classical molecular dynamics and quantum mechanics/molecular mechanics free energy calculations on the d(GpCpGpApApGpC) heptamer, for which a wealth of experiments is available. Our multiscale simulations, consistent with experimental data, reveal a highly complex scenario. The proton either sits on one of the molecules or is fully delocalized on both, depending on the level of hydration of the analytes and the size of the droplets formed during the electrospray experiments. This work complements our previous study of the intramolecular proton transfer on the same heptamer occurring after the processes studied here, and together, they provide a first molecular view of proton transfer in N-ESI/IM-MS.

Electrochemical and Solution Structural Characterization of Fe(III) Azotochelin Complexes: Examining the Coordination Behavior of a Tetradentate Siderophore.

We report an electrochemical setup comprising a boron-doped diamond (BDD) working electrode for the electrochemical study of iron(III) catecholate siderophores. We demonstrate its successful application in the voltammetric investigation of iron(III) azotochelin, an iron complex of a bis(catecholate) siderophore. Cyclic voltammetry results, when complemented by UV-vis and native electrospray ionization-mass spectrometry (ESI-MS) characterization, reveal the formation of a coordinatively unsaturated tetracoordinate 1:1 complex of Fe:azotochelin (M1:L1) at neutral pH, contrary to iron(III) tetradentate siderophore complexes of other classes which favor the hexacoordinate environment of an M2:L3 species. A notable effect of pH and buffer composition on the reduction potential of iron(III) azotochelin is demonstrated. Lower pH values and buffers encompassing primary or secondary amines facilitate a positive potential shift of up to +290 mV and +250 mV vs Ag/AgCl 3 M NaCl, respectively. The study was extended to the investigation of the iron(III) complexes of hexadentate siderophores. For tris(catecholate) siderophores, enterobactin and protochelin, the reduction potentials were found to lie beyond the potential window accessible to the BDD electrode; however, we were successful in observing the electrochemical behavior of a tris(hydroxamate) siderophore, ferricrocin.

Bioaffinity Screening with a Rapid and Sample-Efficient Autosampler for Native Electrospray Ionization Mass Spectrometry.

Fast and efficient handling of ligands and biological targets are required in bioaffinity screening based on native electrospray ionization mass spectrometry (ESI-MS). We use a prototype microfluidic autosampler, called the "gap sampler", to sequentially mix and electrospray individual small molecule ligands together with a target protein and compare the screening results with data from thermal shift assay and surface plasmon resonance. In a first round, all three techniques were used for a screening of 110 ligands against bovine carbonic anhydrase II, which resulted in five mutual hits and some false positives with ESI-MS presumably due to the high ligand concentration or interferences from dimethyl sulfoxide. In a second round, 33 compounds were screened in lower concentrations and in a less complex matrix, resulting in only true positives with ESI-MS. Within a cycle time of 30 s, dissociation constants were determined within an order of magnitude accuracy consuming only 5 pmol of ligand and less than 15 pmol of protein per screened compound. In a third round, dissociation constants of five compounds were accurately determined in a titration experiment. Thus, the gap sampler can rapidly and efficiently be used for high-throughput screening.

Combining Electrospray Mass Spectrometry (ESI-MS) and Computational Techniques in the Assessment of G-Quadruplex Ligands: A Hybrid Approach to Optimize Hit Discovery.

Guanine-rich sequences forming G-quadruplexes (GQs) are present in several genomes, ranging from viral to human. Given their peculiar localization, the induction of GQ formation or GQ stabilization with small molecules represents a strategy for interfering with crucial biological functions. Investigating the recognition event at the molecular level, with the aim of fully understanding the triggered pharmacological effects, is challenging. Native electrospray ionization mass spectrometry (ESI-MS) is being optimized to study these noncovalent assemblies. Quantitative parameters retrieved from ESI-MS studies, such as binding affinity, the equilibrium binding constant, and sequence selectivity, will be overviewed. Computational experiments supporting the ESI-MS investigation and boosting its efficiency in the search for GQ ligands will also be discussed with practical examples. The combination of ESI-MS and in silico techniques in a hybrid high-throughput-screening workflow represents a valuable tool for the medicinal chemist, providing data on the quantitative and structural aspects of ligand–GQ interactions.

Targeting Zinc Finger Proteins with Exogenous Metals and Molecules: Lessons learned from Tristetraprolin, a CCCH type Zinc Finger.

ZF proteins are ubiquitous eukaryotic proteins that play important roles in gene regulation. ZFs contain small domains made up of a combination of four cysteine and histidine residues, and are classified based up on the identity of these residues and their spacing. One emerging class of ZFs are the Cys3His (or CCCH) class of ZFs. These ZFs play key roles in regulating RNA. In this minireview, an overview of the CCCH class of ZFs, with a focus on tristetraprolin (TTP) is provided. TTP regulates inflammation by controlling cytokine mRNAs, and there is an interest in modulating TTP activity to control inflammation. Two methods to control TTP activity are to target with exogenous metals (a 'metals in medicine' approach) or to target with endogenous signaling molecules. Work that has been done to target TTP with Fe, Cu, Cd and Au as well as with H2S is reviewed. This includes attention to new methods that have been developed to monitor metal exchange with the spectroscopically silent ZnII including native electro-spray ionization mass spectrometry (ESI-MS), spin-filter inductively coupled plasma mass spectrometry (ICP-MS) and cryo-electro-spray mass spectrometry (CSI-MS); along with fluorescence anisotropy (FA) to follow RNA binding.

Simultaneous spatial, conformational, and mass analysis of intact proteins and protein assemblies by nano-DESI travelling wave ion mobility mass spectrometry imaging

We have previously demonstrated native nano-desorption electrospray ionization (nano-DESI) mass spectrometry imaging of proteins and protein complexes in thin tissue section of rat kidney. Here, we demonstrate the integration of travelling wave ion mobility spectrometry (TWIMS) into the native nano-DESI MSI workflow. The benefits of TWIMS are twofold: Firstly, arrival time filtering allows subtraction of chemical noise and the resulting ion images show improved specificity. Secondly, the incorporation of TWIMS enables the calculation of collision cross sections, and thus a measure of protein structure, directly from the imaging dataset. Our results show good agreement between the collision cross sections determined from nano-DESI, which requires the use of a heated inlet, and those determined experimentally from liquid extraction surface analysis (LESA) with an ambient temperature inlet, and those available in the literature. Ion images and collision cross sections are presented for a range of proteins and protein assemblies with molecular weights of up to 42.6 kDa.

A new type of flexible CP12 protein in the marine diatom Thalassiosira pseudonana

BackgroundCP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background.MethodsA combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12.ResultsHere, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions.ConclusionsThese findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle.Choregraphy of metabolism by CP12 proteins in Viridiplantae and Heterokonta.While the monomeric CP12 in Viridiplantae is involved in carbon assimilation, regulating phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through the formation of a ternary complex, in Heterokonta studied so far, the dimeric CP12 is associated with Ferredoxin-NADP reductase (FNR) and GAPDH. The Viridiplantae CP12 can bind metal ions and can be a chaperone, the Heterokonta CP12 is more abundant in all stresses (C, N, Si, P limited conditions) and is not specific to a metabolism.5h3nzM1M4bjv4RCQxTYJACVideo

Investigation of labile protein-ligand interactions by native electrospray ionization mass spectrometry

The investigations of noncovalent interaction between proteins (P) and small ligands (L) have played an important role for drug discovery and therapy. Electrospray ionization mass spectrometry (ESI-MS) has been typically applied to analysis of P-L interactions. However, some labile complexes are problematic for determination of association constants (Ka) by ESI-MS due to their in-source dissociation. Here, a strategy based on native MS for analysis of the labile complexes was developed. The reference ligand (Lref) with specific binding to P and a nonspecific binding ligand were employed. The equations for Ka calculation of labile complexes were developed. According to mathematical fitting curves, the Ka values of partially dissociated lysozyme-icariin complex and completely dissociated lysozyme-berberine complex were obtained. The obtained Ka values were consistent with those by fluorescence method. This strategy can be applied to the labile complexes of other small ligands to protein.

Structural and binding characterization of the LacdiNAc-specific adhesin (LabA; HopD) exodomain from Helicobacter pylori.

Helicobacter pylori (H. pylori) uses several outer membrane proteins for adhering to its host's gastric mucosa, an important step in establishing and preserving colonization. Several adhesins (SabA, BabA, HopQ) have been characterized in terms of their three-dimensional structure. A recent addition to the growing list of outer membrane porins is LabA (LacdiNAc-binding adhesin), which is thought to bind specifically to GalNAcβ1-4GlcNAc, occurring in the gastric mucosa. LabA47-496 protein expressed as His-tagged protein in the periplasm of E. coli and purified via subtractive IMAC after TEV cleavage and subsequent size exclusion chromatography, resulted in bipyramidal crystals with good diffraction properties. Here, we describe the 2.06 ​Å resolution structure of the exodomain of LabA from H. pylori strain J99 (PDB ID: 6GMM). Strikingly, despite the relatively low levels of sequence identity with the other three structurally characterized adhesins (20–49%), LabA shares an L-shaped fold with SabA and BabA. The ‘head’ region contains a 4 ​+ ​3 α-helix bundle, with a small insertion domain consisting of a short antiparallel beta sheet and an unstructured region, not resolved in the crystal structure. Sequence alignment of LabA from different strains shows a high level of conservation in the N- and C-termini, and identifies two main types based on the length of the insertion domain (‘crown’ region), the ‘J99-type’ (insertion ~31 ​amino acids), and the H. pylori ‘26695 type’ (insertion ~46 ​amino acids). Analysis of ligand binding using Native Electrospray Ionization Mass Spectrometry (ESI-MS) together with solid phase-bound, ELISA-type assays could not confirm the originally described binding of GalNAcβ1-4GlcNAc-containing oligosaccharides, in line with other recent reports, which also failed to confirm LacdiNAc binding.

A two-quartet G-quadruplex topology of human KIT2 is conformationally selected by a perylene derivative

G-quadruplexes are promising targets for innovative anticancer therapy. Hence, many efforts are being made to find selective ligands. Drug design is often based on the available high-resolution structures, obtained for the thermodynamically stable forms. However, the complexity of the G-quadruplex folding landscape has clearly emerged in recent years, with the discovery of intermediate conformations that persist on the second to the minute time scale. In the case of the KIT2 G-quadruplex forming sequence, found within human c-KIT promoter, we recently identified a long-lived folding intermediate, characterized by guanine stacking in alternating orientation (as determined by circular dichroism). Given the rate of transcriptional processes, a physiological role of this arrangement should not be excluded. In the present study, we applied circular dichroism (CD) spectroscopy, native electrospray ionization mass spectrometry (ESI-MS) and electrophoretic mobility shift assays (EMSA) to show that a perylene derivative (K20) selects this topology. Interestingly, ESI-MS spectra revealed the presence of a single specifically coordinated K+ ion in the structure, which is thus presumably composed of only two consecutive G-quartets. The parent ligand PIPER failed to promote the same conformational selection, which is therefore a process strictly dependent on the perylene side chains composition. The greater affinity of K20 for the two-quartet antiparallel topology, compared to PIPER, was finally corroborated by evaluating their binding to the KIT∗ G-quadruplex, which is also found within the human promoter of c-KIT.

Probing the mechanisms of inhibition for various inhibitors of metallo-β-lactamases VIM-2 and NDM-1

To probe the mechanism of inhibition of several previously-published metallo-β-lactamase (MBL) inhibitors for the clinically-important MBL Verona integron-encoded metallo-β-lactamase 2 (VIM-2), equilibrium dialyses with metal analyses, native state electrospray ionization mass spectrometry (ESI-MS), and UV–Vis spectrophotometry were utilized. The mechanisms of inhibition were analyzed for ethylenediaminetetraacetic acid (EDTA); dipicolinic acid (DPA) and DPA analogs 6-(1H-tetrazol-5-yl)picolinic acid (1T5PA) and 4-(3-aminophenyl)pyridine-2,6-dicarboxylic acid (3AP-DPA); thiol-containing compounds, 2,3-dimercaprol, thiorphan, captopril, and tiopronin; and 5-(pyridine-3-sulfonamido)-1,3-thiazole-4-carboxylic acid (ANT-431). UV–Vis spectroscopy and native-state ESI-MS results showed the formation of ternary complexes between VIM-2 and 1T5PA, ANT-431, thiorphan, captopril, and tiopronin, while a metal stripping mechanism was shown with VIM-2 and EDTA and DPA. The same approaches were used to show the formation of a ternary complex between New Delhi Metallo-β-lactamase (NDM-1) and ANT-431. The studies presented herein show that most of the inhibitors utilize a similar mechanism of inhibition as previously reported for NDM-1. These studies also demonstrate that native mass spectrometry can be used to probe the mechanism of inhibition at lower enzyme/inhibitor concentrations than has previously been achieved.

Interactions of indole alkaloids with myoglobin: A mass spectrometry based spectrometric and computational method.

Interactions of drug molecules and proteins play important roles in physiological and pathological processes in vivo. It is of significance to establish a reliable strategy for studying protein-drug ligand interactions and would be helpful for the design and screening of new drugs in pharmacological research. The interactions between four indole alkaloids (IAs) extracted from Ophiorrhiza japonica (O. japonica) and myoglobin (Mb) protein were investigated using a multi-spectrometric and computational method of native electrospray ionization mass spectrometry (native ESI-MS), hydrogen/deuterium exchange mass spectrometry (HDX-MS), circular dichroism (CD) and molecular docking (MD). The IA-bound Mb complexes were analyzed using native ESI-MS, with the obtained protein-to-ligand stoichiometry at 1:1, 1:2 and 1:3. Binding constants were measured according to the interpretation of MS spectra. MD complemented MS measurements, probing the binding sites and modes of the four IAs to Mb. Analyses involving CD and HDX-MS demonstrated that exposure to IAs could affect the conformation of Mb by decreasing the α-helix content and made Mb more susceptible to HDX at the backbone. A new MS-based integrated analysis method has been developed to successfully study the interactions of Mb and IAs extracted from O. japonica. The experimental and calculation results have good consistency, revealing all of the four IA molecules could bind to Mb to form 1:1, 1:2 and 1:3 Mb-IA complexes. The order of binding ability of these IAs to Mb was ophiorrhine B > compound C > ophiorrhine A > compound D. CD and HDX-MS results indicated that binding with IAs destabilizes Mb. HDX-MS analysis suggests that Mb becomes more susceptible to HDX, indicating that binding with IAs destabilizes the structure of Mb. In addition, the interaction with IAs affected the overall structure of Mb, ascribed to the decrease of α-helix content and less folding of the backbone.

Role of Gold in Inflammation and Tristetraprolin Activity.

The zinc finger protein tristetraprolin (TTP) regulates inflammation by downregulating cytokine mRNAs. Misregulation results in arthritis, sepsis and cancer, and there is an interest in modulating TTP activity with exogenous agents. Gold has anti-inflammatory properties and has recently been shown to modulate the signaling pathway that produces TTP, suggesting that TTP may be a target of gold. The reactivity of [AuIII (terpy)Cl]Cl2 with TTP was investigated by UV/Vis spectroscopy, spin-filter inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy and native electrospray ionization mass spectrometry. AuIII was found to replace zinc in the protein active site in the reduced AuI form, with the AuI ion coordinated to two cysteine residues in a linear geometry. The replacement of ZnII with AuI results in loss of both secondary structure and RNA binding function. In contrast, when ZnII TTP is bound to its RNA target, no replacement of ZnII with AuI is observed, even in the presence of excess AuIII terpy. This discovery of differential reactivity of gold with TTP versus TTP/RNA offers a potential strategy for selective targeting with gold complexes to control inflammation.

Native Electrospray Ionization Mass Spectrometry of RNA-Ligand Complexes.

Native electrospray ionization mass spectrometry (native ESI-MS) is a powerful tool to investigate non-covalent biomolecular interactions. It has been widely used to study protein complexes, but only few examples are described for the analysis of complexes involving RNA-RNA interactions. Here, we provide a detailed protocol for native ESI-MS analysis of RNA complexes. As an example, we present the analysis of the HIV-1 genomic RNA dimerization initiation site (DIS) extended duplex dimer bound to the aminoglycoside antibiotic lividomycin.

Thermokinetic Analysis of Protein Subunit Exchange by Variable-Temperature Native Mass Spectrometry.

Many protein complexes are assembled from a varying number of subunits, which are continuously exchanging with diverse time scales. This structural dynamics is considered to be important for many regulatory and sensory adaptation processes that occur in vivo. We have developed an accurate method for monitoring protein subunit exchange by using native electrospray ionization mass spectrometry (ESI-MS), exemplified here for an extremely stable Rad50 zinc hook (Hk) dimer assembly, Zn(Hk)2. The method has two steps: appropriate protein/peptide mutation and native ESI-MS analysis using a variable-temperature sample inlet. In this work, two Hk mutants were produced, mixed with wild-type Hk, and measured at three different temperatures. A thermokinetic analysis of heterodimer formation allowed us to determine the enthalpy, entropy, and Gibbs free energy of activation for subunit exchange, showing that the reaction is slow and associated with a high enthalpic barrier, consistent with the exceptionally high stability of the Zn(Hk)2 assembly.

Virus-like particle size and molecular weight/mass determination applying gas-phase electrophoresis (native nES GEMMA)

(Bio-)nanoparticle analysis employing a nano-electrospray gas-phase electrophoretic mobility molecular analyzer (native nES GEMMA) also known as nES differential mobility analyzer (nES DMA) is based on surface-dry analyte separation at ambient pressure. Based on electrophoretic principles, single-charged nanoparticles are separated according to their electrophoretic mobility diameter (EMD) corresponding to the particle size for spherical analytes. Subsequently, it is possible to correlate the (bio-)nanoparticle EMDs to their molecular weight (MW) yielding a corresponding fitted curve for an investigated analyte class. Based on such a correlation, (bio-)nanoparticle MW determination via its EMD within one analyte class is possible. Turning our attention to icosahedral, non-enveloped virus-like particles (VLPs), proteinaceous shells, we set up an EMD/MW correlation. We employed native electrospray ionization mass spectrometry (native ESI MS) to obtain MW values of investigated analytes, where possible, after extensive purification. We experienced difficulties in native ESI MS with time-of-flight (ToF) detection to determine MW due to sample inherent characteristics, which was not the case for charge detection (CDMS). nES GEMMA exceeds CDMS in speed of analysis and is likewise less dependent on sample purity and homogeneity. Hence, gas-phase electrophoresis yields calculated MW values in good approximation even when charge resolution was not obtained in native ESI ToF MS. Therefore, both methods-native nES GEMMA-based MW determination via an analyte class inherent EMD/MW correlation and native ESI MS-in the end relate (bio-)nanoparticle MW values. However, they differ significantly in, e.g., ease of instrument operation, sample and analyte handling, or costs of instrumentation.Graphical abstract

Preservation of Protein Zwitterionic States in the Transition from Solution to Gas Phase Revealed by Sodium Adduction Mass Spectrometry.

The structural characterization of proteins and their interaction network mapping in the gas phase highlights the need to preserve their most nativelike conformers in the transition from the solution to gas phase. Zwitterionic interactions in a protein are weak bonds between oppositely charged residues, which make an important contribution to protein stability. However, it is still not clear whether the native zwitterionic states of proteins can be retained or not when it is transferred from the solution to gas phase. Using the nonspecific Na+ adduction as a novel signature, here we show that the zwitterionic states of proteins can be preserved when a moderated droplet desolvation condition (temperature <30 °C) is used in native electrospray ionization mass spectrometry. The very low-level nonspecific metal adduction to proteins under such conditions also enables rapid and direct determination of the binding states of metal-binding proteins and sensitive detection of proteins from solutions containing highly concentrated involatile salts (e.g., 50 mM NaCl). We believe that our findings can be instructive for performing mass spectrometric analysis of proteins and useful for protein ions desalting which simply involves altering the temperature and flow rate of drying gas in the desolvation region.