Unlocking enhanced photocatalytic power: Donor-acceptor synergy in non-metallic g-C3N4 hollow nanospheres.

Selecting safe, non-toxic, and non-metallic semiconductor materials that facilitate the degradation of pollutants in water stands out as an optimal approach to combat environmental pollution. Herein, graphitic carbon nitride (g-C3N4)-based hollow nanospheres nonmetallic photocatalyst modified with covalent organic framework materials named TpMA, based on 1, 3, 5-trimethylchloroglucuronide (Tp) and melamine (MA), was successfully synthesized (abbreviated as CNTP). The ordered electron donor-acceptor structure inherent in TpMA contributed to enhancing the transport efficiency of photogenerated carriers in CNTP. The CNTP photocatalysts exhibited excellent performance in degrading rhodamine B and tetracycline in visible light, with optimal degradation rates reached more than 90% in 60 and 80 min, respectively, which were 5.3 and 3.0 times higher than those of pure CNNS. The increased photocatalytic efficiency observed in CNTP composites could be traced back to the covalently connection between the two molecules, forming a π-conjugated system that facilitated the separative efficiency of photogenerated electron-hole pairs and intensified the utilization of visible light. This study provided a new means to design and fabricate highly efficient and environmentally friendly non-metallic photocatalytic materials.

Anchored VN Quantum Dots Boosting High Capacity and Cycle Durability of Na3V2(PO4)3@NC Cathode for Aqueous Zinc-Ion Battery and Organic Sodium-Ion Battery.

Na3V2(PO4)3 is a promising high-voltage cathode for aqueous zinc-ion batteries (ZIBs) and organic sodium-ion batteries (SIBs). However, the poor rate capability, specific capacity, and cycling stability severely hamper it from further development. In this work, Na3V2(PO4)3 (NVP) with vanadium nitride (VN) quantum dots encapsulated by nitrogen-doped carbon (NC) nanoflowers (NVP/VN@NC) are manufactured as cathode using in situ nitridation, carbon coating, and structural adjustment. The outer NC layer increases the higher electronic conductivity of NVP. Furthermore, VN quantum dots with high theoretical capacity not only improve the specific capacity of pristine NVP, but also serve as abundant "pins" between NVP and NC to strengthen the stability of NVP/VN@NC heterostructure. For Zn-ion storage, these essential characteristics allow NVP/VN@NC to attain a high reversible capacity of 135.4 mAh g-1 at 0.1 A g-1, and a capacity retention of 91% after 2000 cycles at 5 A g-1. Meanwhile, NVP/VN@NC also demonstrates to be a stable cathode material for SIBs, which can reach a high reversible capacity of 124.5 mAh g-1 at 0.1 A g-1, and maintain 92% of initial capacity after 11000 cycles at 5 A g-1. This work presents a feasible path to create innovative high-voltage cathodes with excellent reaction kinetics and structural stability.

Flexible free-standing Fe-CoP-NAs/CC nanoarrays for high-performance full lithium-ion batteries.

In this study, a flexible, free-standing Fe-doped CoP nanoarrays electrode for superior lithium-ion storage has been successfully fabricated. The electrode combines the advantages of a Fe-doping and a flexible carbon cloth (CC) support, resulting in a high specific capacity (1356 mAh/g at 0.2 A/g) and excellent cycling stability (1138 mAh/g after 100 cycles). The cyclic voltammetry (CV) curves at different scan rates investigate the outstanding lithium storage behavior of Fe-CoP-NAs/CC which indicates a combined influence of diffusion behavior and capacitance behavior on the electrochemical process. The galvanostatic intermittent titration technique (GITT) analyzes the diffusion kinetics of Li+ which indicates the fast diffusion kinetics in the Fe-CoP/NAs/CC anode. The assembled Fe-CoP-NAs/CC//LiFePO4 battery exhibits a remarkable capacity of 325.2 mAh/g even at 5 A/g. And the battery also has good cycle stability, and still provides 498.1 mAh/g specific capacity after 200 cycles. Moreover, the Fe-CoP-NAs/CC//LiFePO4 soft-pack battery can continuously power the LEDs when it is bent at various angles which demonstrates its potential for use in wearable devices.

A novel avian intestinal epithelial cell line: its characterization and exploration as an in vitro infection culture model for Eimeria species.

BACKGROUND: The gastrointestinal epithelium plays an important role in directing recognition by the immune system, and epithelial cells provide the host's front line of defense against microorganisms. However, it is difficult to cultivate avian intestinal epithelial cells in vitro for lengthy periods, and the lack of available cell lines limits the research on avian intestinal diseases and nutritional regulation. Chicken coccidiosis is a serious intestinal disease that causes significant economic losses in the poultry industry. In vitro, some cell line models are beneficial for the development of Eimeria species; however, only partial reproduction can be achieved. Therefore, we sought to develop a new model with both the natural host and epithelial cell phenotypes. METHODS: In this study, we use the SV40 large T antigen (SV40T) gene to generate an immortalized cell line. Single-cell screening technology was used to sort positive cell clusters with epithelial characteristics for passage. Polymerase chain reaction (PCR) identification, immunofluorescence detection, and bulk RNA sequencing analysis and validation were used to check the expression of epithelial cell markers and characterize the avian intestinal epithelial cell line (AIEC). AIECs were infected with sporozoites, and their ability to support the in vitro endogenous development of Eimeria tenella was assessed. RESULTS: This novel AIEC consistently expressed intestinal epithelial markers. Transcriptome assays revealed the upregulation of genes associated with proliferation and downregulation of genes associated with apoptosis. We sought to compare E. tenella infection between an existing fibroblast cell line (DF-1) and several passages of AIEC and found that the invasion efficiency was significantly increased relative to that of chicken fibroblast cell lines. CONCLUSIONS: An AIEC will serve as a better in vitro research model, especially in the study of Eimeria species development and the mechanisms of parasite-host interactions. Using AIEC helps us understand the involvement of intestinal epithelial cells in the digestive tract and the immune defense of the chickens, which will contribute to the epithelial innate defense against microbial infection in the gastrointestinal tract.

Band engineering enhances the electrochemical properties by constructing TiO2 NRs-MoS2 NSFs flexible electrode.

Research and development of flexible electrodes with high performance are crucial to largely determine the performance of flexible lithium-ion batteries (FLIBs) to a large extent. In this work, a flexible anode (TiO2 NRs-MoS2 NSFs/CC) is rationally designed and successfully constructed, in which TiO2 nanorods arrays (NRs) vertically grown on CC as a supporting backbone for MoS2 nanosheets flowers (NSFs) to form a TiO2 NRs-MoS2 NSFs heterostructure. The backbone can not only serve as a mechanical support MoS2 and improve its electronic conductivity, but also limit the dissolution of polysulfides issue during cycling. The density functional theory (DFT) analysis manifests that the obvious interaction between O and S at the interface for the TiO2 NRs-MoS2 NSFs heterostructure changes the electronic structure and reduces the band gap of TiO2 NRs-MoS2 NSFs. The small band gap and high electron state at the Fermi level are both beneficial to the transport of electrons, enhancing the kinetics, and giving the long cycling stability at high density and excellent rate capacity. Furthermore, the assembled TiO2 NRs-MoS2 NSFs/CC//NCM622 full cell delivers superior rate capacity and good cycling stability. Meanwhile, the soft-packed cell shows good mechanical flexibility, which can be lighted up successfully and keep brightness when folding with different angles. This result illustrates that it is a highly potential strategy for constructing flexible electrodes with the controlled electronic structure through band engineering to not only improve the electrochemical performance, but also possibly meet the requirements of high-performance FLIBs.

Cryptosporidium uses CSpV1 to activate host type I interferon and attenuate antiparasitic defenses.

Cryptosporidium infects gastrointestinal epithelium and is a leading cause of infectious diarrhea and diarrheal-related death in children worldwide. There are no vaccines and no fully effective therapy available for the infection. Type II and III interferon (IFN) responses are important determinants of susceptibility to infection but the role for type I IFN response remains obscure. Cryptosporidium parvum virus 1 (CSpV1) is a double-stranded RNA (dsRNA) virus harbored by Cryptosporidium spp. Here we show that intestinal epithelial conditional Ifnar1-/- mice (deficient in type I IFN receptor) are resistant to C. parvum infection. CSpV1-dsRNAs are delivered into host cells and trigger type I IFN response in infected cells. Whereas C. parvum infection attenuates epithelial response to IFN-γ, loss of type I IFN signaling or inhibition of CSpV1-dsRNA delivery can restore IFN-γ-mediated protective response. Our findings demonstrate that type I IFN signaling in intestinal epithelial cells is detrimental to intestinal anti-C. parvum defense and Cryptosporidium uses CSpV1 to activate type I IFN signaling to evade epithelial antiparasitic response.

miR-182-5p attenuates Schistosoma japonicum-induced hepatic fibrosis by targeting tristetraprolin.

Egg granuloma formation in the liver is the main pathological lesion caused by Schistosoma japonicum infection, which generally results in liver fibrosis and may lead to death in advanced patients. MicroRNAs (miRNAs) regulate the process of liver fibrosis, but the putative function of miRNAs in liver fibrosis induced by S. japonicum infection is largely unclear. Here, we detect a new miRNA, miR-182-5p, which shows significantly decreased expression in mouse livers after stimulation by soluble egg antigen (SEA) of S. japonicum or S. japonicum infection. Knockdown or overexpression of miR-182-5p in vitro causes the increased or decreased expression of tristetraprolin (TTP), an important immunosuppressive protein in the process of liver fibrosis. Furthermore, knockdown of miR-182-5p in vivo upregulates TTP expression and significantly alleviates S. japonicum-induced hepatic fibrosis. Our data demonstrate that downregulation of miR-182-5p increases the expression of TTP in mouse livers following schistosome infection, which leads to destabilization of inflammatory factor mRNAs and attenuates liver fibrosis. Our results uncover fine-tuning of liver inflammatory reactions related to liver fibrosis caused by S. japonicum infection and provide new insights into the regulation of schistosomiasis-induced hepatic fibrosis.

Tips on Making Tiny Tips: Secrets to Submicron Nanoelectrospray Emitters.

Nanoelectrospray ionization emitters with submicron tip diameters have significant advantages for use in native mass spectrometry, including the ability to produce resolved charge-state distributions for proteins and macromolecular complexes from standard biochemical buffers that contain high concentrations of nonvolatile salts and to prevent nonspecific aggregation that can occur during droplet evaporation. We report on various factors affecting the tip morphology and provide suggestions for producing and using emitters with submicron tips. Effects of pulling parameters for a Sutter Instrument P-87 tip puller on the resulting tip diameter and morphology are shown. The "Pull" parameter has the largest effect on tip diameter, followed by "Velocity", "Pressure", and "Heat", whereas the "Time" parameter has minimal effect beyond a lower threshold. High "Pull" values generate emitters with multiple tapers, whereas high "Velocity" values generate a tip with only a single tapered region. A protocol for producing reproducible emitters in the submicron size range, as well as guidelines and tips for using these emitters with standard biochemical buffers that contain high concentrations of nonvolatile salts, is presented with the aim of expanding their use within the native mass spectrometry community.

Temporal manipulation of Cdkl5 reveals essential postdevelopmental functions and reversible CDKL5 deficiency disorder-related deficits.

CDKL5 deficiency disorder (CDD) is an early onset, neurodevelopmental syndrome associated with pathogenic variants in the X-linked gene encoding cyclin-dependent kinase-like 5 (CDKL5). CDKL5 has been implicated in neuronal synapse maturation, yet its postdevelopmental necessity and the reversibility of CDD-associated impairments remain unknown. We temporally manipulated endogenous Cdkl5 expression in male mice and found that postdevelopmental loss of CDKL5 disrupts numerous behavioral domains, hippocampal circuit communication, and dendritic spine morphology, demonstrating an indispensable role for CDKL5 in the adult brain. Accordingly, restoration of Cdkl5 after the early stages of brain development using a conditional rescue mouse model ameliorated CDD-related behavioral impairments and aberrant NMDA receptor signaling. These findings highlight the requirement of CDKL5 beyond early development, underscore the potential for disease reversal in CDD, and suggest that a broad therapeutic time window exists for potential treatment of CDD-related deficits.

m6A mRNA Methylation Regulates Epithelial Innate Antimicrobial Defense Against Cryptosporidial Infection.

Increasing evidence supports that N6-methyladenosine (m6A) mRNA modification may play an important role in regulating immune responses. Intestinal epithelial cells orchestrate gastrointestinal mucosal innate defense to microbial infection, but underlying mechanisms are still not fully understood. In this study, we present data demonstrating significant alterations in the topology of host m6A mRNA methylome in intestinal epithelial cells following infection by Cryptosporidium parvum, a coccidian parasite that infects the gastrointestinal epithelium and causes a self-limited disease in immunocompetent individuals but a life-threatening diarrheal disease in AIDS patients. Altered m6A methylation in mRNAs in intestinal epithelial cells following C. parvum infection is associated with downregulation of alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 and the fat mass and obesity-associated protein with the involvement of NF-кB signaling. Functionally, m6A methylation statuses influence intestinal epithelial innate defense against C. parvum infection. Specifically, expression levels of immune-related genes, such as the immunity-related GTPase family M member 2 and interferon gamma induced GTPase, are increased in infected cells with a decreased m6A mRNA methylation. Our data support that intestinal epithelial cells display significant alterations in the topology of their m6A mRNA methylome in response to C. parvum infection with the involvement of activation of the NF-кB signaling pathway, a process that modulates expression of specific immune-related genes and contributes to fine regulation of epithelial antimicrobial defense.

Breakage of the oligomeric CaMKII hub by the regulatory segment of the kinase.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is an oligomeric enzyme with crucial roles in neuronal signaling and cardiac function. Previously, we showed that activation of CaMKII triggers the exchange of subunits between holoenzymes, potentially increasing the spread of the active state (Stratton et al., 2014; Bhattacharyya et al., 2016). Using mass spectrometry, we show now that unphosphorylated and phosphorylated peptides derived from the CaMKII-α regulatory segment bind to the CaMKII-α hub and break it into smaller oligomers. Molecular dynamics simulations show that the regulatory segments dock spontaneously at the interface between hub subunits, trapping large fluctuations in hub structure. Single-molecule fluorescence intensity analysis of CaMKII-α expressed in mammalian cells shows that activation of CaMKII-α results in the destabilization of the holoenzyme. Our results suggest that release of the regulatory segment by activation and phosphorylation allows it to destabilize the hub, producing smaller assemblies that might reassemble to form new holoenzymes.

Variation in assembly stoichiometry in non-metazoan homologs of the hub domain of Ca2+ /calmodulin-dependent protein kinase II.

The multi-subunit Ca2+ /calmodulin-dependent protein kinase II (CaMKII) holoenzyme plays a critical role in animal learning and memory. The kinase domain of CaMKII is connected by a flexible linker to a C-terminal hub domain that assembles into a 12- or 14-subunit scaffold that displays the kinase domains around it. Studies on CaMKII suggest that the stoichiometry and dynamic assembly/disassembly of hub oligomers may be important for CaMKII regulation. Although CaMKII is a metazoan protein, genes encoding predicted CaMKII-like hub domains, without associated kinase domains, are found in the genomes of some green plants and bacteria. We show that the hub domains encoded by three related green algae, Chlamydomonas reinhardtii, Volvox carteri f. nagarensis, and Gonium pectoral, assemble into 16-, 18-, and 20-subunit oligomers, as assayed by native protein mass spectrometry. These are the largest known CaMKII hub domain assemblies. A crystal structure of the hub domain from C. reinhardtii reveals an 18-subunit organization. We identified four intra-subunit hydrogen bonds in the core of the fold that are present in the Chlamydomonas hub domain, but not in metazoan hubs. When six point mutations designed to recapitulate these hydrogen bonds were introduced into the human CaMKII-α hub domain, the mutant protein formed assemblies with 14 and 16 subunits, instead of the normal 12- and 14-subunit assemblies. Our results show that the stoichiometric balance of CaMKII hub assemblies can be shifted readily by small changes in sequence.

Native mass spectrometry beyond ammonium acetate: effects of nonvolatile salts on protein stability and structure.

Native mass spectrometry is widely used to probe the structures, stabilities, and stoichiometries of proteins and biomolecular complexes in aqueous solutions, typically containing volatile ammonium acetate or ammonium bicarbonate buffer. In this study, nanoelectrospray emitters with submicron tips are used to produce significantly desalted ions of RNase A and a reduced, alkylated form of this protein, RA-RNase A, from solutions containing 175 mM ammonium acetate, as well as sodium chloride and Tris containing solutions with the same nominal ionic strength and pH. The charge-state distributions formed by nanoelectrospray ionization and tyrosine fluorescence emission data as a function of temperature from these solutions indicate that the folded form of RA-RNase A in solution is stabilized when ammonium acetate is replaced by increasing quantities of NaCl and Tris. Ion mobility data for the 7+ charge state of RA-RNase A indicates that the protein conformation in ammonium acetate changes with increasing concentration of NaCl which stablizes more compact structures. These results are consistent with observations reported 130 years ago by Hofmeister who found that ion identity can affect the stabilities and the structures of proteins in solution. This study indicates the importance of buffer choice when interpreting native mass spectrometry data.

Effect of droplet lifetime on where ions are formed in electrospray ionization.

The location of gaseous ion formation in electrospray ionization under native mass spectrometry conditions was investigated using theta emitters with tip diameters between 317 nm and 4.4 µm to produce droplets with lifetimes between 1 and 50 µs. Mass spectra of ß-lactoglobulin do not depend on instrument source temperatures between 160 and 300 °C with the smallest tips. A high charge-state distribution is observed for larger tips that produce droplets with lifetimes ≥10 µs and this distribution increases at higher source temperatures. These and other results show that gaseous protein ions originating from the smallest droplets are formed outside of the mass spectrometer whereas the majority of protein ions formed from the larger droplets are formed inside of the mass spectrometer where thermal heating of the droplet and concomitant protein unfolding inside of the droplet occurs. These results show that small emitter tips are advantageous in native mass spectrometry by eliminating effects of thermal destabilization of proteins in droplets inside of the mass spectrometer, eliminating the effects of non-specific protein dimerization and aggregation that can occur in larger droplets that contain more than one protein molecule, and significantly reducing salt adduction.

Submicrometer Nanospray Emitters Provide New Insights into the Mechanism of Cation Adduction to Anionic Oligonucleotides.

The electrospray-MS analysis of oligonucleotides is hampered by nonvolatile metal cations, which may produce adducts responsible for signal suppression and loss of resolution. Alternative to replacing metal cations with MS-friendly ammonium, we explored the utilization of nanospray emitters with submicrometer-diameter tips, which was shown to benefit the analysis of protein samples containing elevated salt concentrations. We demonstrated that such benefits are not limited to proteins, but extend also to oligonucleotide samples analyzed in the negative ion mode. At elevated Na+/Mg2+ concentrations, submicrometer tips produced significantly greater signal-to-noise ratios, as well as greatly reduced adducts and salt clusters, than observed when utilizing micrometer tips. These effects were marginally affected by emitter composition (i.e., borosilicate versus quartz), but varied according to salt concentration and number of oligonucleotide phosphates. The results confirmed that adduct formation is driven by the concentrating effects of the desolvation process, which leads to greatly increased solute concentrations as the volume of the droplet decreases. The process promotes cation-phosphate interactions that may not have necessarily existed in the initial sample, but nevertheless shape the observed adduct series. Therefore, such series may not accurately reflect the distribution of counterions surrounding the analyte in solution. No adverse effects were noted on specific metal interactions, such as those present in a model drug-DNA assembly. These observations indicate that the utilization of submicrometer tips represents an excellent alternative to traditional ammonium-replacement approaches, which enables the analysis of oligonucleotides in the presence of Na+/Mg2+ concentrations capable of preserving their structure and functional properties.

Submicrometer Emitter ESI Tips for Native Mass Spectrometry of Membrane Proteins in Ionic and Nonionic Detergents.

Native mass spectrometry (native-MS) of membrane proteins typically requires a detergent screening protocol, protein solubilization in the preferred detergent, followed by protein liberation from the micelle by collisional activation. Here, submicrometer nano-ESI emitter tips are used for native-MS of membrane proteins solubilized in both nonionic and ionic detergent solutions. With the submicrometer nano-ESI emitter tips, resolved charge-state distributions of membrane protein ions are obtained from a 150 mM NaCl, 25 mM Tris-HCl with 1.1% octyl glucoside solution. The relative abundances of NaCl and detergent cluster ions at high m /z are significantly reduced with the submicrometer emitters compared with larger nano-ESI emitters that are commonly used. This technique is beneficial for significantly decreasing the abundances (by two to three orders of magnitude compared with the larger tip size: 1.6 µm) of detergent cluster ions formed from aqueous ammonium acetate solutions containing detergents that can overlap with the membrane protein ion signal. Resolved charge-state distributions of membrane protein ions from aqueous ammonium acetate solutions containing ionic detergents were obtained with the submicrometer nano-ESI emitters; this is the first report of native-MS of membrane proteins solubilized by ionic detergents. Graphical Abstract.

Simultaneous Measurements of Mass and Collisional Cross-Section of Single Ions with Charge Detection Mass Spectrometry.

The masses and mobilities of single multiply charged ions of cytochrome c, ubiquitin, myoglobin, and bovine serum albumin formed by electrospray ionization are measured using charge detection mass spectrometry (CDMS). Single ions are trapped and repeatedly measured as they oscillate inside an electrostatic ion trap with cone electrodes for up to the maximum trapping time set at 500 ms. The histograms of the many single ion oscillation frequencies have resolved peaks that correspond to the different charge states of each protein. The m/z of each ion is determined from the initial oscillation frequency histogram, and the evolution of the ion energy with time is obtained from the changing frequency. A short-time Fourier transform of the time-domain data indicates that the increase in ion frequency occurs gradually with time with occasional sudden jumps in frequency. The frequency jumps are similar for each protein and may be caused by collision-induced changes in the ion trajectory. The rate of the gradual frequency shift increases with protein mass and charge state. This gradual frequency change is due to ion energy loss from collisions with the background gas. The total energy lost by an ion is determined from the latter frequency shifts normalized to a 500 ms lifetime, and these values increase nearly linearly with measured collisional cross-sections for these protein ions. These results show that the mass and collisional cross-section of single multiply charged ions can be obtained from these CDMS measurements by using proteins with known collisional cross-sections for calibration.

Native Mass Spectrometry from Common Buffers with Salts That Mimic the Extracellular Environment.

Nonvolatile salts are essential for the structures and functions of many proteins and protein complexes but can severely degrade performance of native mass spectrometry by adducting to protein and protein complex ions, thereby reducing sensitivity and mass measuring accuracy. Small nanoelectrospray emitters are used to form protein and protein complex ions directly from high-ionic-strength (>150 mm) nonvolatile buffers with salts that mimic the extracellular environment. Charge-state distributions are not obtained for proteins and protein complexes from six commonly used nonvolatile buffers and ≥150 mm Na+ with conventionally sized nanoelectrospray emitter tips but are resolved with 0.5 µm tips. This method enables mass measurements of proteins and protein complexes directly from a variety of commonly used buffers with high concentrations of nonvolatile salts and eliminates the need to buffer exchange into volatile ammonium buffers traditionally used in native mass spectrometry.

Small Emitter Tips for Native Mass Spectrometry of Proteins and Protein Complexes from Nonvolatile Buffers That Mimic the Intracellular Environment.

Salts are often necessary to maintain the native structures and functions of many proteins and protein complexes, but many buffers adversely affect protein analysis by native mass spectrometry (MS). Here, protein and protein complex ions are formed directly from a 150 mM KCl and 25 mM Tris-HCl buffer at pH 7 that is widely used in protein chemistry to mimic the intracellular environment. The protein charge-state distributions are not resolved from electrospray ionization MS using 1.6 µm diameter emitter tips, resulting in no mass information. In contrast, the charge-state distributions are well-resolved using 0.5 µm tips, from which the masses of proteins and protein complexes can be obtained. Adduction of salt to protein ions decreases with decreasing tip size below ∼1.6 µm but not above this size. This suggests that the mechanism for reducing salt adduction is the formation of small initial droplets with on average fewer than one protein molecule per droplet, which lowers the salt:protein ratio in droplets that contain a protein molecule. This is the first demonstration of native mass spectrometry of protein and protein complex ions formed from a buffer containing physiological ionic strengths of nonvolatile salts that mimics the intracellular environment, and this method does not require sample preparation or addition of reagents to the protein solution before or during mass analysis.

Charging of Proteins in Native Mass Spectrometry.

Factors that influence the charging of protein ions formed by electrospray ionization from aqueous solutions in which proteins have native structures and function were investigated. Protein ions ranging in molecular weight from 12.3 to 79.7 kDa and pI values from 5.4 to 9.6 were formed from different solutions and reacted with volatile bases of gas-phase basicities higher than that of ammonia in the cell of a Fourier-transform ion cyclotron resonance mass spectrometer. The charge-state distribution of cytochrome c ions formed from aqueous ammonium or potassium acetate is the same. Moreover, ions formed from these two solutions do not undergo proton transfer to 2-fluoropyridine, which is 8 kcal/mol more basic than ammonia. These results provide compelling evidence that proton transfer between ammonia and protein ions does not limit protein ion charge in native electrospray ionization. Both circular dichroism and ion mobility measurements indicate that there are differences in conformations of proteins in pure water and aqueous ammonium acetate, and these differences can account for the difference in the extent of charging and proton-transfer reactivities of protein ions formed from these solutions. The extent of proton transfer of the protein ions with higher gas-phase basicity bases trends with how closely the protein ions are charged to the value predicted by the Rayleigh limit for spherical water droplets approximately the same size as the proteins. These results indicate that droplet charge limits protein ion charge in native mass spectrometry and are consistent with these ions being formed by the charged residue mechanism. Graphical Abstract ᅟ.