Charge Detection Mass Spectrometry Reveals Conformational Heterogeneity in Megadalton-Sized Monoclonal Antibody Aggregates.

Aggregation of protein-based therapeutics can occur during development, production, or storage and can lead to loss of efficacy and potential toxicity. Native mass spectrometry of a covalently linked pentameric monoclonal antibody complex with a mass of ∼800 kDa reveals several distinct conformations, smaller complexes, and abundant higher-order aggregates of the pentameric species. Charge detection mass spectrometry (CDMS) reveals individual oligomers up to the pentamer mAb trimer (15 individual mAb molecules; ∼2.4 MDa) whereas intermediate aggregates composed of 6-9 mAb molecules and aggregates larger than the pentameric dimer (1.6 MDa) were not detected/resolved by standard mass spectrometry, size exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), or by mass photometry. Conventional quadrupole time-of-flight mass spectrometry (QTOF MS), mass photometry, SEC, and CE-SDS did not resolve partially or more fully unfolded conformations of each oligomer that were readily identified using CDMS by their significantly higher extents of charging. Trends in the charge-state distributions of individual oligomers provides detailed insight into how the structures of compact and elongated mAb aggregates change as a function of aggregate size. These results demonstrate the advantages of CDMS for obtaining accurate masses and information about the conformations of large antibody aggregates despite extensive overlapping m/z values. These results open up the ability to investigate structural changes that occur in small, soluble oligomers during the earliest stages of aggregation for antibodies or other proteins.

Overcoming aggregation with laser heated nanoelectrospray mass spectrometry: thermal stability and pathways for loss of bicarbonate from carbonic anhydrase II.

Variable temperature electrospray mass spectrometry is useful for multiplexed measurements of the thermal stabilities of biomolecules, but the ionization process can be disrupted by aggregation-prone proteins/complexes that have irreversible unfolding transitions. Resistively heating solutions containing a mixture of bovine carbonic anhydrase II (BCAII), a CO2 fixing enzyme involved in many biochemical pathways, and cytochrome c leads to complete loss of carbonic anhydrase signal and a significant reduction in cytochrome c signal above ∼72 °C due to aggregation. In contrast, when the tips of borosilicate glass nanoelectrospray emitters are heated with a laser, complete thermal denaturation curves for both proteins are obtained in <1 minute. The simultaneous measurements of the melting temperature of BCAII and BCAII bound to bicarbonate reveal that the bicarbonate stabilizes the folded form of this protein by ∼6.4 °C. Moreover, the temperature dependences of different bicarbonate loss pathways are obtained. Although protein analytes are directly heated by the laser for only 140 ms, heat conduction further up the emitter leads to a total analyte heating time of ∼41 s. Pulsed laser heating experiments could reduce this time to ∼0.5 s for protein aggregation that occurs on a faster time scale. Laser heating provides a powerful method for studying the detailed mechanisms of cofactor/ligand loss with increasing temperature and promises a new tool for studying the effect of ligands, drugs, growth conditions, buffer additives, or other treatments on the stabilities of aggregation-prone biomolecules.

Ion emission from 1-10 MDa salt clusters: individual charge state resolution with charge detection mass spectrometry.

Salt cluster ions produced by electrospray ionization are used for mass calibration and fundamental investigations into cluster stability and charge separation processes. However, previous studies have been limited to relatively small clusters owing to the heterogeneity associated with large, multiply-charged clusters that leads to unresolved signals in conventional m/z spectra. Here, charge detection mass spectrometry is used to measure both the mass and charge distributions of positively charged clusters of KCl, CaCl2, and LaCl3 with masses between ∼1 and 10 MDa by dynamically measuring the energy per charge, m/z, charge, and mass of simultaneously trapped individual ions throughout a 1 s trapping time. The extent of remaining hydration on the clusters, determined from the change in the frequency of ion motion with time as a result of residual water loss, follows the order KCl < CaCl2 < LaCl3, and is significantly lower than that of a pure water nanodrop, consistent with tighter water binding to the more highly charged cations in these clusters. The number of ion emission events from these clusters also follows this same trend, indicating that water at the cluster surface facilitates charge loss. A new frequency-based method to determine the magnitude of the charge loss resulting from individual ion emission events clearly resolves losses of +1 and +2 ions. Achieving this individual charge state resolution for ion emission events is an important advance in obtaining information about the late stages of bare gaseous ions formation. Future experiments on more hydrated clusters are expected to lead to a better understanding of ion formation in electrospray ionization.

Variable Mixing with Theta Emitter Mass Spectrometry: Changing Solution Flow Rates with Emitter Position.

Two solutions can be rapidly mixed using theta glass emitters, with products measured using electrospray ionization mass spectrometry. The relative flow rates of the two emitter channels can be measured using different calibration compounds in each channel, or the flow rates are often assumed to be the same. The relative flow rates of each channel can be essentially the same when the emitters are positioned directly in front of the capillary entrance of a mass spectrometer, but the relative flow rates can be varied by up to 3 orders of magnitude by moving the position of the emitter tip ±1 cm in a direction that is perpendicular to the inner divider. Results of the emitter position on the different concentrations of reagents in the initially formed electrospray droplets are demonstrated through protein denaturation using a supercharging reagent as well as two different bimolecular reactions. The average charge state of myoglobin changed from +7.8 to +13.8 when 2.5% sulfolane was mixed with a 200 mM ammonium acetate solution containing the protein when the position of the emitter was scanned in front of the mass spectrometer inlet. The conversion ratio of a bimolecular reaction was changed from 0.98 to 0.04 with varying emitter positions. These results show that the relative flow rates must be carefully monitored because the droplet composition depends strongly on the position of the theta glass emitters. This method can be used to measure the dependence of reaction kinetics on different solution concentrations by using a single emitter and only two solutions.

Combined Multiharmonic Frequency Analysis for Improved Dynamic Energy Measurements and Accuracy in Charge Detection Mass Spectrometry.

The ability to determine ion energies in electrostatic ion-trap-based charge detection mass spectrometry (CDMS) experiments is important for the accurate measurement of individual ion m/z, charge, and mass. Dynamic energy measurements throughout the time an ion is trapped take advantage of the relationship between ion energy and the harmonic amplitude ratio (HAR) composed from the fundamental and second harmonic amplitudes in the Fourier transform of the ion signal. This method eliminates the need for energy-filtering optics in CDMS and makes it possible to measure energy lost in collisions and changes in ion masses due to dissociation. However, the accuracy of the energy measurement depends on the signal-to-noise ratio (S/N) of the amplitudes used to determine the HAR. Here, a major improvement to this HAR-based dynamic energy measurement method is achieved using HARs composed of higher-order harmonics in addition to the fundamental and second harmonic to determine ion energies. This combined harmonic amplitude ratios for precision energy refinement (CHARPER) method is applied to the analysis of a 103 nm polystyrene nanoparticle ion (359.7 MDa, m/z = 308,300) and the energy resolution (3140) and effective mass resolution (730) achieved are the best yet demonstrated in electrostatic ion-trap-based CDMS. The CHARPER method applied to an ensemble of several thousand adeno-associated virus ion signals also results in higher mass resolution compared to the basic HAR method, making it possible to resolve additional features in the composite mass histogram.

Dynamic Energy Measurements in Charge Detection Mass Spectrometry Eliminate Adverse Effects of Ion-Ion Interactions.

Ion-ion interactions in charge detection mass spectrometers that use electrostatic traps to measure masses of individual ions have not been reported previously, although ion trajectory simulations have shown that these types of interactions affect ion energies and thereby degrade measurement performance. Here, examples of interactions between simultaneously trapped ions that have masses ranging from ca. 2 to 350 MDa and ca. 100 to 1000 charges are studied in detail using a dynamic measurement method that makes it possible to track the evolution of the mass, charge, and energy of individual ions over their trapping lifetimes. Signals from ions that have similar oscillation frequencies can have overlapping spectral leakage artifacts that result in slightly increased uncertainties in the mass determination, but these effects can be mitigated by the careful choice of parameters used in the short-time Fourier transform analysis. Energy transfers between physically interacting ions are also observed and quantified with individual ion energy measurement resolution as high as ∼950. The mass and charge of interacting ions do not change, and their corresponding measurement uncertainties are equivalent to ions that do not undergo physical interactions. Simultaneous trapping of multiple ions in CDMS can greatly decrease the acquisition time necessary to accumulate a statistically meaningful number of individual ion measurements. These results demonstrate that while ion-ion interactions can occur when multiple ions are trapped, they have negligible effects on mass accuracy when using the dynamic measurement method.

Characterizing Heterogeneous Mixtures of Assembled States of the Tobacco Mosaic Virus Using Charge Detection Mass Spectrometry.

The tobacco mosaic viral capsid protein (TMV) is a frequent target for derivatization for myriad applications, including drug delivery, biosensing, and light harvesting. However, solutions of the stacked disk assembly state of TMV are difficult to characterize quantitatively due to their large size and multiple assembled states. Charge detection mass spectrometry (CDMS) addresses the need to characterize heterogeneous populations of large protein complexes in solution quickly and accurately. Using CDMS, previously unobserved assembly states of TMV, including 16-monomer disks and odd-numbered disk stacks, have been characterized. We additionally employed a peptide-protein conjugation reaction in conjunction with CDMS to demonstrate that modified TMV proteins do not redistribute between disks. Finally, this technique was used to discriminate between protein complexes of near-identical mass but different configurations. We have gained a greater understanding of the behavior of TMV, a protein used across a broad variety of fields and applications, in the solution state.

Laser Heating Nanoelectrospray Emitters for Fast Protein Melting Measurements with Mass Spectrometry.

Temperature-controlled nanoelectrospray ionization has been used to measure heat-induced conformational changes of biomolecules by mass spectrometry, but long thermal equilibration times associated with heating or cooling an entire emitter limit how fast these data can be acquired. Here, the tip of a borosilicate electrospray emitter is heated using 10.6 µm light from an unfocused CO2 laser. At 1.2 W, the solution inside the emitter tip can be heated from room temperature to a steady-state temperature of 78.2 ± 2.5 °C in less than 0.5 s and cools from 82.6 ± 0.6 °C back to room temperature within 4 s. The time required to establish a steady-state temperature is more than 100-fold faster than that required for a resistively heated emitter due to the low thermal mass. Protein unfolding curves measured as a function of laser power can be acquired in ∼40 s compared to a resistively heated apparatus that required ∼21 min to acquire similar data. Laser power is calibrated to temperature by comparisons of the average charge state of the protein cytochrome c measured with laser heating and with resistive heating. This laser heating method is applied to a three-component protein mixture to demonstrate the ability to rapidly acquire melting temperatures of proteins in mixtures. The ability to rapidly assess the thermal stabilities of multiple proteins simultaneously shows significant promise for coupling temperature-controlled electrospray ionization (ESI) to separation techniques, providing a high-throughput method for determining the effects of solution composition, drug binding, or sequence mutations on protein thermal stability.

Effects of Molecular Size on Resolution in Charge Detection Mass Spectrometry.

Instrumental resolution of Fourier transform-charge detection mass spectrometry instruments with electrostatic ion trap detection of individual ions depends on the precision with which ion energy is determined. Energy can be selected using ion optic filters or from harmonic amplitude ratios (HARs) that provide Fellgett's advantage and eliminate the necessity of ion transmission loss to improve resolution. Unlike the ion energy-filtering method, the resolution of the HAR method increases with charge (improved S/N) and thus with mass. An analysis of the HAR method with current instrumentation indicates that higher resolution can be obtained with the HAR method than the best resolution demonstrated for instruments with energy-selective optics for ions in the low MDa range and above. However, this gain is typically unrealized because the resolution obtainable with molecular systems in this mass range is limited by sample heterogeneity. This phenomenon is illustrated with both tobacco mosaic virus (0.6-2.7 MDa) and AAV9 (3.7-4.7 MDa) samples where mass spectral resolution is limited by the sample, including salt adducts, and not by instrument resolution. Nevertheless, the ratio of full to empty AAV9 capsids and the included genome mass can be accurately obtained in a few minutes from 1× PBS buffer solution and an elution buffer containing 300+ mM nonvolatile content despite extensive adduction and lower resolution. Empty and full capsids adduct similarly indicating that salts encrust the complexes during late stages of droplet evaporation and that mass shifts can be calibrated in order to obtain accurate analyte masses even from highly salty solutions.

Effects of Electrospray Droplet Size on Analyte Aggregation: Evidence for Serine Octamer in Solution.

Spraying solutions of serine under a wide variety of conditions results in unusually abundant gaseous octamer clusters that exhibit significant homochiral specificity, but the extent to which these clusters exist in solution or are formed by clustering during droplet evaporation has been debated. Electrospray ionization emitters with tip sizes between 210 nm and 9.2 µm were used to constrain the number of serine molecules that droplets initially contain. Protonated octamer was observed for all tip sizes with 10 mM serine solution, but the abundance decreases from 10% of the serine population at the largest tip size to ∼5.6% for the two smallest tip sizes. At 100 µM, the population abundance of the protonated serine octamer decreases from 1% to 0.6% from the largest to the smallest tip size, respectively. At 100 µM, fewer than 10% of the initial droplets should contain even a single analyte molecule with 210 nm emitter tips. These results indicate that the majority of protonated octamer observed in mass spectra under previous conditions is formed by clustering inside the electrospray droplet, but ≤5.6% and ∼0.6% of serine exists as an octamer complex in 10 mM and 100 µM solutions, respectively. These results show that aggregation occurs in large droplets, but this aggregation can be eliminated using emitters with sufficiently small tips. Use of these emitters with small tips is advantageous for clearly distinguishing between species that exist in solution and species formed by clustering inside droplets as solvent evaporation occurs.

Homochiral preference of serine octamer in solution and formed by dissociation of large gaseous clusters.

The ability of electrospray emitters with submicron tip diameters to significantly reduce and even eliminate aggregation of analyte molecules that can occur inside evaporating droplets was recently demonstrated to show that serine octamer exists in bulk solution, albeit in low abundance. Results using 222 nm emitter tips for D-serine and deuterium labeled L-serine show that the serine octamer that exists in 100 µM solution has a strong homochiral preference. Dissociation of large multiply protonated clusters results in formation of protonated octamer through a doubly protonated decamer intermediate. Remarkably, dissociation of the doubly protonated decamer from solution, which has a heterochiral preference, results in protonated octamer with strong homochiral preference. This homochiral preference is higher when protonated octamer is formed from larger clusters and approaches the chiral preference of the octamer in solution. These results show that the doubly protonated decamer has a different structure when formed from solution than when formed by dissociation of larger clusters. These results indicate that the unusually high abundance of protonated homochiral octamer formed by spray ionization methods that has been reported previously can be largely attributed to aggregation of serine that occurs in rapidly evaporating droplets and from dissociation of large clusters that form abundant protonated octamer at an optimized effective temperature.

Dissociation of large gaseous serine clusters produces abundant protonated serine octamer.

Protonated serine octamer is especially abundant in spray ionization mass spectra of serine solutions under a wide range of conditions. Although serine octamer exists in low abundance in solution, abundant clusters, including octamer, can be formed by aggregation inside evaporating electrospray droplets. A minimum cluster size of 8 and 21 serine molecules was observed for doubly protonated and triply protonated clusters, respectively, formed by electrospray ionization of a 10 mM serine solution. Dissociation of these clusters results in charge separation to produce predominantly protonated serine dimer and some trimer and the complimentary charged ion. Dissociation of clusters significantly larger than the minimum cluster size occurs by sequential loss of serine molecules. Dissociation of all large clusters investigated leads to protonated octamer as the second most abundant cluster (protonated dimer is most abundant) at optimized collision energies. All larger clusters dissociate through a combination of charge separation and neutral serine loss to form small doubly protonated clusters, and the vast majority of protonated octamer is produced by dissociation of the doubly protonated decamer by charge separation. Protonated octamer abundance is optimized at a uniform energy per degrees of freedom for all clusters indicating that simultaneous dissociation of all large clusters will lead to abundant protonated octamer at an optimum ion temperature. These results provide evidence for another route to formation of abundant protonated octamer in spray ionization or other methods that promote formation and subsequent dissociation of large clusters.

How many human proteoforms are there?

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

Multiplexed Charge Detection Mass Spectrometry for High-Throughput Single Ion Analysis of Large Molecules.

Applications of charge detection mass spectrometry (CDMS) for measuring the masses of large molecules, macromolecular complexes, and synthetic polymers that are too large or heterogeneous for conventional mass spectrometry measurements are made possible by weighing individual ions in order to avoid interferences between ions. Here, a new multiplexing method that makes it possible to measure the masses of many ions simultaneously in CDMS is demonstrated. Ions with a broad range of kinetic energies are trapped. The energy of each ion is obtained from the ratio of the intensity of the fundamental to the second harmonic frequencies of the periodic trapping motion making it possible to measure both the m/ z and charge of each ion. Because ions with the exact same m/ z but with different energies appear at different frequencies, the probability of ion-ion interference is significantly reduced. We show that the measured mass of a protein complex consisting of 16 protomers, RuBisCO (517 kDa), is not affected by the number of trapped ions with up to 21 ions trapped simultaneously in these experiments. Ion-ion interactions do not affect the ion trapping lifetime up to 1 s, and there is no influence of the number of ions on the measured charge-state distribution of bovine serum albumin (66.5 kDa), indicating that ion-ion interactions do not adversely affect any of these measurements. Over an order of magnitude gain in measurement speed over single ion analysis is demonstrated, and significant additional gains are expected with this multi-ion measurement method.

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.

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.

Characterizing the Conformationome: Toward a Structural Understanding of the Proteome.

While non-native protein conformations such as folding intermediates are rarely observed in solution such species are often stabilized as gaseous ions during electrospray ionization for mass spectrometry. This opens the possibility of large scale efforts to capture information about many non-native structures such as folding intermediates or malformed conformations having deleterious effects: studies of the conformationome.

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.

Competition between salt bridge and non-zwitterionic structures in deprotonated amino acid dimers.

Structures of deprotonated Cys, Asp, Glu, Phe, Pro, His homo dimers as well as [2Cys - 3H]-, [Asp + Glu - H]- and [2Glu - 2H + Na]- are investigated with infrared multiple-photon dissociation (IRMPD) spectroscopy between 650 and 1850 cm-1 and theory. The IRMPD spectra of all investigated complexes but [2His - H]-, [2Phe - H]- and [2Pro - H]- indicate that the structures consist of a neutral non-zwitterionic (NZ) and a deprotonated form of the amino acids. In contrast, the spectrum of [2His - H]- is complex and indicates the presence of multiple isomers and/or interactions between His and [His - H]-, so that its structure differs from that of the other deprotonated amino acid dimers. For [2Phe - H]- and especially for [2Pro - H]-, some IRMPD bands can only be explained by the presence of salt bridge (SB) structures in the dimer in which a deprotonated amino acid interacts with a zwitterionic neutral amino acid. Computational results indicate that SB structures are lower in energy at 298 K than corresponding NZ structures for neutral-anion complexes in which SB formation is not disrupted by amino acid side chains or conformational constraints, such as in [2Glu - H]- and [2Cys - 3H]- for which NZ structures are most consistent with experimental results. For deprotonated amino acid dimers in which these interfering interactions are absent, such as in [2Phe - H]- and [2Pro - H]-, the higher number of hydrogen bonds in SB compared to NZ structures stabilize the formation of zwitterionic neutral amino acids and consequently SB structures in agreement with results from IRMPD spectroscopy. These results suggest that SB structures likely occur in deprotonated peptide or protein ions at hydrophobic sites, such as protein-protein interfaces or in the interior of proteins, where interfering functional groups will not disrupt SB formation.

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.