Is Native Mass Spectrometry in Ammonium Acetate Really Native? Protein Stability Differences in Biochemically Relevant Salt Solutions.

Ammonium acetate is widely used in native mass spectrometry to provide adequate ionic strength without adducting to protein ions, but different ions can preferentially stabilize or destabilize the native form of proteins in solution. The stability of bovine serum albumin (BSA) was investigated in 50 mM solutions of a variety of salts using electrospray emitters with submicron tips to desalt protein ions. The charge-state distribution of BSA is narrow (+14 to +18) in ammonium acetate (AmmAc), whereas it is much broader (+13 to +42) in solutions containing sodium acetate (NaAc), ammonium chloride (AmmCl), potassium chloride (KCl), and sodium chloride (NaCl).

High-Throughput Single-Particle Characterization of Aggregation Pathways and the Effects of Inhibitors for Large (Megadalton) Protein Oligomers.

Protein aggregation is involved in many human diseases, but characterizing the sizes and shapes of intermediate oligomers (∼10-100 nm) that are important to the formation of macroscale aggregates like amyloid fibrils is a significant analytical challenge. Here, charge detection mass spectrometry (CDMS) is used to characterize individual conformational states of bovine serum albumin oligomers with up to ∼225 molecules (15 MDa). Elongated, partially folded, and globular conformational families for each oligomer can be readily distinguished based on the extent of charging.

Are Hydroxyl Radicals Spontaneously Generated in Unactivated Water Droplets?

Spontaneous ionization/breakup of water at the surface of aqueous droplets has been reported with evidence ranging from formation of hydrogen peroxide and hydroxyl radicals, indicated by ions at m/z 36 attributed to OH⋅-HO or (HO-OH)⋅ as well as oxidation products of radical scavengers in mass spectra of water droplets formed by pneumatic nebulization. Here, aqueous droplets are formed both by nanoelectrospray, which produces highly charged nanodrops with initial diameters ~100 nm, and a vibrating mesh nebulizer, which produces 2-20 μm droplets that are initially less highly charged. The lifetimes of these droplets range from 10s of μs to 560 ms and the surface-to-volume ratios span ~100-fold range.

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.

Characterization of Mass, Diameter, Density, and Surface Properties of Colloidal Nanoparticles Enabled by Charge Detection Mass Spectrometry.

A variety of scattering-based, microscopy-based, and mobility-based methods are frequently used to probe the size distributions of colloidal nanoparticles with transmission electron microscopy (TEM) often considered to be the "gold standard". Charge detection mass spectrometry (CDMS) is an alternative method for nanoparticle characterization that can rapidly measure the mass and charge of individual nanoparticle ions with high accuracy. Two low polydispersity, ∼100 nm diameter nanoparticle size standards with different compositions (polymethyl methacrylate/polystyrene copolymer and 100% polystyrene) were characterized using both TEM and CDMS to explore the merits and complementary aspects of both methods.

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 CO fixing enzyme involved in many biochemical pathways, and cytochrome leads to complete loss of carbonic anhydrase signal and a significant reduction in cytochrome 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.

Optimal trade-off control in machine learning-based library design, with application to adeno-associated virus (AAV) for gene therapy.

Adeno-associated viruses (AAVs) hold tremendous promise as delivery vectors for gene therapies. AAVs have been successfully engineered-for instance, for more efficient and/or cell-specific delivery to numerous tissues-by creating large, diverse starting libraries and selecting for desired properties. However, these starting libraries often contain a high proportion of variants unable to assemble or package their genomes, a prerequisite for any gene delivery goal.

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 / spectra. Here, charge detection mass spectrometry is used to measure both the mass and charge distributions of positively charged clusters of KCl, CaCl, and LaCl with masses between ∼1 and 10 MDa by dynamically measuring the energy per charge, /, charge, and mass of simultaneously trapped individual ions throughout a 1 s trapping time.

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 /, 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.

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.

Dynamics of Rayleigh Fission Processes in ∼100 nm Charged Aqueous Nanodrops.

Fission of micron-size charged droplets has been observed using optical methods, but little is known about fission dynamics and breakup of smaller nanosize droplets that are important in a variety of natural and industrial processes. Here, spontaneous fission of individual aqueous nanodrops formed by electrospray is investigated using charge detection mass spectrometry. Fission processes ranging from formation of just two progeny droplets in 2 ms to production of dozens of progeny droplets over 100+ ms are observed for nanodrops that are charged above the Rayleigh limit.

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.

Lighting Up at High Potential: Effects of Voltage and Emitter Size in Nanoelectrospray Ionization.

Effects of electrospray voltage on cluster size and abundance formed from aqueous CsI were investigated with emitter tip diameters between 260 ± 7 nm and 2.45 ± 0.30 μm.

The role of analyte concentration in accelerated reaction rates in evaporating droplets.

Accelerated reactions in microdroplets have been reported for a wide range of reactions with some microdroplet reactions occurring over a million times faster than the same reaction in bulk solution. Unique chemistry at the air-water interface has been implicated as a primary factor for accelerated reaction rates, but the role of analyte concentration in evaporating droplets has not been as well studied. Here, theta-glass electrospray emitters and mass spectrometry are used to rapidly mix two solutions on the low to sub-microsecond time scale and produce aqueous nanodrops with different sizes and lifetimes.

Accurate Sizing of Nanoparticles Using a High-Throughput Charge Detection Mass Spectrometer without Energy Selection.

The sizes and shapes of nanoparticles play a critical role in their chemical and material properties. Common sizing methods based on light scattering or mobility lack individual particle specificity, and microscopy-based methods often require cumbersome sample preparation and image analysis. A promising alternative method for the rapid and accurate characterization of nanoparticle size is charge detection mass spectrometry (CDMS), an emerging technique that measures the masses of individual ions.

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.

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 CO laser.

Apodization Specific Fitting for Improved Resolution, Charge Measurement, and Data Analysis Speed in Charge Detection Mass Spectrometry.

Short-time Fourier transforms with short segment lengths are typically used to analyze single ion charge detection mass spectrometry (CDMS) data either to overcome effects of frequency shifts that may occur during the trapping period or to more precisely determine the time at which an ion changes mass or charge, or enters an unstable orbit. The short segment lengths can lead to scalloping loss unless a large number of zero-fills are used, making computational time a significant factor in real-time analysis of data. Apodization specific fitting leads to a 9-fold reduction in computation time compared to zero-filling to a similar extent of accuracy.

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 /) and thus with mass.

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.

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.

Direct observation of ion emission from charged aqueous nanodrops: effects on gaseous macromolecular charging.

Mechanistic information about how gaseous ions are formed from charged droplets has been difficult to establish because direct observation of nanodrops in a size range relevant to gaseous macromolecular ion formation by optical or traditional mass spectrometry methods is challenging owing to their small size and heterogeneity. Here, the mass and charge of individual aqueous nanodrops between 1-10 MDa (15-32 nm diameter) with ∼50-300 charges are dynamically monitored for 1 s using charge detection mass spectrometry. Discrete losses of minimally solvated singly charged ions occur, marking the first direct observation of ion emission from aqueous nanodrops in late stages of droplet evaporation relevant to macromolecular ion formation in native mass spectrometry.

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.

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.

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

Ca/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.