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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. We demonstrate that for a simple bimolecular reaction where surface chemistry does not appear to play a role, reaction rate acceleration factors are between 102 and 107 for different initial solution concentrations, and these values do not depend on nanodrop size. A rate acceleration factor of 107 is among the highest reported and can be attributed to concentration of analyte molecules, initially far apart in dilute solution, but brought into close proximity in the nanodrop through evaporation of solvent from the nanodrops prior to ion formation. These data indicate that analyte concentration phenomenon is a significant factor in reaction acceleration where droplet volume throughout the experiment is not carefully controlled.

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.

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

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. This makes possible real-time data analysis using a standard desktop computer. Rectangular apodization leads to higher resolution than the more commonly used Gaussian or Hann apodization and makes it possible to separate ions with similar frequencies, a significant advantage for experiments in which the masses of many individual ions are measured simultaneously. Equally important is a20% increase in S/N obtained with rectangular apodization compared to Gaussian or Hann, which directly translates to a corresponding improvement in accuracy of both charge measurements and ion energy measurements that rely on the amplitudes of the fundamental and harmonic frequencies. Combined with computing the fast Fourier transform in a lower-level language, this fitting procedure eliminates computational barriers and should enable real-time processing of CDMS data on a laptop computer.

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

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.

Clusters consisting of 20 water molecules and a single cesium ion are especially stable due to their clathrate structure that is composed exclusively of three-coordinate water molecules. Clathrate stability was investigated using infrared photodissociation (IRPD) spectroscopy in the free-OH stretching region (∼3600-3800 cm-1) at ion cell temperatures between 135 and 355 K. At 275 K and colder, IRPD spectra of Cs+(H2O)20 have just one acceptor-acceptor-donor band. At higher temperatures, a higher-energy acceptor-donor band emerges and grows in intensity. Non-clathrate Na+(H2O)20 structures contain both of these bands, which do not change significantly in intensity over the temperature range. These results indicate a rapid onset in the conversion from clathrate to non-clathrate structures with temperature and suggest that some clathrate population remains even at the highest temperatures investigated. These results provide new insights into the role of entropy in clathrate stability.

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.

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.

Weighing single ions with charge detection mass spectrometry (CDMS) makes it possible to obtain the masses of molecules of essentially unlimited size even in highly heterogeneous samples, but producing a mass histogram that is representative of all of the components in a mixture requires substantial measurement time. Multiple ions can be trapped to reduce analysis time but ion signals can overlap. To determine the maximum gains in analysis speed possible with current instrumentation with multiple ion trapping, simulations calculating the frequency and overlap rate of ions with different mass, charge, and energy ranges were performed. For an analyte with a broad mass distribution, such as long chain polyethylene glycol (PEG, 8 MDa), gains in analysis speed of up to 160 times that of prior CDMS experiments are possible. For signals from homogeneous samples, ions with the same m/z have frequencies that overlap and interfere, reducing the effectiveness of multiplexing in experiments where ions have the same energy per charge. We show that by maximizing the decoupling of ion m/z from frequency using a broad range of ion energies, the rate of signal overlap is significantly reduced making it possible to trap more ions. Under optimum decoupling conditions, a measurement speed nearly 50 times greater than that of prior CDMS experiments is possible for RuBisCO (517 kDa). The reduction in overlap due to decoupling also results in more accurate quantitation in samples that contain multiple analytes with different concentrations.

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.

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.

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.

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. Nanodrop charge depends on the identity of constituent ions, with pure water nanodrops charged slightly above the Rayleigh limit and aqueous solutions containing alkali metal ions charged progressively below the Rayleigh limit with increasing cation size. MS2 capsid ions (∼3.5 MDa; ∼27 nm diameter) are more highly charged from aqueous ammonium acetate than from its biochemically preferred, 100 mM NaCl/10 mM Na phosphate solution, consistent with ion emission reducing the nanodrop and resulting capsid charge. The extent of charging indicates that the capsid partially collapses inside the nanodrops prior to the charging and formation of the dehydrated gaseous ions. These results demonstrate that ion emission can affect macromolecular charging and that conformational changes to macromolecular structure can occur in nanodrops prior to the formation of naked gaseous ions., Ion evaporation from aqueous nanodrops is measured for the first time using charge detection mass spectrometry, and the origin of solute ion dependent charging of large (MDa) macromolecules is revealed.

Clusters consisting of 20 water molecules and a single cesium ion are especially stable due to their clathrate structure that is composed exclusively of three-coordinate water molecules. Clathrate stability was investigated using infrared photodissociation (IRPD) spectroscopy in the free-OH stretching region (∼3600-3800 cm

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 β-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.

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.

A method to correct for the effect of ion energy on charge measurements of individual ions trapped and weighed with charge detection mass spectrometry (CDMS) is demonstrated. Ions with different energies induce different signal patterns inside an electrostatic ion trap. The sum of the amplitudes of the fundamental and second harmonic frequencies in the Fourier transform of the induced signal, which has been used to obtain the ion charge, depends on both ion energy and charge. The amplitudes of the fundamental frequencies of ions increase over time as ions lose energy by collisions with background gas and solvent loss from larger ions. Model ion signals are simulated with the same time-domain amplitude at different energies and frequencies and the resulting fundamental frequency amplitudes are used to normalize real ion signals for energy and frequency effects. The fundamental frequency amplitude decreases dramatically below 20 kHz and increases by ~ 17% from the highest energy to lowest energy that is stable with a given trap potential at all frequencies. Normalizing the fundamental frequency amplitude with the modeled amplitudes removes the systematic changes in the charge measurement of polyethylene glycol (PEG) and other ions and makes it possible to signal average the amplitude over long times, which reduces the charge uncertainty to 0.04% for a PEG ion for a 500-ms measurement. This method improves charge measurement accuracy and uncertainty, which are important for high-accuracy mass measurement with CDMS. Graphical abstract ᅟ.

The electrospray-MS analysis of oligonucleotides is hampered by non-volatile 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(+)/Mg(2+) 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 counter-ions 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(+)/Mg(2+) concentrations capable of preserving their structure and functional properties.

Ubiquitin confined within nanodroplets was irradiated with a variable-power CO(2) laser. Mass spectrometry analysis shows evidence for a protein “melting”-like transition within droplets prior to solvent evaporation and ion formation. Ion mobility spectrometry reveals that structures associated with early steps of denaturation are trapped because of short droplet lifetimes.

Theta glass electrospray emitters can rapidly mix solutions to investigate fast reactions that occur as quickly as 1 μs, but emitters with submicron tips have the unusual properties of desalting protein ions and affecting the observed abundances of some proteins as a result of protein-surface interactions. The role of protein physical properties on ion signal was investigated using 1.7 ± 0.1 μm and 269 ± 7 nm emitters and 100 mM aqueous ammonium acetate or ammonium bicarbonate solutions. Protein ion desalting occurs for both positive and negative ions. The signal of a mixture of proteins with the 269 nm tips is time-dependent and the order in which ions of each protein is observed is related to the expected strengths of the protein-surface interactions. These results indicate that it is not just the high surface-to-volume ratio that plays a role in protein adsorption and reduction or absence of initial ion signal, but the small diffusion distance and extremely low flow rates of the smaller emitters can lead to complete adsorption of some proteins and loss of signal until the adsorption sites are filled and the zeta potential is significantly reduced. After about 30 min, signals for a protein mixture from the two different size capillaries are similar. These results show the advantages of submicron emitters but also indicate that surface effects must be taken into account in experiments using such small tips or that coating the emitter surface to prevent adsorption should be considered. Graphical Abstract.

Synchrotron-radiation-based Fourier transform infrared (SR-FTIR or sFTIR) facilities worldwide are probing both live cells and tissues non-destructively under native-like conditions. From spectrosc...

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.

A new charge detection mass spectrometer that combines array detection and electrostatic ion trapping to repeatedly measure the masses of single ions is described. This instrument has four detector tubes inside an electrostatic ion trap with conical electrodes (cone trap) to provide multiple measurements of an ion on each pass through the trap resulting in a signal gain over a conventional trap with a single detection tube. Simulations of a cone trap and a dual ion mirror trap design indicate that more passes through the trap per unit time are possible with the latter. However, the cone trap has the advantages that ions entering up to 2 mm off the central axis of the trap are still trapped, the trapping time is less sensitive to the background pressure, and only a narrow range of energies are trapped so it can be used for energy selection. The capability of this instrument to obtain information about the molecular weight distributions of heterogeneous high molecular weight samples is demonstrated with 8 MDa polyethylene glycol (PEG) and 50 and 100 nm amine modified polystyrene nanoparticle samples. The measured mass distribution of the PEG sample is centered at 8 MDa. The size distribution obtained from mass measurements of the 100 nm nanoparticle sample is similar to the size distribution obtained from transmission electron microscopy (TEM) images, but most of the smaller nanoparticles observed in TEM images of the 50 nm nanoparticles do not reach a sufficiently high charge to trigger the trap on a single pass and be detected by the mass spectrometer. With the maximum trapping time set to 100 ms, the charge uncertainty is as low as ±2 charges and the mass uncertainty is approximately 2% for PEG and polystyrene ions.

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.

Protein-protein interactions (PPIs) play critical functional and regulatory roles in virtually all cellular processes. They are essential for the formation of macromolecular complexes, which in turn constitute the basis for extended protein interaction networks that determine the functional state of a cell. We and others have previously shown that chromatographic fractionation of native protein complexes in combination with bottom-up mass spectrometric analysis of consecutive fractions supports the multiplexed characterization and detection of state-specific changes of protein complexes.In this study, we describe a computational approach that extends the analysis of data from the co-fractionation / mass spectrometric analysis of native complexes to the level of PPI networks, thus enabling a qualitative and quantitative comparison of the proteome organization between samples and states. The Size-Exclusion Chromatography Algorithmic Toolkit (SECAT) implements a novel, network-centric strategy for the scalable and robust differential analysis of PPI networks. SECAT and its underlying statistical framework elucidate differential quantitative abundance and stoichiometry attributes of proteins in the context of their PPIs. We validate algorithm predictions using publicly available datasets and demonstrate that SECAT represents a more scalable and effective methodology to assess protein-network state and that our approach thus obviates the need to explicitly infer individual protein complexes. Further, by differential analysis of PPI networks of HeLa cells in interphase and mitotic state, respectively, we demonstrate the ability of the algorithm to detect PPI network differences and to thus suggest molecular mechanisms that differentiate cellular states.

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.

Charge detection mass spectrometry is used to measure the mass, charge, MSn and mobility of an individual ion produced by electrospray ionization of a 8 MDa polyethylene glycol sample. The charge detection mass spectrometer is an electrostatic ion trap that uses cone electrodes and a single tube detector and can detect ions for up to the full trapping time of 4.0 s. The time-domain signal induced on the detector tube by a single multiply charged ion can be complex owing to sequential fragmentation of the original precursor ion as well as increasing oscillation frequencies of the single ion owing to collisions with background gas that reduce the kinetic energy of the ion inside the trap. Simulations show that the ratio of the time for the ion to turn around inside the cone region of the trap to the time for the ion to travel through the detector tube is constant with m/z and increases with the ion energy per charge. By measuring this ratio, the kinetic energy of an ion can be obtained with good precision (∼1%) and this method to measure ion kinetic energies eliminates the necessity of ion energy selection prior to trapping for high precision mass measurement of large molecules in complex mixtures. This method also makes it possible to measure the masses of each sequential fragment ion formed from the original precursor ion. MS7 of a single multiply charged PEG molecule is demonstrated, and from these ion energy measurements and effects of collisions on the ion motion inside the trap, information about the ion mobility of the precursor ion and its fragments is obtained.

An important but understudied class of human exposures is comprised of reactive electrophiles that cannot be measured in vivo because they are short-lived. An avenue for assessing these meaningful exposures focuses on adducts from reactions with nucleophilic loci of blood proteins, particularly Cys34 of human serum albumin, which is the dominant scavenger of reactive electrophiles in serum. We developed an untargeted analytical scheme and bioinformatics pipeline for detecting, quantitating, and annotating Cys34 adducts in tryptic digests of human serum/plasma. The pipeline interrogates tandem mass spectra to find signatures of the Cys34-containing peptide, obtains accurate masses of putative adducts, quantitates adduct levels relative to a "housekeeping peptide", and annotates modifications based on a combination of retention time, accurate mass, elemental composition, and database searches. We used the adductomics pipeline to characterize 43 adduct features in archived plasma from healthy human subjects and found several that were highly associated with smoking status, race, and other covariates. Since smoking is a strong risk factor for cancer and cardiovascular disease, our ability to discover adducts that distinguish smokers from nonsmokers with untargeted adductomics indicates that the pipeline is suitable for use in epidemiologic studies. In fact, adduct features were both positively and negatively associated with smoking, indicating that some adducts arise from reactions between Cys34 and constituents of cigarette smoke (e.g., ethylene oxide and acrylonitrile) while others (Cys34 oxidation products and disulfides) appear to reflect alterations in the serum redox state that resulted in reduced adduct levels in smokers.

Electrothermal supercharging (ETS) with electrospray ionization produces highly charged protein ions from buffered aqueous solutions in which proteins have native folded structures. ETS increases the charge of ribonuclease A by 34%, whereas only a 6% increase in charge occurs for a reduced-alkylated form of this protein, which is unfolded and its structure is ~66% random coil in this solution. These results indicate that protein denaturation that occurs in the ESI droplets is the primary mechanism for ETS. ETS does not affect the extent of solution-phase hydrogen-deuterium exchange (HDX) that occurs for four proteins that have significantly different structures in solution, consistent with a droplet lifetime that is considerably shorter than observable rates of HDX. Rate constants for HDX of ubiquitin are obtained with a spatial resolution of ~1.3 residues with ETS and electron transfer dissociation of the 10+ charge-state using a single capillary containing a few μL of protein solution in which HDX continuously occurs. HDX protection at individual residues with ETS HDX is similar to that with reagent supercharging HDX and with solution-phase NMR, indicating that the high spray potentials required to induce ETS do not lead to HD scrambling. Graphical Abstract ᅟ.

The multi‐subunit Ca(2+)/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.

Gas phase infrared dissociation spectra of the radical cation, deprotonated and protonated forms of the hormone melatonin, and its complexes with alkali (Li+, Na+, and K+) and alkaline earth metal ions (Mg2+, Ca2+, and Sr2+) are measured in the spectral range 800–1800 cm−1. Minimum energy geometries calculated at the B3LYP/LACVP++** level are used to assign structural motifs to absorption bands in the experimental spectra. The melatonin anion is deprotonated at the indole-N. The indole-C linking the amide chain is the most favored protonation site. Comparisons between the experimental and calculated spectra for alkali and alkaline earth metal ion complexes reveal that the metal ions interact similarly with the amide and methoxy oxygen atoms. The amide I band undergoes a red shift with increasing charge density of the metal ion and the amide II band shows a concomitant blue shift. Another binding motif in which the metal ions interact with the amide-O and the π-electron cloud of the aromatic group is identified but is higher in energy by at least 18 kJ/mol. Melatonin is deprotonated at the amide-N with Mg2+ and the metal ion coordinates to the amide-N and an indole-C or the methoxy-O. These results provide information about the intrinsic binding of metal ions to melatonin and combined with future studies on solvated melatonin-metal ion complexes may help elucidate the solvent effects on metal ion binding in solution and the biochemistry of melatonin. These results also serve as benchmarks for future theoretical studies on melatonin-metal ion interactions.

A general method for in situ measurements of the energy of individual ions trapped and weighed using charge detection mass spectrometry (CDMS) is described. Highly charged (> 300 e), individual polyethylene glycol (PEG) ions are trapped and oscillate within an electrostatic trap, producing a time domain signal. A segmented Fourier transform (FT) of this signal yields the temporal evolution of the fundamental and harmonic frequencies of ion motion throughout the 500-ms trap time. The ratio of the fundamental frequency and second harmonic (HAR) depends on the ion energy, which is an essential parameter for measuring ion mass in CDMS. This relationship is calibrated using simulated ion signals, and the calibration is compared to the HAR values measured for PEG ion signals where the ion energy was also determined using an independent method that requires that the ions be highly charged (> 300 e). The mean error of 0.6% between the two measurements indicates that the HAR method is an accurate means of ion energy determination that does not depend on ion size or charge. The HAR is determined dynamically over the entire trapping period, making it possible to observe the change in ion energy that takes place as solvent evaporates from the ion and collisions with background gas occur. This method makes it possible to measure mass changes, either from solvent evaporation or from molecular fragmentation (MSn), as well as the cross sections of ions measured using CDMS.

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.

The 1.5 μs and

A new experimental setup for spatially resolved ambient infrared laser ablation-mass spectrometry (AIRLAB-MS) that uses an infrared microscope with an infinity-corrected reflective objective and a continuous flow solvent probe coupled to a Fourier transform ion cyclotron resonance mass spectrometer is described. The efficiency of material transfer from the sample to the electrospray ionization emitter was determined using glycerol/methanol droplets containing 1 mM nicotine and is ∼50%. This transfer efficiency is significantly higher than values reported for similar techniques. Laser desorption does not induce fragmentation of biomolecules in droplets containing bradykinin, leucine enkephalin and myoglobin, but loss of the heme group from myoglobin occurs as a result of the denaturing solution used. An application of AIRLAB-MS to biological materials is demonstrated for tobacco leaves. Chemical components are identified from the spatially resolved mass spectra of the ablated plant material, including nicotine and uridine. The reproducibility of measurements made using AIRLAB-MS on plant material was demonstrated by the ablation of six closely spaced areas (within 2 × 2 mm) on a young tobacco leaf, and the results indicate a standard deviation of10% in the uridine signal obtained for each area. The spatial distribution of nicotine was measured for selected leaf areas and variation in the relative nicotine levels (15-100%) was observed. Comparative analysis of the nicotine distribution was demonstrated for two tobacco plant varieties, a genetically modified plant and its corresponding wild-type, indicating generally higher nicotine levels in the mutant.

Complexes of CuOH + (H 2 O) n where n = 2–9 are examined using infrared photodissociation spectroscopy (IRPD) with frequencies between ~2700 and 3900 cm −1 . Structural characterization is achieved through comparison between experimental and theoretical spectra. Geometry optimizations and frequency calculations are performed on a myriad of possible low-energy structures at the B3LYP/6-311+G(d,p) level. Subsequent single- point energy calculations are performed at the B3LYP, B3P86, M06, and MP2(full) levels of theory using a 6-311+G(2d,2p) basis set to obtain relative free energies. The IRPD spectra of all complexes where n ≥ 3 are consistent with structures having a coordination number (CN) of 4 although broad features in the CuOH + (H 2 O) 5 and CuOH + (H 2 O) 6 spectra accommodate structures having both CN = 4 and CN = 5. For CuOH + (H 2 O) 7 , spectral bands in the free-OH region narrow, revealing fine structure that confirms the presence of both CN = 4 and CN = 5 isomers. Conformational assignments are made on the basis of asymmetric stretching frequencies of free-OH water molecules specific to each CN. Relative intensities of these bands are generally in good agreement with relative energies predicted by MP2(full) and not those of the other levels. MP2(full) and M06 single-point energies for the 4 and 5-coordinate isomers are typically within 5 kJ/mol of each other suggesting possible equilibration between the two if isomerization barriers are low.

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

The structures of Gly3·X−, Ala3·X− and Leu3·X− (X = Cl, Br and I) are investigated with computational chemistry and infrared multiple-photon dissociation (IRMPD) spectroscopy. Low-energy structures calculated at the B3LYP/6-31+G** level of theory (or with the CRENBL basis set and effective core potential implemented for Br and I) for these complexes have similar structural motifs in which the halide anion binds to the peptide via hydrogen bonds at amide, amine, and/or carboxylic acid H atoms. The IRMPD spectra do not depend significantly on anion identity. Comparisons between measured spectra and those calculated for low-energy structures of each of the chloridated complexes indicate that all three complexes have similar binding motifs. These results suggest that the size of the alkyl side chain does not significantly influence how halide anions bind to these peptides. The coordination geometries of Gly3·X− and Ala3·X− are “inverted” compared to those for the Na+ cationized peptides, where the peptides coordinate to Na+ via lone pair electrons of O and N atoms. The “inversion” in structures between Ala3·Na+ and Ala3·X− results in greater steric hindrance for some geometries of the latter. There is a subtle blue shift in the C-terminal C O stretch frequency with increasing halide anion size for each peptide, consistent with contributions from Stark and charge transfer effects. In contrast, the N H bends red shift with increasing halide anion size, which can only be attributed to the charge transfer effect. This is the first report of IR spectra of peptides complexed with anions, and these results provide insights into anion-peptide binding interactions.

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.

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.

International audience; In celebration of Jose Riveros' many essential contributions to gas phase ion chemistry. Keywords: Hydrated sulfate IRPD spectroscopy AMOEBA polarizable force field Second hydration shell Mixed quantum classical modelling O H stretching frequency a b s t r a c t Infrared photodissociation (IRPD) spectra of SO 4 2− (H 2 O) n , n = 9-13, recorded in the cooled cell of a Fourier-transform ion cyclotron resonance mass spectrometer, between 2900 and 3800 cm −1 , are reported. The structures, energetics and infrared spectra of n = 9 and 11-13 were investigated by a combination of classical polarizable molecular dynamics and static quantum chemical calculations. Low-energy structures are mainly determined by the strong structuring effect of the sulfate ion, however, the highest cohesion is achieved when strong water-water interactions are present as well. As a result, the sulfate ion in the most stable structures for n = 9, 11 and 12 is on the surface of the water cluster. While SO 4 2− (H 2 O) 9 involves a mixture of isomers, the other sizes are found to be described by a single structural family, with the most stable structures of SO 4 2− (H 2 O) 11 and SO 4 2− (H 2 O) 13 deriving from that of SO 4 2− (H 2 O) 12 by removal and addition of a water molecule, respectively, without substantial reorganization. An important feature of these structures is that the number of water molecules in the second solvation sphere increases with cluster size, up to 3 for n = 12 and 4 for n = 13. This is directly reflected in the IRPD spectra. All spectra display two main features in the 3150-3350 and 3350-3650 cm −1 range, plus a small band near 3100 cm −1. The 3350-3650 cm −1 massif, which includes most bands arising from second sphere molecules, acquires larger intensity relative to that at 3150-3350 cm −1 which is mainly composed of stretches in first sphere molecules. Whereas most water molecules have ADD coordination (where A stands for acceptor and D stands for donor of a hydrogen bond), special cases, including DD and AADD account for bands at the red and blue ends of the spectra. Computed IR spectra are able to account for most experimental features, especially when anharmonicities are taken into account for the largest red shifts. Finally, the higher abundance of n = 12 relative to other sizes is related to a lower water evaporation rate constant, in good agreement with the water binding energy which is computed to be larger for n = 12 than for 13.

The effects of ion charge, polarity and size on the surface morphology of size-selected aqueous nanodrops containing a single ion and up to 550 water molecules are investigated with infrared photodissociation (IRPD) spectroscopy and theory. IRPD spectra of M(H2O) n where M = La3+, Ca2+, Na+, Li+, I-, SO42- and supporting molecular dynamics simulations indicate that strong interactions between multiply charged ions and water molecules can disrupt optimal hydrogen bonding (H-bonding) at the nanodrop surface. The IRPD spectra also reveal that "free" OH stretching frequencies of surface-bound water molecules are highly sensitive to the ion's identity and the OH bond's local H-bond environment. The measured frequency shifts are qualitatively reproduced by a computationally inexpensive point-charge model that shows the frequency shifts are consistent with a Stark shift from the ion's electric field. For multiply charged cations, pronounced Stark shifting is observed for clusters containing ∼100 or fewer water molecules. This is attributed to ion-induced solvent patterning that extends to the nanodrop surface, and serves as a spectroscopic signature for a cation's ability to influence the H-bond network of water located remotely from the ion. The Stark shifts measured for the larger nanodrops are extrapolated to infinite dilution to obtain the free OH stretching frequency of a surface-bound water molecule at the bulk air-water interface (3696.5-3701.0 cm-1), well within the relatively wide range of values obtained from SFG measurements. These cluster measurements also indicate that surface curvature effects can influence the free OH stretching frequency, and that even nanodrops without an ion have a surface potential that depends on cluster size.