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.
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.
Raptors provide critical ecosystem services, yet there is currently no systematic, global synthesis of their conservation status or threats. We review the International Union for the Conservation of ...Nature's Red List to examine the conservation status, distributions, threats, and conservation recommendations for all 557 raptor species. We further assess the significance of Important Bird and Biodiversity Areas (IBAs) for raptor conservation. We also determine which countries contain the most species listed under the Memorandum of Understanding on the Conservation of Migratory Birds of Prey in Africa and Eurasia (Raptors MoU). Raptors, especially Old World vultures, are more threatened than birds in general. Eighteen percent of raptors are threatened with extinction and 52% of raptors have declining global populations. South and Southeast Asia have the highest richness and the largest number of threatened raptor species. By country, Indonesia has the highest richness of raptor species (119) and most declining species (63). China and Russia contain the most Raptors MoU species, although they are not yet signatories to the agreement. Raptor species that require forest are more likely to be threatened and declining than those that do not. Agriculture and logging are the most frequently identified threats, although poisoning is especially detrimental to Old World vultures. Of the 10 most important IBAs for raptors, six are in Nepal. Highest priority conservation actions to protect raptors include preventing mortality and conserving key sites and priority habitats. Improved long-term monitoring would allow for conservation to be appropriately targeted and effectiveness of interventions to be assessed.
Theta-glass emitters are used to rapidly mix two solutions to induce either protein folding or unfolding during nanoelectrospray (nanoESI). Mixing acid-denatured myoglobin with an aqueous ammonium ...acetate solution to increase solution pH results in protein folding during nanoESI. A reaction time and upper limit to the droplet lifetime of 9 ± 2 μs is obtained from the relative abundance of the folded conformer in these rapid mixing experiments compared to that obtained from solutions at equilibrium and a folding time constant of 7 μs. Heme reincorporation does not occur, consistent with the short droplet lifetime and the much longer time constant for this process. Similar mixing experiments with acid-denatured cytochrome c and the resulting folding during nanoESI indicate a reaction time of between 7 and 25 μs depending on the solution composition. The extent of unfolding of holo-myoglobin upon rapid mixing with theta-glass emitters is less than that reported previously (Fisher et al. Anal. Chem. 2014, 86, 4581−4588 ), a result that is attributed to the much smaller, ∼1.5 μm, average o.d. tips used here. These results indicate that the time frame during which protein folding or unfolding can occur during nanoESI depends both on the initial droplet size, which can be varied by changing the emitter tip diameter, and on the solution composition. This study demonstrates that protein folding or unfolding processes that occur on the ∼10 μs time scale can be readily investigated using rapid mixing with theta-glass emitters combined with mass spectrometry.
Nonvolatile salts are essential for the structures and functions of many proteins and protein complexes but can severely degrade performance of native mass spectrometry by adducting to protein and ...protein complex ions, thereby reducing sensitivity and mass measuring accuracy. Small nanoelectrospray emitters are used to form protein and protein complex ions directly from high‐ionic‐strength (>150 mm) nonvolatile buffers with salts that mimic the extracellular environment. Charge‐state distributions are not obtained for proteins and protein complexes from six commonly used nonvolatile buffers and ≥150 mm Na+ with conventionally sized nanoelectrospray emitter tips but are resolved with 0.5 μm tips. This method enables mass measurements of proteins and protein complexes directly from a variety of commonly used buffers with high concentrations of nonvolatile salts and eliminates the need to buffer exchange into volatile ammonium buffers traditionally used in native mass spectrometry.
Small 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 not obtained with conventional‐sized nanoelectrospray emitter tips are resolved with 0.5 μm tips.
Double-barrel wire-in-a-capillary electrospray emitters prepared from theta-glass capillaries were used to mix solutions during the electrospray process. The relative flow rate of each barrel was ...continuously monitored with internal standards. The complexation reaction of 18-crown-6 and K+, introduced from opposite barrels, reaches equilibrium during the electrospray process, suggesting that complete mixing also occurs. A simplified diffusion model suggests that mixing occurs in less than a millisecond, and contributions of turbulence, estimated from times of coalescing ballistic microdroplets, suggest that complete mixing occurs within a few microseconds. This mixing time is 2 orders of magnitude less than in any mixer previously coupled to a mass spectrometer. The reduction of 2,6-dichloroindophenol by l-ascorbic acid was performed using the theta-glass emitters and monitored using mass spectrometry. On the basis of the rate constant of this reaction in bulk solution, an apparent reaction time of 274 ± 60 μs was obtained. This reaction time is an upper limit to the droplet lifetime because the surface area to volume ratio and the concentration of reagents increase as the droplet evaporates and some product formation occurs in the Taylor cone prior to droplet formation. On the basis of increases in reaction rates measured by others in droplets compared to rates in bulk solution, the true droplet lifetime is likely 1–3 orders of magnitude less than the upper limit, i.e., between 27 μs and 270 ns. The rapid mixing and short droplet lifetime achieved in these experiments should enable the monitoring of many different fast reactions using mass spectrometry.
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.
The use of theta-glass emitters and mass spectrometry to monitor reactions that occur as fast as one μs is demonstrated. Acidified aqueous solutions containing unfolded proteins are mixed with ...aqueous ammonium acetate solutions to increase the solution pH and induce protein folding during nanoelectrospray ionization. Protein charge-state distributions show the extent to which folding occurs, and reaction times are obtained from known protein folding time constants. Shorter reaction times are obtained by decreasing the solution flow rate, and reaction times between 1.0 and 22 μs are obtained using flow rates between 48 and 2880 pL/s, respectively. Remarkably similar reaction times are obtained for three different proteins (Trp-cage, myoglobin, and cytochrome c) with folding time constants that differ by more than an order of magnitude (4.1, 7, and 57 μs, respectively), indicating that the reaction times obtained using rapid mixing from theta-glass emitters are independent of protein identity. A folding time constant of 2.2 μs is obtained for the formation of a β-hairpin structure of renin substrate tetradecapeptide, which is the fastest folding event measured using a rapid mixing technique. The 1.0 μs reaction time obtained here is about an order of magnitude lower than the shortest reaction time probed using a conventional mixer (8 μs). Moreover, this fast reaction time is obtained with a 48 pL/s flow rate, which is 2000-times less than the flow rate required to obtained the 8 μs reaction time using a conventional mixer. These results indicate that rapid mixing with theta-glass emitters can be used to access significantly faster reaction times while consuming substantially less sample than in conventional mixing apparatus.
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 10
2
and 10
7
for different initial solution concentrations, and these values do not depend on nanodrop size. A rate acceleration factor of 10
7
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.
Reactant concentration increases in evaporating droplets, leading to an apparent reaction acceleration up to 10 million fold, and this effect is a significant factor in reaction acceleration in droplets where volume is not carefully controlled.
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.
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