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Malware that attack the electrical power grid consist of exploits and operations modules. The exploits are similar to those of traditional malware. These malware hack into an industrial computer and subsequently deploy operational modules. Some operational modules penetrate the operating system of the compromised industrial computer to take over computing functions and hence facilitate further attacks. Examples include interception of cryptographic keys, and generation of deceptive status data that indicate normal operation of a power transformer, while in reality the transformer is in distress due to the attacks. Other operational modules are designed to recognize and disrupt the physics of the physical equipment. We refer to these operations modules as physics-centric modules. The subject of this research is how physics-centric modules of malware can cause physical damage to power grid equipment. This research simulates a power transformer and a set of its protection algorithms. We make several contributions in this research, namely: i) we emulate in Python the protection algorithms that run on an industrial computer and monitor and protect a power transformer from a variety of faults; ii) we leverage these emulations to analyze the cyberattack surface of a power transformer; iii) with these insights at hand, we devise attack modus operandi that malware could use against a power transformer; and iv) we emulate these cyberattacks in Python to empirically observe and quantify their destructive effects on a power transformer. Our overall research findings in this paper serve the purpose of informing better defense against the physics-centric modules of malware that attack the electrical power grid.

Water-soluble Fe 4 L 4 4- cages can be synthesized in a multicomponent self-assembly process exploiting functionalized trigonal ligands, Fe II salts, and water-soluble sulfonated formylpyridine components. The cages are soluble in purely aqueous solution and display an overall 4- charge, but are capable of binding suitably sized non-coordinating anions in the host cavity despite their anionic nature. Anions such as PF 6 - or AsF 6 - occupy the internal cavity, whereas anions that are too small (BF 4 - ) or too large (NTf 2 - ) are not encapsulated. The external anionic charge and sterically blocked ligand cores limit the exchange rate of bound anions, as no exchange is seen over a period of weeks with the anion-filled cages, and internalization of added PF 6 - by an empty cage takes multiple weeks, despite the strong affinity of the cavity for PF 6 - ions. In the future, this recognition mechanism could be used to control release of anions for environmental applications., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Cysteine is the most reactive naturally occurring amino acid due to the presence of a free thiol, presenting a tantalizing handle for covalent modification of peptides/proteins. Although many mass spectrometry experiments could benefit from site-specific modification of Cys, the utility of direct arylation has not been thoroughly explored. Recently, Spokoyny and co-workers reported a Au(III) organometallic reagent that robustly arylates Cys and tolerates a wide variety of solvents and conditions. Given the chromophoric nature of aryl groups and the known susceptibility of carbon-sulfur bonds to photodissociation, we set out to identify an aryl group that could efficiently cleave Cys carbon-sulfur bonds at 266 nm. A streamlined workflow was developed to facilitate rapid examination of a large number of aryls with minimal sample using a simple test peptide, RAAACGVLK. We were able to identify several aryl groups that yield abundant homolytic photodissociation of the adjacent Cys carbon-sulfur bonds with short activation times (<10 ms). In addition, we characterized the radical products created by photodissociation by subjecting the product ions to further collisional activation. Finally, we tested Cys arylation with human hemoglobin, identified reaction conditions that facilitate efficient modification of intact proteins, and evaluated the photochemistry and activation of these large radical ions.

Isomerized amino acid residues have been identified in many peptides extracted from tissues or excretions of humans and animals. These isomerized residues can play key roles by affecting biological activity or by exerting an influence on the process of aging. Isomerization occurs spontaneously and does not result in a mass shift. Thus, identifying and localizing isomerized residues in biological samples is challenging. Herein, we introduce a fast and efficient method using tandem mass spectrometry (MS) to locate isomerized residues in peptides. Although MS 2 spectra are useful for identifying peptides that contain an isomerized residue, they cannot reliably localize isomerization sites. We show that this limitation can be overcome by utilizing MS 3 experiments to further evaluate each fragment ion from the MS 2 stage. Comparison at the MS 3 level, utilizing statistical analyses, reveals which MS 2 fragments differ between samples and, therefore, must contain the isomerized sites. The approach is similar to previous work relying on ion mobility to discriminate MS 2 product ions by collision cross-section. The MS 3 approach can be implemented using either ion-trap or beam-type collisional activation and is compatible with the quantification of isomer mixtures when coupled to a calibration curve. The method can also be implemented in combination with liquid chromatography in a targeted approach. Enabling the identification and localization of isomerized residues in peptides with an MS-only methodology will expand accessibility to this important information.

Spacious M 4 L 6 tetrahedra can act as catalytic inhibitors for base-mediated reactions. Upon adding only 5 % of a self-assembled Fe 4 L 6 cage complex, the conversion of the conjugate addition between ethylcyanoacetate and β-nitrostyrene catalyzed by proton sponge can be reduced from 83 % after 75 mins at ambient temperature to <1 % under identical conditions. The mechanism of the catalytic inhibition is unusual: the octacationic Fe 4 L 6 cage increases the acidity of exogenous water in the acetonitrile reaction solvent by favorably binding the conjugate acid of the basic catalyst. The inhibition only occurs for Fe 4 L 6 hosts with spacious internal cavities: minimal inhibition is seen with smaller tetrahedra or Fe 2 L 3 helicates. The surprising tendency of the cationic cage to preferentially bind protonated, cationic ammonium guests is quantified via the comprehensive modeling of spectrophotometric titration datasets., (© 2023 Wiley-VCH GmbH.)

Top-down mass spectrometry (MS) is a versatile tool that has been employed to investigate both protein sequence and structure. Although a variety of different fragmentation methods are available in top-down MS that can potentially yield structural information, quantifying differences between spectra remains challenging. Herein, we show that subtle differences in spectra produced by a variety of fragmentation methods are surprisingly sensitive to protein structure and/or charge localization, even in highly unfolded proteins observed in high charge states. In addition to exposing information about the protein structure, differences in fragmentation also reveal insight into the mechanisms underlying the dissociation methods themselves. The results further reveal that small changes in experimental parameters (such as the addition of methanol instead of acetonitrile) lead to changes in structure that are reflected in statistically reproducible differences in dissociation. Collisional annealing of structurally dissimilar ions in the gas phase eventually leads to dissociation spectra that are indistinguishable, suggesting that structural differences can be erased by sufficient thermal activation. Additional experiments illustrate that identical charge states of the same protein can be distinguished if those produced directly by electrospray are compared to ions manipulated by in vacuo proton-transfer charge reduction. Overall, the results show that subtle differences in both three-dimensional structure and charge-site localization can influence the abundance of fragment ions produced by top-down MS, including dissociation methods not typically thought to be structurally sensitive.

Fucose is a signaling carbohydrate that is attached at the end of glycan processing. It is involved in a range of processes, such as the selectin-dependent leukocyte adhesion or pathogen-receptor interactions. Mass-spectrometric techniques, which are commonly used to determine the structure of glycans, frequently show fucose-containing chimeric fragments that obfuscate the analysis. The rearrangement leading to these fragments-often referred to as fucose migration-has been known for more than 25 years, but the chemical identity of the rearrangement product remains unclear. In this work, we combine ion-mobility spectrometry, radical-directed dissociation mass spectrometry, cryogenic IR spectroscopy of ions, and density-functional theory calculations to deduce the product of the rearrangement in the model trisaccharides Lewis x and blood group H2. The structural search yields the fucose moiety attached to the galactose with an α(1→6) glycosidic bond as the most likely product., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Isomeric molecules are important analytes in many biological and chemical arenas, yet their similarity poses challenges for many analytical methods, including mass spectrometry (MS). Tandem-MS provides significantly more information about isomers than intact mass analysis, but highly similar fragmentation patterns are common and include cases where no unique m / z peaks are generated between isomeric pairs. However, even in such situations, differences in peak intensity can exist and potentially contain additional information. Herein, we present a framework for comparing mass spectra that differ only in terms of peak intensity and include calculation of a statistical probability that the spectra derive from different analytes. This framework allows for confident identification of peptide isomers by collision-induced dissociation, higher-energy collisional dissociation, electron-transfer dissociation, and radical-directed dissociation. The method successfully identified many types of isomers including various d/l amino acid substitutions, Leu/Ile, and Asp/IsoAsp. The method can accommodate a wide range of changes in instrumental settings including source voltages, isolation widths, and resolution without influencing the analysis. It is shown that quantification of the composition of isomeric mixtures can be enabled with calibration curves, which were found to be highly linear and reproducible. The analysis can be implemented with data collected by either direct infusion or liquid-chromatography MS. Although this framework is presented in the context of isomer characterization, it should also prove useful in many other contexts where similar mass spectra are generated.

Radical-directed dissociation (RDD) is a fragmentation technique in which a radical created by selective 213/266 nm photodissociation of a carbon-iodine bond is reisolated and collisionally activated. In previous RDD experiments, collisional activation was effected by ion-trap collision-induced dissociation (CID). Higher-energy collisional dissociation (HCD) differs from CID both in terms of how ions are excited and in the number, type, or abundance of fragments that are observed. In this paper, we explore the use of HCD for activation in RDD experiments. While RDD-CID favors fragments produced from radical-directed pathways such as a/z-ions and side chain losses regardless of the activation energy employed, RDD-HCD spectra vary considerably as a function of activation energy, with lower energies favoring RDD while higher energies favor products resulting from cleavage directed by mobile protons (b/y-ions). RDD-HCD therefore affords more tunable fragmentation based on the HCD energy provided. Importantly, the abundance of radical products decreases as a function of increasing HCD energy, confirming that RDD generally proceeds via lower-energy barriers relative to mobile-proton-driven dissociation. The dominance of b/y-ions at higher energies for RDD-HCD can therefore be explained by the higher survivability of fragments not containing the radical after the initial or subsequent dissociation events. Furthermore, these results confirm previous suspicions that HCD spectra differ from CID spectra due to multiple dissociation events.

Appending functional groups to the exterior of Zn 4 L 4 self-assembled cages allows gated control of anion binding. While the unfunctionalized cages contain aryl groups in the ligand that can freely rotate, attaching inert functional groups creates a "doorstop", preventing rotation and slowing the guest exchange rate, even though the interiors of the host cavities are identically structured. The effects on anion exchange are subtle and depend on multiple factors, including anion size, the nature of the leaving anion, and the electron-withdrawing ability and steric bulk of the pendant groups. Multiple exchange mechanisms occur, and the nature of the external groups controls associative and dissociative exchange processes: these bulky groups affect both anion egress and ingress, introducing an extra layer of selectivity to the exchange. Small changes can have large effects: affinities for anions as similar as PF 6 - and SbF 6 - can vary by as much as 400-fold between identically sized cavities., (© 2022 Wiley-VCH GmbH.)

Long-lived proteins (LLPs), although less common than their short-lived counterparts, are increasingly recognized to play important roles in age-related diseases such as Alzheimer's. In particular, spontaneous chemical modifications can accrue over time that serve as both indicators of and contributors to disrupted autophagy. For example, isomerization in LLPs is common and occurs in the absence of protein turnover while simultaneously interfering with the protein turnover by impeding proteolysis. In addition to the biological implications this creates, isomerization may also interfere with its own analysis. To clarify, bottom-up proteomics experiments rely on protein digestion by proteases, most commonly trypsin, but the extent to which isomerization might interfere with trypsin digestion is unknown. Here, we use a combination of liquid chromatography and mass spectrometry to examine the effect of isomerization on proteolysis by trypsin and chymotrypsin. Isomerized aspartic acid and serine residues (which represent the most common sites of isomerization in LLPs) were placed at various locations relative to the preferred protease cleavage point to evaluate the influence on digestion efficiency. Trypsin was found to be relatively tolerant of isomerization, except when present at the residue immediately C-terminal to Arg/Lys. For chymotrypsin, the influence of isomerization on digestion was less predictable, resulting in long-range interference for some isomer/peptide combinations. Given the trypsin- and chymotrypsin-like behaviors of the 20S proteasome, and to further establish the biological relevance of isomerization in LLPs, substrates with isomerized sites were also tested against proteasomal degradation. Significant disruption of 20S proteolysis was observed, suggesting that if LLPs persist long enough to isomerize, it will be difficult for the cells to digest them.

Radical-directed dissociation (RDD) is a powerful technique for structural characterization of peptides in mass spectrometry experiments. Prior to analysis, a radical precursor must typically be appended to facilitate generation of a free radical. To explore the use of a radical precursor that can be easily attached in a single step, we modified peptides using a "click" reaction with iodophenyl isothiocyanate. Coupling with amine functional groups proceeds with high yields, producing stable iodophenylthiourea-modified peptides. Photodissociation yields were recorded at 266 and 213 nm for the 2-, 3-, and 4-iodo isomers of the modifier and found to be highest for the 4-iodo isomer in nearly all cases. Fragmentation of the modified peptides following collisional activation revealed favorable losses of the tag, and electronic structure calculations were used to evaluate a potential mechanism involving hydrogen transfer within the thiourea group. Examination of RDD data revealed that 4-iodobenzoic acid, 4-iodophenylthiourea, and 3-iodotyrosine yield similar fragmentation patterns for a given peptide, although differences in fragment abundance are noted. Iodophenyl isothiocyanate labeling in combination with RDD can be used to differentiate isomeric amino acids within peptides, which should facilitate simplified evaluation of isomers present in complex biological samples.

Self-assembled Fe 4 L 6 cage complexes with variable internal functions can be synthesized from a 2,7-dibromocarbazole ligand scaffold, which orients six functional groups to the cage interior. Both ethylthiomethylether and ethyldimethylamino groups can be incorporated. The cages show strong ligand-centered fluorescence emission and a broad range of guest binding properties. Coencapsulation of neutral organic guests is favored in the larger, unfunctionalized cage cavity, whereas the thioether cage has a more sterically hindered cavity that favors 1 : 1 guest binding. Binding affinities up to 10 6 M -1 in CH 3 CN are seen. The dimethylamino cage is more complex, as the internal amines display partial protonation and can be deprotonated by amine bases. This amine cage displays affinity for a broad range of neutral organic substrates, with affinities and stoichiometries comparable to that of the similarly sized thioether cage. These species show that simple variations in ligand backbone allow variations in the number and type of functions that can be displayed towards the cavity of self-assembled hosts, which will have applications in biomimetic sensing, catalysis and molecular recognition.

Defects in autophagy are implicated in many age-related diseases that cause neurodegeneration including both Alzheimer's and Parkinson's. Within autophagy, the lysosome plays a crucial role by enabling the breakdown and recycling of a wide range of biomolecular species. Herein, the effects of isomerization of aspartic acid (Asp) on substrate recognition and degradation are investigated for a collection of lysosomal cathepsins using liquid chromatography coupled to mass spectrometry. By examining a series of synthetic peptides with sequences derived from long-lived proteins known to undergo Asp isomerization, we demonstrate that isomerized forms of Asp significantly perturb cathepsin activity by impeding digestion and shifting preferential sites of proteolysis. Although the sensitivity to isomerization varies for each cathepsin, none of the cathepsins were capable of digesting sites within several residues of the C-terminal side of the isomerized Asp. Under physiological conditions, the peptide fragments left behind after such incomplete digestion would not be suitable substrates for transporter recognition and could precipitate autophagic malfunction in the form of lysosomal storage.

Purpose: Accurate diagnosis of amyloidosis remains a significant clinical challenge and unmet need for patients. The amyloid-reactive peptide p5+14 radiolabeled with iodine-124 has been developed for the detection of amyloid by PET/CT imaging. In a first-in-human evaluation, the dosimetry and tissue distribution of 124 I-p5+14 peptide in patients with systemic amyloidosis. Herein, we report the dosimetry and dynamic distribution in the first three enrolled patients with light chain-associated (AL) amyloidosis., Procedures: The radiotracer was assessed in a single-site, open-label phase 1 study (NCT03678259). The first three patients received a single intravenous infusion of 124 I-p5+14 peptide (≤37 MBq). Serial PET/CT imaging was performed during the 48 h post-infusion. Dosimetry was determined as a primary endpoint for each patient and gender-averaged mean values were calculated. Pharmacokinetic parameters were estimated from whole blood radioactivity measurements and organ-based time activity data. Lastly, the biodistribution of radiotracer in major organs was assessed visually and compared to clinically appreciated organ involvement., Results: Infusion of the 124 I-p5+14 was well tolerated with rapid uptake in the heart, kidneys, liver, spleen, pancreas, and lung. The gender-averaged whole-body effective radiation dose was estimated to be 0.23 (± 0.02) mSv/MBq with elimination of the radioactivity via renal and gastrointestinal routes. The whole blood elimination t 1/2 of 21.9 ± 7.6 h. Organ-based activity concentration measurements indicated that AUC last tissue:blood ratios generally correlated with the anticipated presence of amyloid. Peptide uptake was observed in 4/5 clinically suspected organs, as noted in the medical record, as well as six anatomic sites generally associated with amyloidosis in this population., Conclusion: Peptide 124 I-p5+14 rapidly distributes to anatomic sites consistent with the presence of amyloid in patients with systemic AL. The dosimetry estimates established in this cohort are acceptable for whole-body PET/CT imaging. Pharmacokinetic parameters are heterogeneous and consistent with uptake of the tracer in an amyloid compartment. PET/CT imaging of 124 I-p5+14 may facilitate non-invasive detection of amyloid in multiple organ systems., (© 2021. World Molecular Imaging Society.)

Rationale: The function of a protein or the binding affinity of an antibody can be substantially altered by the replacement of leucine (Leu) with isoleucine (Ile), and vice versa, so the ability to identify the correct isomer using mass spectrometry can help resolve important biological questions. Tandem mass spectrometry approaches for Leu/Ile (Xle) discrimination have been developed, but they all have certain limitations., Methods: Four model peptides and two wild-type peptide sequences containing either Leu or Ile residues were subjected to charge transfer dissociation (CTD) mass spectrometry on a modified three-dimensional ion trap. The peptides were analyzed in both the 1+ and 2+ charge states, and the results were compared to conventional collision-induced dissociation spectra of the same peptides obtained using the same instrument., Results: CTD resulted in 100% sequence coverage for each of the studied peptides and provided a variety of side-chain cleavages, including d, w and v ions. Using CTD, reliable d and w ions of Xle residues were observed more than 80% of the time. When present, d ions are typically greater than 10% of the abundance of the corresponding a ions from which they derive, and w ions are typically more abundant than the z ions from which they derive., Conclusions: CTD has the benefit of being applicable to both 1+ and 2+ precursor ions, and the overall performance is comparable to that of other high-energy activation techniques like hot electron capture dissociation and UV photodissociation. CTD does not require chemical modifications of the precursor peptides, nor does it require additional levels of isolation and fragmentation., (© 2021 John Wiley & Sons Ltd.)

The ability to understand the function of a protein often relies on knowledge about its detailed structure. Sometimes, seemingly insignificant changes in the primary structure of a protein, like an amino acid substitution, can completely disrupt a protein's function. Long-lived proteins (LLPs), which can be found in critical areas of the human body, like the brain and eye, are especially susceptible to primary sequence alterations in the form of isomerization and epimerization. Because long-lived proteins do not have the corrective regeneration capabilities of most other proteins, points of isomerism and epimerization that accumulate within the proteins can severely hamper their functions and can lead to serious diseases like Alzheimer's disease, cancer and cataracts. Whereas tandem mass spectrometry (MS/MS) in the form of collision-induced dissociation (CID) generally excels at peptide characterization, MS/MS often struggles to pinpoint modifications within LLPs, especially when the differences are only isomeric or epimeric in nature. One of the most prevalent and difficult-to-identify modifications is that of aspartic acid between its four isomeric forms: L-Asp, L-isoAsp, D-Asp, and D-isoAsp. In this study, peptides containing isomers of Asp were analyzed by charge transfer dissociation (CTD) mass spectrometry to identify spectral features that could discriminate between the different isomers. For the four isomers of Asp in three model peptides, CTD produced diagnostic ions of the form c n +57 on the N-terminal side of iso-Asp residues, but not on the N-terminal side of Asp residues. Using CTD, the L- and D forms of Asp and isoAsp could also be differentiated based on the relative abundance of y - and z ions on the C-terminal side of Asp residues. Differentiation was accomplished through a chiral discrimination factor, R , which compares an ion ratio in a spectrum of one epimer or isomer to the same ion ratio in the spectrum of a different epimer or isomer. The R values obtained using CTD are as robust and statistically significant as other fragmentation techniques, like radical directed dissociation (RDD). In summary, the extent of backbone and side-chain fragments produced by CTD enabled the differentiation of isomers and epimers of Asp in a variety of peptides.

A self-assembled Fe II 4 L 6 cage was synthesized with 12 internal amines in the cavity. The cage forms as the dodeca-ammonium salt, despite the cage carrying an overall 8+ charge at the metal centers, extracting protons from displaced water in the reaction. Despite this, the basicity of the internal amines is lower than their counterparts in free solution. The 12 amines have a sliding scale of basicity, with a ≈6 pK a unit difference between the first and last protons to be removed. This moderation of side-chain basicity in an active site is a hallmark of enzymatic catalysis., (© 2022 Wiley-VCH GmbH.)

Isomerization of individual residues in long-lived proteins (LLPs) is a subject of growing interest in connection with many age-related human diseases. When isomerization occurs in LLPs, it can lead to deleterious changes in protein structure, function, and proteolytic degradation. Herein, we present a novel labeling technique for rapid identification of l-isoAsp using the enzyme protein l-isoaspartyl methyltransferase (PIMT) and Tris. The succinimide intermediate formed during reaction of l-isoAsp-containing peptides with PIMT and S -adenosyl methionine (SAM) is reactive with Tris base and results in a Tris-modified aspartic acid residue with a mass shift of +103 Da. Tris-modified aspartic acid exhibits prominent and repeated neutral loss of water when subjected to collisional activation. In addition, another dissociation pathway regenerates the original peptide following loss of a characteristic mass shift. Furthermore, it is demonstrated that Tris modification can be used to identify sites of isomerization in LLPs from biological samples such as the lens of the eye. This approach simplifies identification by labeling isomerization sites with a tag that causes a mass shift and provides characteristic loss during collisional activation.

One of the potential benefits of using data-independent acquisition (DIA) proteomics protocols is that information not originally targeted by the study may be present and discovered by subsequent analysis. Herein, we reanalyzed DIA data originally recorded for global proteomic analysis to look for isomerized peptides, which occur as a result of spontaneous chemical modifications to long-lived proteins. Examination of a large set of human brain samples revealed a striking relationship between Alzheimer's disease (AD) status and isomerization of aspartic acid in a peptide from tau. Relative to controls, a surprising increase in isomer abundance was found in both autosomal dominant and sporadic AD samples. To explore potential mechanisms that might account for these observations, quantitative analysis of proteins related to isomerization repair and autophagy was performed. Differences consistent with reduced autophagic flux in AD-related samples relative to controls were found for numerous proteins, including most notably p62, a recognized indicator of autophagic inhibition. These results suggest, but do not conclusively demonstrate, that lower autophagic flux may be strongly associated with loss of function in AD brains. This study illustrates that DIA data may contain unforeseen results of interest and may be particularly useful for pilot studies investigating new research directions. In this case, a promising target for future investigations into the therapy and prevention of AD has been identified.

Aggregation of amyloid-β (Aβ) into extracellular plaques is a well-known hallmark of Alzheimer's disease (AD). Similarly, autophagic vacuoles, autophagosomes, and other residual bodies within dystrophic neurites, though more difficult to detect, are characteristic features of AD. To explore the potential intersection between these observations, we conducted experiments to assess whether Aβ fibril formation disrupts proteolysis by lysosomal enzymes. Fibrils constituted by either Aβ 1-40 or Aβ 1-42 were grown under both neutral and acidic pH. The extent of proteolysis by individual cathepsins (L, D, B, and H) was monitored by both thioflavin T fluorescence and liquid chromatography combined with mass spectrometry. The results show that all Aβ fibril morphologies are resistant to cathepsin digestion, with significant amounts of the undigested material remaining for samples grown in either neutral or acidic pH. Further analysis revealed that the neutral-grown fibrils are proteolytically resistant throughout the sequence, while the acid-grown fibrils prevented digestion primarily in the C-terminal portion of the sequence. Fibrils grown from Aβ 1-42 are generally more resistant to degradation compared to Aβ 1-40. Overall, the results indicate that Aβ fibrils formed in the neutral pH environments found in intracellular or extracellular spaces may pose the greatest difficulty for complete digestion by the lysosome, particularly when the fibrils are comprised of Aβ 1-42., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Top-down mass spectrometry (TD-MS) of intact proteins results in fragment ions that can be correlated to the protein primary sequence. Fragments generated can either be terminal fragments that contain the N- or C-terminus or internal fragments that contain neither termini. Traditionally in TD-MS experiments, the generation of internal fragments has been avoided because of ambiguity in assigning these fragments. Here, we demonstrate that in TD-MS experiments internal fragments can be formed and assigned in collision-based, electron-based, and photon-based fragmentation methods and are rich with sequence information, allowing for a greater extent of the primary protein sequence to be explained. For the three test proteins cytochrome c , myoglobin, and carbonic anhydrase II, the inclusion of internal fragments in the analysis resulted in approximately 15-20% more sequence coverage, with no less than 85% sequence coverage obtained. Combining terminal fragment and internal fragment assignments results in near complete protein sequence coverage. Hence, by including both terminal and internal fragment assignments in TD-MS analysis, deep protein sequence analysis, allowing for the localization of modification sites more reliably, can be possible.

Cathepsin B is an important protease within the lysosome, where it helps recycle proteins to maintain proteostasis. It is also known to degrade proteins elsewhere but has no other known functionality. However, by carefully monitoring peptide digestion with liquid chromatography and mass spectrometry, we observed the synthesis of novel peptides during cathepsin B incubations. This ligation activity was explored further with a variety of peptide substrates to establish mechanistic details and was found to operate through a two-step mechanism with proteolysis and ligation occurring separately. Further explorations using varied sequences indicated increased affinity for some substrates, though all were found to ligate to some extent. Finally, experiments with a proteolytically inactive form of the enzyme yielded no ligation, indicating that the ligation reaction occurs in the same active site but in the reverse direction of proteolysis. These results clearly establish that in its native form cathepsin B can act as both a protease and ligase, although protease action eventually dominates over longer periods of time., Competing Interests: Conflicts of interest The authors declare no conflicts of interest in regard to this manuscript.

Solvochromatic effects are most frequently associated with solution-phase phenomena. However, in the gas phase, the absence of solvent leads to intramolecular solvation that can be driven by strong forces including hydrogen bonds and ion-dipole interactions. Here we examine whether isomerization of a single residue in a peptide results in structural changes sufficient to shift the absorption of light by an appended chromophore. By carrying out the experiments inside a mass spectrometer, we can easily monitor photodissociation yield as a readout for chromophore excitation. A series of peptides of different lengths, charge states, and position and identity of the isomerized residue were examined by excitation with both 266 and 213 nm light. The results reveal that differences in intramolecular solvation do lead to solvochromatic shifts in many cases. In addition, the primary product following photoexcitation is a radical. Ion-molecule reactions with this radical and adventitious oxygen were monitored and also found to vary as a function of isomeric state. In this case, differences in intramolecular solvation alter the availability of the reactive radical. Overall, the results reveal that small changes in a single amino acid can influence the overall structural ensemble sufficient to alter the efficiency of multiple gas-phase reactions.

Although most peptide bonds in proteins exist in the trans configuration, when cis peptide bonds do occur, they can have major impact on protein structure and function. The rapid identification of cis peptide bonds is therefore an important task. Peptide bonds containing proline are more likely to adopt the cis configuration because the ring connecting the side chain and backbone in proline flattens the energetic landscape relative to amino acids with free side chains. Examples of cis proline isomers have been identified in both solution and in the gas phase by a variety of structure-probing methods. Mass spectrometry is an attractive potential method for identifying cis proline due to its speed and sensitivity; however, the question remains of whether cis/trans proline isomers originating in solution are preserved during ionization and manipulation within a mass spectrometer. Herein, we investigate the gas-phase stability of isolated solution-phase cis and trans proline isomers using a synthetic peptide sequence with a Tyr-Pro-Pro motif. A variety of dissociation methods were explored to evaluate their potential to distinguish cis/trans configuration, including collision-induced dissociation, radical-directed dissociation, and photodissociation. Only photodissociation employed in conjunction with extremely gentle electrospray and charge solvation by 18-crown-6 ether was able to distinguish cis/trans isomers for our model peptide, suggesting that any thermal activation during transfer or while in the gas phase leads to isomer scrambling. Furthermore, the necessity for 18-crown-6 suggests that intramolecular charge solvation taking place during electrospray ionization can override cis/trans isomer homogeneity. Overall, the results suggest that solution-phase cis/trans proline isomers are fragile and easily lost during electrospray, requiring careful selection of instrument parameters and consideration of charge solvation to prevent cis/trans scrambling.

Recent studies have illuminated connections between spontaneous chemical reactions that cause isomerization at specific protein residues and various age-related diseases including cataracts and Alzheimer's. These discoveries provide impetus for better analytical methods to detect and characterize isomerization in proteins, which will enable a more complete understanding of the underlying relationship between these modifications and biology. Herein we employ a two-dimensional approach for identification of peptides isomers that also includes pinpointing of the modified residue. Collision-induced dissociation is used to fragment ions in the first dimension, followed by separation of the fragments with travelling-wave ion mobility. By comparing data obtained from both isomers, differences in either fragment-ion intensities or arrival-time distributions can be used to identify isomeric forms and the specific site of modification within the peptides. Synthetic peptide standards with sequences derived from long-lived proteins in the eye lens and isomerization at serine, aspartic acid, and glutamic acid were examined. Although both dimensions are capable of isomer identification, ion mobility is much better at determining the site of modification. In general, separation of isomeric forms by ion mobility is possible but does not follow predictable trends dictated by sequence or fragment-ion length. In most cases, however, the site of isomerization can be precisely determined.

Spontaneous chemical modifications play an important role in human disease and aging at the molecular level. Deamidation and isomerization are known to be among the most prevalent chemical modifications in long-lived human proteins and are implicated in a growing list of human pathologies, but the relatively minor chemical change associated with these processes has presented a long standing analytical challenge. Although the adoption of high-resolution mass spectrometry has greatly aided the identification of deamidation sites in proteomic studies, isomerization (and the isomeric products of deamidation) remain exceptionally challenging to characterize. Herein, we present a liquid chromatography/mass spectrometry-based approach for rapidly characterizing the isomeric products of Gln deamidation using diagnostic fragments that are abundantly produced and capable of unambiguously identifying both Glu and isoGlu. Importantly, the informative fragment ions are produced through orthogonal fragmentation pathways, thereby enabling the simultaneous detection of both isomeric forms while retaining compatibility with shotgun proteomics. Furthermore, the diagnostic fragments associated with isoGlu pinpoint the location of the modified residue. The utility of this technique is demonstrated by characterizing the isomeric products generated during in vitro aging of a series of glutamine-containing peptides. Sequence-dependent product profiles are obtained, and the abundance of deamidation-linked racemization is examined. Finally, comparisons are made between Gln deamidation, which is relatively poorly understood, and asparagine deamidation, which has been more thoroughly studied.

Mass spectrometry affords rapid and sensitive analysis of peptides and proteins. Coupling spectroscopy with mass spectrometry allows for the development of new methods to enhance biomolecular structure determination. Herein, we demonstrate two new energy acceptors that can be utilized for action-excitation energy transfer experiments. In the first system, C-S bonds in methionine act as energy acceptors from native chromophores, including tyrosine, tryptophan, and phenylalanine. Comparison among chromophores reveals that tyrosine transfers energy most efficiently at 266 nm, but phenylalanine and tryptophan also transfer energy with comparable efficiencies. Overall, the C-S bond dissociation yields following energy transfer are low for methionine, which led to an investigation of selenomethionine, a common analog that is found in many naturally occurring proteins. Sulfur and selenium are chemically similar, but C-Se bonds are weaker than C-S bonds and have lower lying σ* anti-bonding orbitals. Excitation of peptides containing tyrosine and tryptophan results in efficient energy transfer to selenomethionine and abundant C-Se bond dissociation. A series of helical peptides were examined where the positions of the donor or acceptor were systematically scanned to explore the influence of distance and helix orientation on energy transfer. The distance was found to be the primary factor affecting energy transfer efficiency, suggesting that selenomethionine may be a useful acceptor for probing protein structure in the gas phase.

Proteinaceous aggregation is a well-known observable in Alzheimer's disease (AD), but failure and storage of lysosomal bodies within neurons is equally ubiquitous and actually precedes bulk accumulation of extracellular amyloid plaque. In fact, AD shares many similarities with certain lysosomal storage disorders though establishing a biochemical connection has proven difficult. Herein, we demonstrate that isomerization and epimerization, which are spontaneous chemical modifications that occur in long-lived proteins, prevent digestion by the proteases in the lysosome (namely, the cathepsins). For example, isomerization of aspartic acid into l-isoAsp prevents digestion of the N-terminal portion of Aβ by cathepsin L, one of the most aggressive lysosomal proteases. Similar results were obtained after examination of various target peptides with a full series of cathepsins, including endo-, amino-, and carboxy-peptidases. In all cases peptide fragments too long for transporter recognition or release from the lysosome persisted after treatment, providing a mechanism for eventual lysosomal storage and bridging the gap between AD and lysosomal storage disorders. Additional experiments with microglial cells confirmed that isomerization disrupts proteolysis in active lysosomes. These results are easily rationalized in terms of protease active sites, which are engineered to precisely orient the peptide backbone and cannot accommodate the backbone shift caused by isoaspartic acid or side chain dislocation resulting from epimerization. Although Aβ is known to be isomerized and epimerized in plaques present in AD brains, we further establish that the rates of modification for aspartic acid in positions 1 and 7 are fast and could accrue prior to plaque formation. Spontaneous chemistry can therefore provide modified substrates capable of inducing gradual lysosomal failure, which may play an important role in the cascade of events leading to the disrupted proteostasis, amyloid formation, and tauopathies associated with AD., Competing Interests: The authors declare no competing financial interest.

Global salinization trends present an urgent need for methods to monitor aquatic ecosystem health and characterize known and emerging stressors for water bodies that are becoming increasingly saline. Environmental metabolomics methods that combine quantitative measurements of metabolite levels and multivariate statistical analysis are powerful tools for ascertaining biological impacts and identifying potential biomarkers of exposure. We propose the use of the saltwater aquatic crustacean, Artemia franciscana, as a model organism for environmental metabolomics in saltwater ecosystems. Artemia are a good choice for ecotoxicity assays and metabolomics analysis because they have a short life cycle, their hemolymph is rich in metabolites and they tolerate a wide salinity range. In this work we explore the potential of Artemia franciscana for environmental metabolomics through exposure to the broad-spectrum herbicide, glyphosate. The LC 50 for a 48 h exposure of Roundup ® was determined to be 237 ± 23 ppm glyphosate in the Roundup ® formulation. Artemia cysts were hatched and exposed to sub-lethal glyphosate concentrations of 1.00, 10.0, 50.0, or 100 ppm glyphosate in Roundup ® . We profiled 48 h old Artemia extracts using 1 H NMR and GC-MS. Dose-dependent metabolic perturbation was evident for several metabolites using univariate and multivariate analyses. Metabolites significantly affected by Roundup ® exposure included aspartate, formate, betaine, glucose, tyrosine, phenylalanine, gadusol, and isopropylamine. Biochemical pathway analysis with the KEGG database suggests impairment of carbohydrate and energy metabolism, folate-mediated one-carbon metabolism, Artemia molting and development, and microbial metabolism., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Photoinitiated radical chemistry has proven to be useful for breaking covalent bonds within many biomolecules in the gas phase. Herein, we demonstrate that radical chemistry is useful for bond synthesis in the gas phase. Single peptides containing two cysteine residues capped with propylmercaptan (PM) often form disulfide bonds following ultraviolet excitation at 266 nm and loss of both PM groups. Similarly, noncovalently bound peptide pairs where each peptide contains a single cysteine residue can be induced to form disulfide bonds. Comparison with disulfide bound species sampled directly from solution yields identical collisional activation spectra, suggesting that native disulfide bonds have been recapitulated in the gas phase syntheses. Another approach utilizing radical chemistry for covalent bond synthesis involves creation of a reactive diradical that can first abstract hydrogen from a target peptide, creating a new radical site, and then recombine the second radical with the new radical to form a covalent bond. This chemistry is illustrated with 2-(hydroxymethyl-3,5-diiodobenzoate)-18-crown-6 ether, which attaches noncovalently to protonated primary amines in peptides and proteins. Following photoactivation and crosslinking, the site of noncovalent adduct attachment can frequently be determined. The ramifications of these observations on peptide structure and noncovalent attachment of 18-crown-6-based molecules is discussed.

Long-lived proteins are subject to spontaneous degradation and may accumulate a range of modifications over time, including subtle alterations such as side-chain isomerization. Recently, tandem MS has enabled identification and characterization of such peptide isomers, including those differing only in chirality. However, the structural and functional consequences of these perturbations remain largely unexplored. Here, we examined the impact of isomerization of aspartic acid or epimerization of serine at four sites mapping to crucial oligomeric interfaces in human αA- and αB-crystallin, the most abundant chaperone proteins in the eye lens. To characterize the effect of isomerization on quaternary assembly, we utilized synthetic peptide mimics, enzyme assays, molecular dynamics calculations, and native MS experiments. The oligomerization of recombinant forms of αA- and αB-crystallin that mimic isomerized residues deviated from native behavior in all cases. Isomerization also perturbs recognition of peptide substrates, either enhancing or inhibiting kinase activity. Specifically, epimerization of serine (αASer-162) dramatically weakened inter-subunit binding. Furthermore, phosphorylation of αBSer-59, known to play an important regulatory role in oligomerization, was severely inhibited by serine epimerization and altered by isomerization of nearby αBAsp-62. Similarly, isomerization of αBAsp-109 disrupted a vital salt bridge with αBArg-120, a contact that when broken has previously been shown to yield aberrant oligomerization and aggregation in several disease-associated variants. Our results illustrate how isomerization of amino acid residues, which may seem to be only a minor structural perturbation, can disrupt native structural interactions with profound consequences for protein assembly and activity., (© 2019 Lyon et al.)

Isomerization and epimerization are spontaneously occurring modifications that represent common pathways for age-related damage in proteins. These modifications accumulate over time and are known to disrupt both structure and function, making their detection and identification an important goal in proteomic characterization. Unfortunately, because these modifications lead to no change in mass, they are typically overlooked in traditional proteomics. Herein we present protocols for mass-spectrometric identification and quantification of protein isomerization and epimerization. Illustrative examples are provided for analysis of crystallin proteins extracted from the eye lens. Crystallins provide an attractive model system for studying the age-related modifications of long-lived proteins due to the lack of protein turnover in the eye lens. We first outline procedures for tissue processing and protein extraction. We then describe methods for data acquisition and analysis for differentiation and identification of isomerized residues, including methods for quantifying isomers which cannot be chromatographically resolved., (© 2019 Elsevier Inc. All rights reserved.)

Small changes in steric bulk at the terminus of bis-iminopyridine ligands can effect large changes in the spin state of self-assembled Fe(II)-iminopyridine cage complexes. If the added bulk is properly matched with ligands that are either sufficiently flexible to allow twisted octahedral geometries at the Fe centers or can assemble with unusual mer configurations at the metals, room temperature high spin Fe(II) cages can be synthesized. These complexes maintain their high spin state in solution at low temperatures and have been characterized by X-ray crystallographic and computational methods. The high spin M 2 L 3 meso-helicate and M 4 L 6 cage complexes display longer N-Fe bond distances and larger interligand N-Fe-N bond angles than their diamagnetic counterparts, and these structural changes invert the ligand selectivity in narcissistic self-sorting and accelerate subcomponent exchange rates. The paramagnetic cages can be easily converted to diamagnetic cages by subcomponent exchange under mild conditions, and the intermediates of the exchange process can be visualized in situ by NMR analysis.

Disulfide heterogeneity and other non-native crosslinks introduced during therapeutic antibody production and storage could have considerable negative effects on clinical efficacy, but tracking these modifications remains challenging. Analysis must also be carried out cautiously to avoid introduction of disulfide scrambling or reduction, necessitating the use of low pH digestion with less specific proteases. Herein we demonstrate that 213 nm ultraviolet photodissociation streamlines disulfide elucidation through bond-selective dissociation of sulfur-sulfur and carbon-sulfur bonds in combination with less specific backbone dissociation. Importantly, both types of fragmentation can be initiated in a single MS/MS activation stage. In addition to disulfide mapping, it is also shown that thioethers and trisulfides can be identified by characteristic fragmentation patterns. The photochemistry resulting from 213 nm excitation facilitates a simplified, two-tiered data processing approach that allows observation of all native disulfide bonds, scrambled disulfide bonds, and non-native sulfur-based linkages in a pepsin digest of Rituximab. Native disulfides represented the majority of bonds according to ion count, but the highly solvent-exposed heavy/light interchain disulfides were found to be most prone to modification. Production and storage methods that facilitate non-native links are discussed. Due to the importance of heavy and light chain connectivity for antibody structure and function, this region likely requires particular attention in terms of its influence on maintaining structural fidelity.

Glycans are fundamental biological macromolecules, yet despite their prevalence and recognized importance, a number of unique challenges hinder routine characterization. The multiplicity of OH groups in glycan monomers easily afford branched structures and alternate linkage sites, which can result in isomeric structures that differ by minute details. Herein, radical chemistry is employed in conjunction with mass spectrometry to enable rapid, accurate, and high throughput identification of a challenging series of closely related glycan isomers. The results are compared with analysis by collision-induced dissociation, higher-energy collisional dissociation, and ultraviolet photodissociation (UVPD) at 213 nm. In general, collision-based activation struggles to produce characteristic fragmentation patterns, while UVPD and radical-directed dissociation (RDD) can distinguish all isomers. In the case of RDD, structural differentiation derives from radical mobility and subsequent fragmentation. For glycans, the energetic landscape for radical migration is flat, increasing the importance of the three-dimensional structure. RDD is therefore a powerful and straightforward method for characterizing glycan isomers.

Ultraviolet photodissociation or UVPD is an increasingly popular option for tandem-mass spectrometry experiments. UVPD can be carried out at many wavelengths, and it is important to understand how the results will be impacted by this choice. Here, we explore the utility of 213 nm photons for initiating bond-selective fragmentation. It is found that bonds previously determined to be labile at 266 nm, including carbon-iodine and sulfur-sulfur bonds, can also be cleaved with high selectivity at 213 nm. In addition, many carbon-sulfur bonds that are not subject to direct dissociation at 266 nm can be selectively fragmented at 213 nm. This capability can be used to site-specifically create alaninyl radicals that direct backbone dissociation at the radical site, creating diagnostic d-ions. Furthermore, the additional carbon-sulfur bond fragmentation capability leads to signature triplets for fragmentation of disulfide bonds. Absorption of amide bonds can enhance dissociation of nearby labile carbon-sulfur bonds and can be used for stochastic backbone fragmentation typical of UVPD experiments at shorter wavelengths. Several potential applications of the bond-selective fragmentation chemistry observed at 213 nm are discussed. Graphical Abstract ᅟ.

Self-assembly of a carboxylic acid-containing ligand into an Fe 4 L 6 iminopyridine cage allows endohedral positioning of the acid groups while maintaining a robust cage structure. The cage is an effective supramolecular catalyst, providing up to 1000-fold rate enhancement of acetal solvolysis. This enhanced reactivity allows a tandem deprotection/cage-to-cage interconversion that cannot be achieved with other acid catalysts. The combination of rate enhancements and sequestration of the reactive function confers both activity and selectivity on the process, mimicking enzymatic behavior.

Although it is well-known that protein turnover essentially stops in mature lens fiber cells, mapping out the ensuing protein degradation and its effects on lens function over time remains challenging. In particular, isomerization is a common, spontaneous post-translational modification that occurs over long timescales and generates products invisible to most analytical methods. Nevertheless, isomerization can significantly impact protein structure, function, and solubility, which are all necessary to maintain clarity and proper refractive index within the lens. Herein, we examine the degree of isomerization occurring in crystallin proteins in the human eye lens as a function of both age and location within the lens. A novel mass spectrometric technique leveraging radical chemistry enables detailed characterization of proteins extracted from the cortex and nucleus of the lens. It is observed that the degree of isomerization increases significantly between the cortex and nucleus and between water-soluble and water-insoluble fractions. Interestingly, the abundance of L-isoAsp is low in the water-soluble cortex despite being the dominant product generated by isomerization of Asp in vitro, suggesting that Protein L-isoaspartyl methyltransferase (PIMT) is active in the cortex and suppresses the accumulation of L-isoAsp. The abundance of L-isoAsp increases dramatically in the nucleus, revealing that PIMT activity decreases over time in the center of the lens. In addition, the growth of L-isoAsp in the nuclear fraction suggests protein isomerization continues within the nucleus, despite the fact that most of the protein within the nucleus has become insoluble. Additionally, it is demonstrated that sequential Asp residues lead to isomerization hotspots in human crystallin proteins and that the isomerization profiles for αA and αB crystallin are notably different. Although αA is more prone to isomerization, αB loses solubility more rapidly upon modification. These differences are likely related to the distribution of Asp residues within αA and αB, which are in turn connected to refractive index. The high Asp content of αA is a hazard in terms of isomerization and aging, but it serves to enhance the refractive index of αA relative to αB, and may explain why αA is only found in the eye., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A strained, "springloaded" Fe 2 L 3 iminopyridine mesocate shows highly variable reactivity upon postassembly reaction with competitive diamines. The strained assembly is reactive toward transimination in minutes at ambient temperature and allows observation of kinetically trapped intermediates in the self-assembly pathway. When diamines are used that can only form less favored cage products upon full equilibration, trapped ML 3 fragments with pendant, "hanging" NH 2 groups are selectively formed instead. Slight variations in diamine structure have large effects on the product outcome: less rigid diamines convert the mesocate to more favored self-assembled cage complexes under mild conditions and allow observation of heterocomplex intermediates in the displacement pathway. The mesocate allows control of equilibrium processes and direction of product outcomes via small, iterative changes in added subcomponent structure and provides a method of accessing metal-ligand cage structures not normally observed in multicomponent Fe-iminopyridine self-assembly.

Analysis of whole proteins by mass spectrometry, or top-down proteomics, has several advantages over methods relying on proteolysis. For example, proteoforms can be unambiguously identified and examined. However, from a gas-phase ion-chemistry perspective, proteins are enormous molecules that present novel challenges relative to peptide analysis. Herein, the statistics of cleaving the peptide backbone multiple times are examined to evaluate the inherent propensity for generating internal versus terminal ions. The raw statistics reveal an inherent bias favoring production of terminal ions, which holds true regardless of protein size. Importantly, even if the full suite of internal ions is generated by statistical dissociation, terminal ions are predicted to account for at least 50% of the total ion current, regardless of protein size, if there are three backbone dissociations or fewer. Top-down analysis should therefore be a viable approach for examining proteins of significant size. Comparison of the purely statistical analysis with actual top-down data derived from ultraviolet photodissociation (UVPD) and higher-energy collisional dissociation (HCD) reveals that terminal ions account for much of the total ion current in both experiments. Terminal ion production is more favored in UVPD relative to HCD, which is likely due to differences in the mechanisms controlling fragmentation. Importantly, internal ions are not found to dominate from either the theoretical or experimental point of view. Graphical abstract ᅟ.

Deamidation of asparagine is a spontaneous and irreversible post-translational modification associated with a growing list of human diseases. While pervasive, deamidation is often overlooked because it represents a relatively minor chemical change. Structural and functional characterization of this modification is complicated because deamidation of asparagine yields four isomeric forms of Asp. Herein, radical directed dissociation (RDD), in conjunction with mass spectrometry, is used to identify and quantify all four isomers in a series of model peptides that were subjected to various deamidation conditions. Although primary sequence significantly influences the rate of deamidation, it has little impact on the relative proportions of the product isomers. Furthermore, the addition of ammonia can be used to increase the rate of deamidation without significantly perturbing isomer populations. Conversely, external factors such as buffer conditions and temperature alter product distributions but exhibit less dramatic effects on the deamidation rate. Strikingly, the common laboratory and biologically significant bicarbonate buffer is found to strongly promote racemization, yielding increased amounts of d-Asp and d-isoAsp. These outcomes following deamidation have broad implications in human aging and should be considered during the development of protein-based therapeutics.

Self-assembled Fe-iminopyridine cage complexes containing doubly benzylic methylene units such as fluorene and xanthene can be selectively oxidized at the ligand backbone with t BuOOH, with no competitive oxidation observed at the metal centers. The self-assembled cage structure controls the reaction outcome, yielding oxidation products that are favored by the assembly, not by the reactants or functional groups. Whereas uncomplexed xanthene and fluorene control ligands are solely oxidized to the ketone equivalents with t BuOOH, the unfavorability of the self-assembled ketone cages forces the reaction to form the t butyl peroxide and alcohol-containing oxidation products, respectively. In addition, the oxidation is diastereoselective, with only single isomers of the cage assemblies formed, despite the presence of as many as 10 stereocenters in the final product. The self-assembled structures exploit self-complementary hydrogen bonding and geometrical constraints to direct the postassembly reactions to outcomes not observed in free solution. This selectivity is reminiscent of the fine control of post-translational modification seen in biomacromolecules.

Structural characterization of proteins in the gas phase is becoming increasingly popular, highlighting the need for a greater understanding of how proteins behave in the absence of solvent. It is clear that charged residues exert significant influence over structures in the gas phase due to strong Coulombic and hydrogen-bonding interactions. The net charge for a gaseous ion is easily identified by mass spectrometry, but the presence of zwitterionic pairs or salt bridges has previously been more difficult to detect. We show that these sites can be revealed by photoinduced electron transfer dissociation, which produces characteristic c and z ions only if zwitterionic species are present. Although previous work on small molecules has shown that zwitterionic pairs are rarely stable in the gas phase, we now demonstrate that charge-separated states are favored in larger molecules. Indeed, we have detected zwitterionic pairs in peptides and proteins where the net charge equals the number of basic sites, requiring additional protonation at nonbasic residues. For example, the small protein ubiquitin can sustain a zwitterionic conformer for all charge states up to 14+, despite having only 13 basic sites. Virtually all of the peptides/proteins examined herein contain zwitterionic sites if both acidic and basic residues are present and the overall charge density is low. This bias in favor of charge-separated states has important consequences for efforts to model gaseous proteins via computational analysis, which should consider not only charge state isomers that include salt bridges but also protonation at nonbasic residues.

Traditional electron-transfer dissociation (ETD) experiments operate through a complex combination of hydrogen abundant and hydrogen deficient fragmentation pathways, yielding c and z ions, side-chain losses, and disulfide bond scission. Herein, a novel dissociation pathway is reported, yielding homolytic cleavage of carbon-iodine bonds via electronic excitation. This observation is very similar to photodissociation experiments where homolytic cleavage of carbon-iodine bonds has been utilized previously, but ETD activation can be performed without addition of a laser to the mass spectrometer. Both loss of iodine and loss of hydrogen iodide are observed, with the abundance of the latter product being greatly enhanced for some peptides after additional collisional activation. These observations suggest a novel ETD fragmentation pathway involving temporary storage of the electron in a charge-reduced arginine side chain. Subsequent collisional activation of the peptide radical produced by loss of HI yields spectra dominated by radical-directed dissociation, which can be usefully employed for identification of peptide isomers, including epimers. Graphical Abstract ᅟ.

The eye lens crystallins represent an ideal target for studying the effects of aging on protein structure. Herein we examine separately the water-soluble (WS) and water-insoluble (WI) crystallin fractions and identify sites of isomerization and epimerization. Both collision-induced dissociation and radical-directed dissociation are needed for detection of these non-mass-shifting post-translational modifications. Isomerization levels differ significantly between the WS and the WI fractions from sheep, pig, and cow eye lenses. Residues that are most susceptible to isomerization are identified site-specifically and are found to reside in structurally disordered regions. However, isomerization in structured domains, although less common, often yields more dramatic effects on solubility. Numerous isomerization hotspots were also identified and occur in regions with aspartic acid and serine repeats. For example, 128 KMEIVDDDVPSLW 140 in βB3 crystallin contains three sequential aspartic acid residues and is isomerized heavily in the WI fractions, while it is not modified at all in the WS fractions. Potential causes for enhanced isomerization at sites with acidic residue repeats are presented. The importance of acidic residue repeats extends beyond the lens, as they are found in many other long-lived proteins associated with disease.

Small changes in the electron donating ability of coordinating groups have substantial effects on the multicomponent self-assembly of Fe (II)-iminopyridine-based meso-helicate complexes. Both the nature of the internal diamine core and the terminal formylpyridine reactants control the rate of the assembly process, the thermodynamic favorability of the meso-helicate products, and the selective incorporation of different aldehyde termini into the assembly. Steric congestion at the coordinating ligands can prevent assembly altogether, and favorable incorporation of electron-rich aldehyde termini is observed, even though the rate of reaction is accelerated by the use of electron-poor aldehyde reactants. NMR and electrospray ionization mass spectrometry analyses were employed to determine the synergistic nature of narcissistic self-sorting in this system, which depends on both the rigidity of the central core and the electronic donor ability of the aldehyde terminus. These experiments illustrate that significant control of self-sorting and self-assembly is possible upon extremely small variations in ligand structure, rigidity, and donor ability.

Significant effort is being employed to utilize the inherent speed and sensitivity of mass spectrometry for rapid structural determination of proteins; however, a thorough understanding of factors influencing the transition from solution to gas phase is critical for correct interpretation of the results from such experiments. It was previously shown that combined use of action excitation energy transfer (EET) and simulated annealing can reveal detailed structural information about gaseous peptide ions. Herein, we utilize this method to study microsolvation of charged groups by retention of 18-crown-6 (18C6) in the gas phase. In the case of GTP (CEGNVRVSRE LAGHTGY), solvation of the 2+ charge state leads to reduced EET, whereas the opposite result is obtained for the 3+ ion. For the mini-protein C-Trpcage, solvation by 18C6 leads to dramatic increase in EET for the 3+ ion. Examination of structural details probed by molecular dynamics calculations illustrate that solvation by 18C6 alleviates the tendency of charged side chains to seek intramolecular solvation, potentially preserving native-like structures in the gas phase. These results suggest that microsolvation may be an important tool for facilitating examination of native-like protein structures in gas phase experiments. Graphical Abstract ᅟ.

Recent advances in mass spectrometry and lasers have facilitated the development of novel experiments combining the benefits of both technologies. This minireview focuses on the coupling of visible/ultraviolet photons with mass spectrometry for analysis of peptide and protein three-dimensional structure. Practical aspects of instrument design and the relationship between experiment and theory are discussed. Experiments utilizing spectroscopy, action spectroscopy, excitation energy transfer, photodissociation, and photoactivated radical chemistry are described. The strengths and weaknesses of each approach are discussed in relation to the type of information typically obtained. A significant body of data suggests that under appropriate source conditions, kinetically trapped structures are observed in these experiments rather than true gas phase minima, suggesting retention of solution phase structural features is possible. Further refinement and exploration of these methods promises to accelerate protein structure discovery in the near future.