Statistical Framework for Identifying Differences in Similar Mass Spectra: Expanding Possibilities for Isomer Identification.

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.

Analysis of Glutamine Deamidation: Products, Pathways, and Kinetics.

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.

Evaluating sub-lethal stress from Roundup® exposure in Artemia franciscana using 1H NMR and GC-MS.

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 LC50 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 1H 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.

Structural and functional consequences of age-related isomerization in α-crystallins.

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.

Small Structural Variations Have Large Effects on the Assembly Properties and Spin State of Room Temperature High Spin Fe(II) Iminopyridine Cages.

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 M2L3 meso-helicate and M4L6 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.

Tandem Reactivity of a Self-Assembled Cage Catalyst with Endohedral Acid Groups.

Self-assembly of a carboxylic acid-containing ligand into an Fe4L6 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.

A Springloaded Metal-Ligand Mesocate Allows Access to Trapped Intermediates of Self-Assembly.

A strained, "springloaded" Fe2L3 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 ML3 fragments with pendant, "hanging" NH2 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.

Differences in α-Crystallin isomerization reveal the activity of protein isoaspartyl methyltransferase (PIMT) in the nucleus and cortex of human lenses.

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.

The Ups and Downs of Repeated Cleavage and Internal Fragment Production in Top-Down Proteomics.

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.

Stereoselective Postassembly CH Oxidation of Self-Assembled Metal-Ligand Cage Complexes.

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 tBuOOH, 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 tBuOOH, the unfavorability of the self-assembled ketone cages forces the reaction to form the tbutyl 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.

Photoelectron Transfer Dissociation Reveals Surprising Favorability of Zwitterionic States in Large Gaseous Peptides and Proteins.

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.

Leveraging Electron Transfer Dissociation for Site Selective Radical Generation: Applications for Peptide Epimer Analysis.

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.

Identification of Sequence Similarities among Isomerization Hotspots in Crystallin Proteins.

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, 128KMEIVDDDVPSLW140 in ßB3 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.

Electronic Effects on Narcissistic Self-Sorting in Multicomponent Self-Assembly of Fe-Iminopyridine meso-Helicates.

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.

Photolytic determination of charge state for large proteins and fragments in an ion trap mass spectrometer.

RATIONALE: One of the major shortcomings of linear ion trap mass spectrometers is poor resolution. Failure to resolve isotopic peaks makes charge state determination for large proteins very difficult, hindering the ability to perform top-down proteomics. METHODS: Peptides, proteins and corresponding fragments modified with para-iodobenzoate were trapped and irradiated with 266 nm light from an Nd:YAG laser. Loss of iodine due to photodissociation was then used to assign charge states by measuring the corresponding m/z shifts. RESULTS: Initial experiments on small peptides illustrate the feasibility of the method. Further studies performed on larger proteins in higher charge states yielded similar results, revealing that fragment ions over a significant mass range either remain in or are quickly cooled to the laser overlap region of the ion trap. CONCLUSIONS: Rapid charge state assignment for both whole molecules and collision-induced dissociation (CID) fragments can be obtained by photoactivation of chromophores with labile carbon-iodine bonds.

Narcissistic self-sorting in self-assembled cages of rare Earth metals and rigid ligands.

Highly selective, narcissistic self-sorting can be achieved in the formation of self-assembled cages of rare earth metals with multianionic salicylhydrazone ligands. The assembly process is highly sensitive to the length of the ligand and the coordination geometry. Most surprisingly, high-fidelity sorting is possible between ligands of identical coordination angle and geometry, differing only in a single functional group on the ligand core, which is not involved in the coordination. Supramolecular effects allow discrimination between pendant functions as similar as carbonyl or methylene groups in a complex assembly process.