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.

Gated, Selective Anion Exchange in Functionalized Self-Assembled Cage Complexes.

Appending functional groups to the exterior of Zn4 L4 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 PF6 - and SbF6 - can vary by as much as 400-fold between identically sized cavities.

Catalytic Inhibition of Base-Mediated Reactivity by a Self-Assembled Metal-Ligand Host.

Spacious M4 L6 tetrahedra can act as catalytic inhibitors for base-mediated reactions. Upon adding only 5 % of a self-assembled Fe4 L6 cage complex, the conversion of the conjugate addition between ethylcyanoacetate and ß-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 Fe4 L6 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 Fe4 L6 hosts with spacious internal cavities: minimal inhibition is seen with smaller tetrahedra or Fe2 L3 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.

Decoding the Fucose Migration Product during Mass-Spectrometric analysis of Blood Group Epitopes.

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.

Does Data-Independent Acquisition Data Contain Hidden Gems? A Case Study Related to Alzheimer's Disease.

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.

Influence of Asp Isomerization on Trypsin and Trypsin-like Proteolysis.

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.

Differentiation of leucine and isoleucine residues in peptides using charge transfer dissociation mass spectrometry (CTD-MS).

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.

Differentiating aspartic acid isomers and epimers with charge transfer dissociation mass spectrometry (CTD-MS).

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 cn+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.

Moderated Basicity of Endohedral Amine Groups in an Octa-Cationic Self-Assembled Cage.

A self-assembled FeII4 L6 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 pKa 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.

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.

Two-dimensional identification and localization of isomers in crystallin peptides using TWIM-MS.

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.

Differentiation of peptide isomers by excited-state photodissociation and ion-molecule interactions.

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.

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.

Synthesis of New S-S and C-C Bonds by Photoinitiated Radical Recombination Reactions in the Gas Phase.

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.

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.

Glycan Isomer Identification Using Ultraviolet Photodissociation Initiated Radical Chemistry.

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.

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.

Simplified identification of disulfide, trisulfide, and thioether pairs with 213 nm UVPD.

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.

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.

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.