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.

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

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.

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 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.

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.

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.

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.

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.