Localizing Isomerized Residue Sites in Peptides with Tandem Mass Spectrometry.

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 MS2 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 MS3 experiments to further evaluate each fragment ion from the MS2 stage. Comparison at the MS3 level, utilizing statistical analyses, reveals which MS2 fragments differ between samples and, therefore, must contain the isomerized sites. The approach is similar to previous work relying on ion mobility to discriminate MS2 product ions by collision cross-section. The MS3 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.

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.

Fragment Ion Abundance Reveals Information about Structure and Charge Localization in Highly Charged Proteins.

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.

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.

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.

RDD-HCD Provides Variable Fragmentation Routes Dictated by Radical Stability.

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.

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.

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.

Modifying the internal substituents of self-assembled cages controls their molecular recognition and optical properties.

Self-assembled Fe4L6 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 106 M-1 in CH3CN 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.

PIMT-Mediated Labeling of l-Isoaspartic Acid with Tris Facilitates Identification of Isomerization Sites in Long-Lived Proteins.

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.

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.

LC-MS Reveals Isomeric Inhibition of Proteolysis by Lysosomal Cathepsins.

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.

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.

First in Human Evaluation and Dosimetry Calculations for Peptide 124I-p5+14-a Novel Radiotracer for the Detection of Systemic Amyloidosis Using PET/CT Imaging.

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 124I-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 124I-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 124I-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 t1/2 of 21.9 ± 7.6 h. Organ-based activity concentration measurements indicated that AUClast 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 124I-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 124I-p5+14 may facilitate non-invasive detection of amyloid in multiple organ systems.

Efficient Isothiocyanate Modification of Peptides Facilitates Structural Analysis by Radical-Directed Dissociation.

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.

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.

Proteolysis of Amyloid ß by Lysosomal Enzymes as a Function of Fibril Morphology.

Aggregation of amyloid-ß (Aß) 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 Aß fibril formation disrupts proteolysis by lysosomal enzymes. Fibrils constituted by either Aß 1-40 or Aß 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 Aß 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 Aß 1-42 are generally more resistant to degradation compared to Aß 1-40. Overall, the results indicate that Aß 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 Aß 1-42.

A two-trick pony: lysosomal protease cathepsin B possesses surprising ligase activity.

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.

Internal Fragments Generated from Different Top-Down Mass Spectrometry Fragmentation Methods Extend Protein Sequence Coverage.

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.