Isoaspartate-containing galanin in rat hypothalamus

Okyem, Samuel; Mast, David H.; Romanova, Elena V.; Rubakhin, Stanislav S.; Sweedler, Jonathan V.

doi:10.1038/s42004-025-01475-5

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Published: 08 March 2025

Isoaspartate-containing galanin in rat hypothalamus

Communications Chemistry volume 8, Article number: 72 (2025) Cite this article

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Abstract

The isoaspartate residue is a spontaneous, time-dependent post-translational modification (PTM) of proteins and peptides, associated with in vivo protein aggregation and changes in molecule lifetime. While this is considered a slow modification impacting long lived proteins, surprisingly, we observed this PTM at high levels within the relatively short-lived neuropeptide galanin (Gal). The combination of liquid chromatography-trapped ion mobility mass spectrometry and protein Isoaspartyl methyltransferase assays demonstrated that 20 ± 2% of the mature Gal contain L-Isoaspartate residue in the hypothalamus of Rattus norvegicus. Aspartate in Gal isomerizes spontaneously under mildly acidic conditions within 48 h in vitro, much faster than previously assumed. Gal with the L-isoaspartate PTM significantly enhanced fibril formation. Transmission electron microscopy revealed differences in morphology of fibrils formed by D17Isoasparte Gal compared to the unmodified peptide. Observed characteristics of D17Isoasparte Gal suggest a potential mechanism for the development of in vivo Gal fibril deposits previously reported in the brain.

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Introduction

The formation of L-Isoaspartate (IsoAsp) residue in proteins and peptides is a spontaneous nonenzymatic post-translational modification (PTM) occurring under physiological conditions^1,2. This modification can significantly impact protein and peptide structure, function, stability, and the ability to form fibrills^{2,3,4,5,6,7,8}. The enzyme Isoaspartyl O-methyl transferase (PIMT), also known as L-Isoaspartyl/D-aspartyl protein carboxyl methyltransferase (PCMT), catalyzes repair of IsoAsp-containing proteins and peptides by transfer of methyl residue to the carboxyl group side chain consecutively leading to the formation of aspartate^9,10. Although IsoAsp accumulation in proteins is recognized as an age-dependent PTM², PIMT-deficient organisms without aging symptoms exhibit early physiological abnormalities, disrupted calcium signaling, and epileptic seizures, ultimately resulting in premature death^11,12,13. Administration of an adenovirus expressing PIMT partially alleviates some symptoms and extends the organism’s lifespan¹⁴. In a young PIMT-deficient mouse, IsoAsp levels increased ninefold¹³, indicating the important role of PIMT in the adjustment of this PTM concentration. However, PIMT activity declines with age due to reduced co-factor S-adenosyl methionine (SAM) levels¹⁵, a methyl donor in converting IsoAsp to a methyl ester, and subsequent formation of Asp residue.

The known age-dependent accumulation of IsoAsp residues has traditionally focused research on its occurrence in aging subjects and relatively long-lived proteins^{8,16,17,18,19,20}. However, the PIMT involvement in developmental processes and the substantial accumulation of IsoAsp in PIMT-deficient organisms^14,21 demonstrates that IsoAsp formation occurs throughout an organism’s lifetime. Here, we explore IsoAsp formation in a short-lived peptide Gal.

Neuroendocrine peptides serve as intercellular signaling molecules, pivotal in several physiological processes, including development²². A notable example is the expression of Gal in the ventrobasal thalamus of mice, which coincides with the formation of the whisker map and influences subcortical circuit wiring²³. Consequently, alterations to the mature peptide sequence, such as the isomerization to IsoAsp or D-amino acids, can profoundly impact the peptide’s functionality and stability. Despite this, the presence of IsoAsp in short-lived neuroendocrine peptides and its potential physiological and pathological implications remains largely unexplored.

We identified the presence of IsoAsp residues in galanin (Gal) peptides using liquid chromatography trap ion mobility mass spectrometry (LC-TIMS-MS), PIMT, and endoproteinase AspN. Significant IsoAsp residues were characterized in matured Gal peptides extracted from the rat hypothalamus. Our findings show that IsoAsp facilitates amyloid fibril formation in rat Gal. Notably, the action of PIMT significantly reduces or even prevents Gal fibrillization.

Recent studies reveal that Gal and prolactin are stored as amyloid fibrils in the secretory granules of female rats²¹. Given that IsoAsp residues are known to promote amyloid fibril formation, the occurrence of IsoAsp in Gal may contribute to its fibrillization within secretory granules.

The discovery of IsoAsp in Gal expands our understanding of aspartate isomerization as an age-related PTM affecting long-lived proteins to its occurrence in short-lived neuropeptides, potentially influencing their biological function, stability, and accumulation. Our findings help explain the physiological abnormalities experienced by PIMT-knocked-out organisms since neuropeptides play a vital role in development.

Results

LC-IMS-MS Identification of IsoAsp-Gal

Rat Gal (GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH₂), isomers were initially identified in hypothalamic extracts by LC-TIMS-MS. Two well-resolved chromatographic peaks (Fig. 1a) formed by ions with identical molecular mass but demonstrating noticeable mobility differences (Fig. 1b, c, e & f) were analyzed by tandem mass spectrometry. The fragment ions for both peaks confirmed that they represent Gal. The sequence of endogenous Gal was validated against the fragment ions of synthetic Gal standard. The elution of identical peptides at different retention times and showing different ion mobility suggests a possible structural difference between them due to a zero-Dalton modification²⁴. We hypothesized that one of the two Gal peptides contains an isomerized amino acid resulting from aspartate isomerization. In rat Gal, aspartate residues are located at positions 17 and 24, while asparagine residues are found at positions 5 and 18. These residues can potentially isomerize under harsh extraction conditions²⁵; hereafter, an isotopically labeled Gal synthetic standard (molecular mass difference +9 Da) was spiked into a rat hypothalamic tissue homogenate during analyte extraction. A single chromatographic peak was observed for the isotopically labeled synthetic standard at m/z 793.90, z = 4 + . In contrast, the occurrence of Gal peptide at multiple retention times (rt) previously observed in the hypothalamic extract remained (Fig. 1d), suggesting that the observation of Gal isomeric peaks is not caused by our analyte extraction protocol.

**Fig. 1: Identification of Gal peptide isomers in rat hypothalamic extract by LC-TIMS-MS.**

Dehydration and cyclization of aspartate residues lead to the formation of four isomers: L-Asp, L-isoAsp, D-Asp, and D-isoAsp²⁶. Various analytical methods were employed to determine the specific isomeric form of Gal present in the extract. Electron transfer dissociation (ETD) generates diagnostic fragment ions, specifically C_n-1 + 57 and Z_n-57, where n represents the position of the isomerized residue^27,28. ETD analysis of the two-Gal peaks revealed the presence of a C₁₆ + 57 fragment ion for the second eluting Gal peak, whereas no such ion was detected for the first eluting Gal peak (Supplementary Fig. 1g, h). These findings suggest that the second Gal peak corresponds to isoAsp Gal possibly at the D17 residue, ruling out D-Gal. As PIMT (vide infra) exhibits several thousand-fold lower activity on D-isoAsp residues compared to L-isoAsp²⁶, the observed degradation of the second Gal peak upon enzymatic treatment confirms that this peak corresponds to an L-isoAsp residue. To confirm the identity of the unmodified and isomerized Gal peaks, chromatographic retention times and ion mobility profiles were compared with synthetic isotopically labeled Gal and D17IsoAsp-Gal standards (Fig.1g, h). Hence, unless otherwise stated, all references to isoAsp hereafter specifically refer to L-isoAsp. and D17IsoAsp-Gal standards (Fig. 1g, h). Spiking of the D17IsoAsp-Gal standard into hypothalamic extracts resulted in an increased intensity of the second Gal peak (Fig. 1g, bottom panel). Also, the ion mobilities of the D17IsoAsp-Gal standard and the second endogenous Gal signal matched (Fig. 1h). Accordingly, the first and most abundant peak was assigned unmodified Gal. The second eluting peak at 22% relative abundance was assigned as IsoAsp-Gal.

Localization of IsoAsp residues in Gal by PIMT and ASPN

PIMT, an endogenous enzyme that catalyzes the methylation of IsoAsp residues in peptides^29,30,31, was used to confirm and determine the localization of IsoAsp residues in Gal. PIMT converts IsoAsp to aspartate in IsoAsp-containing Gal. Treatment of hypothalamic extracts with PIMT resulted in a remarkable decrease in the IsoAsp-containing Gal signal at rt = 41.5 min (Fig. 2a). In contrast, the chromatogram of the two Gal isomers remained unchanged in the extract not treated by PIMT. Also, elevated levels of the succinimide intermediate for D17 and D24 residue of Gal were observed in the PIMT-treated extract compared to the no PIMT control (Fig. 2c), and assignment of the succinimide intermediate was done using MS2 spectra.

**Fig. 2: Characterization of IsoAsp residues in Gal using PIMT and ASPN enzymes.**

Further analysis of the PIMT reaction product showed three methylated forms of Gal (Fig. 2b). The methylation site was confirmed by analysis of the fragment ions of methylated Gal (supporting Fig. 2a–d). A mass shift of +14 Da was evident for the y₁₇-y₂₃ Gal fragment ions. In contrast, no mass shift was observed for the y₅, y₆, and y₁₁ product ions of the MS2 spectra acquired at rt = 41.20 min (Supplementary Fig. 1b). Thus, the second methylated Gal peak (Fig. 2b) was assigned to the peptide containing the methylated D17 residue. Likewise, the MS2 spectra of the first methylated Gal had a mass shift of +14 Da for the y₇-y₂₃ fragment ions (Supplementary Fig. 1c), which indicates methylation at the D24 residue. Additionally, a + 15 Da mass shift in y₁₂-y₂₅ fragment ions for the MS2 spectra acquired at rt = 41.43 mins (Supplementary Fig. 1d) suggests asparagine deamidation followed by methylation. The chromatogram of the D17 and D24 methylated Gal was baseline resolved. In contrast, the deamidated N18 methyl Gal coeluted with the D17 methyl Gal (Fig. 2b). However, further deconvolution of the D17 and N18 species was accomplished using their distinct ion mobility values, detailed in the supplementary Fig. 1e, f. Also, the ion mobilities of the D17 and D24 methyl Gal ions at m/z 795.15, z = 4+ were baseline resolved with values of 0.91 and 0.96 V·s·cm^-2 respectively (Fig. 2b, right panel).

Endoproteinase AspN, a zinc metalloproteinase, preferentially cleaves at the N-terminus to aspartic acid compared to IsoAsp^32,33. Leveraging this specificity, we used AspN to enrich the IsoAsp-containing fragments of Gal. We hypothesized that the full length of IsoAsp-Gal or its cleavage product harboring the IsoAsp residue would be enriched while AspN digests the unmodified form of Gal. LC-IMS-MS analysis of post-enzymatic treated extracts showed a noticeable enrichment of the Gal fragment formed by cleavage at the D24 position (GWTNSAGYLLGPHAID17NHRSF). This fragment detected at rt = 42 mins (Fig. 2d) was significantly enriched compared to the portion resulting from cleavage at the D17 aspartate residue (GWTNSAGYLLGPHAI, rt = 49 mins). Collision-induced dissociation (CID) analysis of the enriched D17 containing Gal peptide formed during AspN enzymatic treatment (GWTNSAGYLLGPHAID17NHRSF) (Supplementary Fig. 2) revealed an IsoAsp characteristic fragment ion (y₇-46) at m/z 816.37 Da, (Fig. 2e) indicating that the enriched D17-containing fragment contains an IsoAsp. The above observations further confirm that Gal has an IsoAsp located at the D17 residue and is the predominant IsoAsp form of Gal among the others observed from the PIMT assay. This observation further confirms the presence of IsoAsp in matured Gal peptides.

In-vitro isomerization of Gal

Isomerization of aspartate to IsoAsp is traditionally considered a slow process, typically associated with long-lived proteins^2,18,34. However, the presence of unusually high levels of IsoAsp-containing proteins in young PIMT-deficient^14,35 organisms suggests that the accumulation of IsoAsp in proteins in vivo is mitigated by PIMT activity, which helps to reduce the levels of these molecules. The molecular sequence, environment, 3D-molecular structure, solvent, and enzyme accessibility to aspartate residues likely influence the isomerization rate. Thus, neuropeptides like Gal, due to their relatively simple three-dimensional structures, may be more prone to IsoAsp formation compared to larger proteins. After confirming the presence of IsoAsp in Gal extracted from the hypothalamus of rats, we investigated the isomerization rate in vitro.

Gal standards were prepared in a phosphate buffer at pH 5.8 and incubated at 37 °C. The pH of 5.8 was chosen to mimic the average pH of dense core vesicles where neuropeptides are stored^36,37. At each time point, 10 µL of the mixture was sampled, desalted, and analyzed by LC-MS. A shoulder peak, indicating IsoAsp formation, appeared between 5 and 12 h of incubation at approximately 5–9% relative abundance of the major peak. After 48 h, two baseline-resolved Gal peaks were observed (Fig. 3a), with 15–18% of IsoAsp-Gal formed from unmodified Gal (Fig. 3b). The percentage of IsoAsp-Gal remained constant for 14 days but increased to 36–40% at 28 days of incubation. Multiple Gal peaks emerged after day 14 (Supplementary Fig. 3a). These results demonstrate that significant amounts of IsoAsp can accumulate in Gal-containing solution under mildly acidic conditions within hours and days, contrasting with the slower isomerization observed in larger proteins^38,39. Considering that the mean age of dense core vesicles is 8–24 h^40,41,42, our findings suggest the likelihood of accumulation of IsoAsp in Gal within dense core vesicles.

**Fig. 3: In vitro isomerization of Gal.**

Aspartate hydrolysis and asparagine deamidation lead to L-IsoAsp, D-IsoAsp, and D-Asp formation^25,43. While PIMT can revert these isomers to L-Asp, its affinity for L-IsoAsp is 7000 times higher than for D-Asp^43,44,45. After 14 days of incubation, the sample was treated with PIMT to investigate isomerization in Gal. Most isomerized peaks degraded within 40 minutes of incubation (supplementary Fig. 3a), indicating that the peaks were predominantly L-IsoAsp.

Following PIMT treatment, four methylated forms of Gal were identified (supplementary Fig. 3a; insert). Peaks at retention times of ~37 and ~38 min had a monoisotopic mass of 791.15 (z = 4 + ), showing a + 14 Da mass shift consistent with methylation at aspartate residues. Peaks at rt ~34.5 and ~38.3 min had a monoisotopic mass of 795.40 (z = 4 + ), indicating a + 15 Da shift due to deamidation followed by methylation at asparagine residues.

DIA-PASEF and tandem MS analysis was used to pinpoint methylation sites. As shown in supplementary Fig. 3b, a + 14 Da mass shift was observed for the y₆-y₁₃ fragments at rt ~37 min, and a similar shift was detected for the y₁₃-y₁₆ fragments, whereas only y₁₃-y₁₆ fragments had a mass shift of +14 Da at rt ~38 min. These observations indicate methylation at D24 and D17 for peaks at rt ~37 and ~38 min, respectively. Fragment analysis of peaks at rt ~34.5 and ~38.3 min revealed deamidation and subsequent methylation at N5 and N18, respectively (Supporting Fig. 6a, inset).

Effect of IsoAsp on Gal amyloid fibril formation

Recent studies have demonstrated that Gal fibrillization can be enhanced by the glycosaminoglycan heparin, a polysaccharide repeating unit that can facilitate the packaging of proteins into secretory granules^21,46. Also, the co-aggregation of Gal and prolactin into amyloid fibrils has been reported²¹, suggesting a possible mechanism for their storage in secretory granules. Herein, we investigated the potential role of IsoAsp in Gal in enhancing amyloid fibril formation. Unmodified Gal and D17IsoAsp-Gal synthetic standards were incubated at pH 5.8 with and without heparin for 7–14 days. The amyloidogenic nature of the fibrils formed from the Gal standards was monitored using Thioflavin T (Thio T) amyloid binding assay. This fluorescent dye increases in fluorescence intensity when bound to amyloid fibrils.

In the presence of heparin, we observed a significantly higher Thio-T fluorescence for the D17IsoAsp-Gal compared to the unmodified form (Fig. 4a). Interestingly, D17IsoAsp-Gal preparation exhibited a notable formation of amyloid fibrils even in the absence of heparin, with a Thio-T fluorescence intensity comparable to that of the unmodified Gal-heparin mixture (Fig. 4a). However amyloid fibrils were detected for the unmodified Gal without heparin addition. LC-MS analysis of the unmodified Gal-heparin mixture showed the presence of 23% IsoAsp-Gal (Fig. 4c), similar to the relative percentage of IsoAsp observed in our hypothalamic extract. Our findings suggest that IsoAsp modification is essential for Gal fibrillization and may influence the initiation of amyloid fibril formation in vivo, potentially through preferential binding to granule helper molecules or a change in peptide conformation that favors β-sheet formation.

**Fig. 4: Characterization of Gal and D17IsoAsp-Gal fibrillization.**

Transmission electron microscopy (TEM) was also used to examine amyloid fibrils formed by Gal and IsoAsp-Gal. Fibrils were seen for the unmodified and IsoAsp-Gal heparin mixtures shown in Fig. 4e. No fibrils were observed for unmodified Gal without heparin, whereas IsoAsp-Gal formed fibrils even without heparin (Fig. 4e; D17IsoAspGal). Variations in fibril orientation, thickness, and length were observed between the unmodified and IsoAsp-Gal. In the presence of heparin, IsoAsp-Gal showed highly condensed, intertwined, and lengthy bundles of fibrils (Fig. 4e; D17IsoAsp-Gal + Hep). Additionally, the IsoAsp-Gal fibrils were thicker compared to unmodified Gal. To assess the impact of the observed amount of IsoAsp Gal on fibrillization, a mixture containing 20% D17 IsoAsp Gal and 80% unmodified Gal was incubated under similar conditions without heparin. As shown in Fig. 4e (20% D17 IsoAsp Gal), long, thin fibrils with lower density were observed compared to the D17 IsoAsp + Hep condition. These fibrils appeared more elongated than those in the D17 IsoAsp Gal-only mixture. Our findings indicate that IsoAsp Gal can seed the fibrillization of unmodified Gal. To this end, we propose that IsoAsp Gal may influence the formation of amyloid fibrils observed in secretory granules of female rats and may be an important mechanism in the basal forebrain of AD patients.

Impact of PIMT on gal fibrilization

After establishing the crucial role of IsoAsp in Gal’s fibrillization process, we examined the impact of PIMT on Gal fibril formation. Gal was incubated with heparin for 7–15 days, with and without PIMT, followed by Thio T fluorescence amyloid fibril quantification. In the presence of PIMT, we observed a significant decrease (p = 0.005) in Thio T fluorescence, indicating a reduced amyloid fibril formation. This contrasted significantly with the substantial Thio T binding observed in the Gal-heparin mixture without PIMT, as shown in Fig. 4b. The impact of PIMT on Gal and IsoAsp-Gal fibrillization was also examined using TEM. No substantial amyloid fibrils were seen in the presence of PIMT for unmodified Gal (Fig. 4e; Gal + Hep + PIMT). However, the IsoAsp-Gal highly dense, tangled fibrils were significantly reduced and transformed into long, thin fibrils (Fig. 4e; D17IsoAspGal + Hep + PIMT) with similar morphology as the 20%D17isoAspGal and Gal mixture. This observation is not surprising since approximately 80% of D17isoAspGal is converted to Gal after the PIMT reaction (supplementary Fig. 5). Moreover, there is a correlation between the intensity recorded in the Thio T fluorescence experiment (Fig. 4a, b) and the fibril density observed from the TEM images. These findings further confirm that IsoAsp plays an important role in Gal fibrillization.

The specific mechanisms of amyloid fibril formation depend on the peptide or protein physicochemical properties and properties of the surrounding environment; however, most amyloid fibrillization proceeds through nucleation, elongation, and secondary nucleation mechanisms⁴⁷. Hence, the time course of the fibrillization process can be modeled by a sigmoidal growth curve (e.g. Fig. 4d)⁴⁸. This curve can be characterized by the lag time (tlag), which is defined as the start time of fibrillization, the aggregation half-time (t₅₀), the time taken^41,42 for the fibrillization to reach half its maximum value, and the maximum fibril growth rate (rmax) is calculated as the slope of the inflection point. Using the Thio T fluorescence assay, we studied Gal and D17IsoAsp-Gal amyloid fibril formation kinetics and the effects of PIMT on kinetic parameters. For this assay, we used mixtures of Gal and heparin, D17IsoAspGal and heparin with and without PIMT, and D17IsoAspGal without heparin. Thio T was added to each mixture and equilibrated for 40 min before kinetics measurement. All measurement was performed in triplicates. From our kinetic studies, the lag time of the D17IsoAsp-Gal fibrillization was eliminated when incubated with heparin (Fig. 4d). However, D17IsoAsp-Gal has an average lag time of 20 h without heparin. Similar observations were reported for the effect of heparin on the amyloid fibrillization kinetics of Apo myoglobin⁴⁹. The lag time of amyloid fibril formation for most proteins and peptides is inversely related to the nucleation and elongation reaction rate. Glycosaminoglycans such as heparin are known to stabilize proteins mainly through binding interactions⁵⁰ therefore, the substantial decrease in the lag time of D17IsoAsp-GAL by heparin can be attributed to heparin either increasing nucleation or elongation reaction rate constant of fibrilization by lowering the activation energy for the nucleation process through the assembly of monomers or facilitating the assembly of the protofibrils to form matured fibrils.

The fibrilization lag time for D17IsoAsp-GAL-heparin drastically increased to 48 h when incubated with PIMT (Fig. 4d). The density of fibril formed was significantly reduced for the PIMT-treated mixture. Comparable observations were made by Chatterjee T. et al. when Aβ-42 amyloid peptide and an IsoAsp-containing hexapeptide were grown in the presence of PIMT^51,52. Consequently, we determined that PIMT has a pronounced effect on the onset and extent of D17isoap-Gal and unmodified Gal amyloid fibril formation.

Discussion

The detection of IsoAsp residues formed in peptides and proteins by asparagine deamidation became possible with increased mass resolution^33,53,54; yet, since isomerization does not alter molecular mass, measuring IsoAsp produced from Aspartate residues still presents difficulties. Here, we leveraged the synergy of ion mobility with high-resolution mass measurements and enzymatic reaction kinetics to unambiguously localize IsoAsp residues in Gal peptide. We optimized our PIMT assay to allow the detection of the methylated IsoAsp species by mass spectrometry and investigated novel IsoAsp forms of Gal in the rat hypothalamus. The described approach provides a general framework for locating and identifying IsoAsp residues in proteins and peptides.

Aspartate and asparagine residues are more prone to hydrolysis and deamidation leading to accumulation of IsoAsp residue when followed by glycine, serine, or histidine³⁹. Using synthetic standards, we found that, in slightly acidic conditions, Gal accumulated a notable amount of IsoAsp within 48 h (Fig. 3). In rat Gal, the two aspartate residues have asparagine and lysine at the N + 1 position and histidine at the N + 2 position, where N is the aspartate residue’s location, implying that isomerization can still occur in a short time even if the aspartate residues are not contiguous to these residues. The relatively quick isomerization of Gal indicates that other characteristics, such as the charge and electron density around the Aspartate residue, can influence the rate of isomerization. These factors may contribute to aspartate isomerization by stabilizing the cyclic oxoanionic imide intermediate, a critical stage in the isomerization process. Our findings suggest that a more detailed mechanistic approach is needed to understand aspartate isomerization in peptides.

Although IsoAsp residue is known to have a negative effect on protein function^6,16,17, in some cases, its occurrence in proteins and peptides can be advantageous. For example, this PTM can promote the activation of extracellular matrix protein, initiate integrin binding, and promote fibrillization^7,46,55,56 and cellular adhesion⁵⁷. IsoAsp in Gal can, therefore, have several physiological and biochemical effects, including modulation of its stability, storage, transport, and cellular adhesion. Presumably, the presence of an IsoAsp can effect GPRC activation; while this has yet to be shown for the IsoAsp modification, we have reported an endogenous d-Amino acid-containing neuropeptide that interacts with a GPCR that is not activated by the all-L peptide and this modified peptides impacts behavior⁵⁸; it is interesting to speculate whether this is true for IsoAsp containing peptides.

Peptide hormones, including Gal, can be stored in secretory granules as functional amyloid fibrils⁴⁶. Our results show that IsoAsp significantly accelerates the fibrillization of IsoAsp-Gal, which may affect its storage and release from the granules. Likewise, we demonstrate that IsoAsp-Gal serves as a PIMT substrate where PIMT influences initiation, rate, and extent of Gal fibrillization by controlling the amount of IsoAsp form of Gal. Also, our findings indicate that the IsoAsp-containing form of Gal is essential for Gal amyloid fibril formation; thus, the extent of fibrilization may be affected by the fraction of IsoAsp-Gal in the mixture. When the same molar ratio of D17IsoAsp-Gal and PIMT was incubated at 37 ^οC for the time of equilibration (40 min) before the kinetic assay, 80% of D17IsoAsp-Gal was converted to methyl-Gal by PIMT as shown in Supplementary Fig. 5. Thus, the fraction of D17IsoAsp-Gal in the reaction mixture is reduced by at least 80% before the Thio T kinetics measurement. The lag time for a nucleation elongation mechanism depends on the primary nucleation’s rate constant, which increases as the fraction of the amyloid-forming monomer increases. A decrease in lag time is observed for most amyloid-forming peptides and proteins when there is a higher fraction of amyloid-forming monomer⁴⁸. Accordingly, the increase in the lag time for the D17IsoAsp-Gal-heparin mixture when incubated with PIMT can be attributed to a substantial reduction in free IsoAsp-Gal concentration. These results demonstrate the need for a critical concentration of IsoAsp-Gal to initiate Gal amyloid fibrillization.

PIMT is ubiquitously expressed throughout the brain, including the hypothalamus⁵⁹. This widespread expression suggests that both cytosolic and extracellular levels of IsoAsp-Gal can be regulated by PIMT activity. However, in conditions where PIMT activity may be reduced, such as Alzheimer’s disease (AD) and other age-related disorders⁶⁰, elevated levels of extracellular IsoAsp-Gal may persist. To this end, the question of whether elevated levels of Gal fibril in AD patients’ basal forebrains represent neural defense against excitotoxicity or worsened cognitive impairment^61,62 can be partially addressed. By considering the recent hypothesis linking elevated global levels of IsoAsp to decreased PIMT activity in AD⁶⁰ we propose that the increased levels of Gal fibrils in the basal forebrain and other brain regions of AD patients may be significantly influenced by IsoAsp-Gal levels. Our findings emphasize the importance of employing innovative molecular characterization techniques to distinguish isomeric forms of peptides. Such an approach is crucial for enhancing our understanding of these peptides’ physiological functions.

Conclusions

By employing LC-IMS-MS, PIMT, ASPN, and synthetic standards, we have demonstrated the occurrence of IsoAsp residues in Gal from rat hypothalamus. D17, N18, and D24 are identified as IsoAsp-forming sites in Gal. We have shown that the isomerization of aspartate residues in Gal to IsoAsp can occur within 48 h under mildly acidic conditions. The role of IsoAsp in the fibrillization of Gal was also assessed. We found that IsoAsp formation is essential for Gal fibrillization. Thus, a substantial increase in the rate and extent of Gal fibrillization was observed when the D17 residue was substituted with IsoAsp. D17IsoAsp-Gal was employed in the fibrillization studies because we determined it to be the most isomerized residue among the others identified. We also show that PIMT can decrease the Gal amyloid fibril formation rate by reducing the amount of IsoAsp-Gal participating in the fibrillization. Likewise, PIMT inhibits Gal fibril formation induced by heparin.

Gal and PIMT have been implicated in several diseases, including Alzheimer’s disease and cancer^60,63. Our observations of IsoAsp in matured Gal peptides, along with the rapid accumulation of IsoAsp within 48 h, highlight the necessity of studying aspartate isomerization in human Gal. Investigating this process is essential for understanding its physiological effects and the potential impact on disease progression.

Now that IsoAsp modifications have been found in a Gal, future work will determine how common the IsoAsp modification is in other peptides. Recent results have shown that in another zero Dalton PTM, the racemization of an amino acid to the D-form also occurs in neuropeptides⁶⁴ and that this modification can be required for bioactivity⁶⁵; complete characterization of endogenous brain peptides requires additional characterization steps that have not been commonly employed in peptidomics workflows⁶⁶.

Methods

Peptide extraction & desalting

Rats used for these experiments were purchased from Charles River Laboratories (Wilmington, MA). All animal experiments were performed in compliance with local and federal regulations and in accordance with the animal use protocols approved by the Illinois Institutional Animal Care and Use Committee.

The hypothalami of 1–3-month-old rats were surgically dissected and heat-treated to prevent peptide degradation. Tissues were manually homogenized using a mechanical homogenizer equipped with a piston. Peptide extraction was done using 10% glacial acetic acid and 1% water in methanol. Homogenates were centrifuged at 16000 g for 15–20 min at 4 °C. After centrifuging, the supernatant was collected into a new centrifuge tube. The pelleted residual tissue was mixed with LC-MS grade water to form a homogenate and centrifuged at 16000 g for 10 min at 4 °C. Supernatants from the water extraction were combined with those of the acidified methanol extraction and preconcentrated using an Eppendorf Vacufuge.

To evaluate the effect of extraction conditions on Gal isomerization, 2 µl of 1 picomole isotopically labeled Gal ( + 9 Da) was added to a hypothalamic Gal tissue homogenate. The resulting solution was taken through the peptide extraction protocol described above. Preconcentrated peptide extract was desalted with a Pierce C-18 spin column. Pierce C-18, spin column sorbent, was initially activated with 50% methanol in water and equilibrated with 5% methanol in 0.5% Formic acid water. The spin columns were loaded with 50 µL of extract and centrifuged at 1500 g for 1 min. The flow-through was recovered and re-loaded onto the column to ensure efficient binding. Washing was performed using 5% methanol and 0.5% formic acid in water. A 70% methanol, 0.1% FA solution was used to elute peptides from the column. LC-MS grade solvents (Thermo Fisher Scientific, Rockford, IL) were used in all cases. Eluent was dried and stored at -80 °C until further analysis.

LC-IMS-MS analysis

LC-IMS-MS analysis was performed on Bruker nano Elute UHPLC (Ultra High-Pressure Liquid Chromatography) system coupled to Bruker timsTOF Pro, a trap-ion-mobility quadrupole time-of-flight (TIMS-Q-TOF) mass spectrometer. A captive spray ion source was used as an interface, and the data was acquired in a positive ion mode. Peptides were separated by PepSep series nano column (Bruker, Billerica MA) with 75 μm internal diameter, 15 cm length, 1.9 μm particle size, and 100 Ǻ pore size. Mobile phase solvent comprised 0.1% FA in water (Solvent A) and 0.1% FA in acetonitrile (solvent B). A mobile phase gradient of 4–40% B delivered at a flow rate of 300 nl/min for a 90 or 60 min time range was used for peptide separation. MS data was acquired in DDA and DIA-PASEF mode using a 100–1700 mz full scan range in a mobility range of 0.6–1.6 Vs/cm^-2. Ions were accumulated at 100 ms, and the mobility separation was performed with a custom resolution setting employing a ramp time of 100 ms at a scan rate of 1.1 Hz. Ten PASEF MS/MS frames were performed on isolated precursors at a target threshold of 20000 counts. Collisional cross-section and mass calibration were performed using an Agilent tuning mix (Agilent, Santa Clara, CA). The tunnel-in and tunnel-out pressures were set at 0.81 and 2.60 mbar, respectively.

PIMT reaction

PIMT reaction was performed by mixing 3 µg of human recombinant PIMT (Abcam Cambridge, UK) with 30 µg of peptide extract and 10 µL of 1 mM S-adenosyl methionine. The resulting mixture was diluted to a final volume of 50 µL using 100 mM Tris-HCl buffer at pH 7.5 and incubated at 37 ^οC for 40 min. The reaction was quenched by adding 10 µL potassium phosphate and centrifuging at 1000 g for 7 min. For the control experiments, 3 µL of water was added in place of PIMT. The final quenched reaction mixture was acidified with 0.5% FA and desalted using C-18 Zip-Tips (Millipore Sigma Burlington, MA) before LC-IMS-MS analysis.

IsoAsp containing peptides enrichment reaction ASPN digestion

The IsoAsp enrichment reaction used an LC-MS sequencing grade of endo-proteinase ASPN (Promega Madison, WI). 0.2 μg of ASPN was added to Gal containing LC fraction of peptides extracted from rat hypothalamus. The reaction mixture constituted a final ASPN: peptide ratio of 1:20 (w/w). The peptide digestion was performed in a 100 mM Tris-HCl buffer pH 8. The reaction mixture was incubated at 37 ^οC for 12 h. Peptide digest was desalted using C-18 Zip Tips before LC-IMS-MS analysis.

IsoAsp-Gal synthetic standards spike-in

The D17IsoAsp-Gal standard was diluted in mobile phase A to 100 fmole/μL. Two μL of the D17IsoAsp-Gal standard was spiked into a hypothalamic extract to a final 10 fmol/μL concentration.

Gal isomerization studies

In vitro isomerization studies of Gal were conducted in a 10 mM phosphate buffer containing 100 mM NaCl (pH 5.8). A 1 pmol/μL Gal solution was prepared from a synthetic standard obtained from CPC Scientific (Sunnyvale, CA) and incubated at 37 °C for 28 days. At each time point, a 10 μL aliquot was acidified with formic acid and desalted using a C-18 ZipTip. The cleaned samples were then dried and reconstituted in 0.1% formic acid in water for LC-IMS-MS analysis.

Gal and D17IsoAsp-Gal amyloid fibril formation studies

Gal amyloid fibril formation was performed using a published protocol with some modifications²¹. Briefly, a mixture of 400 µM of heparin and Gal standards prepared in a 10 mM phosphate buffer and 100 mM NaCl (pH 5.8) was incubated at 37 ^οC for 7–15 days. Similar unmodified Gal and D17IsoAsp-Gal standard solutions were prepared in the same buffer without heparin and incubated at similar conditions. To evaluate the effect of PIMT on Gal amyloid formation, a 400 μM solution of Gal and heparin was incubated with PIMT under the same experimental conditions. S-adenosyl-methionine was added to the reaction mixture to serve as a methyl donor. This reaction’s final Gal to PIMT molar ratio was 1 to 0.1.

Thio T Fluorescence and Kinetics of fibrillization of GAL and D17isaoasp-Gal

Fluorescence measurements were performed using a BioTek Cytation 5 multimode reader at an excitation wavelength of 450 nm and an emission wavelength of 480 nm. A slit width of 5 nm was employed for both excitation and emission. The final concentration of each sample and Thio T was 10 μM and 0.02 μM, respectively. A freshly prepared 10 μM Gal and D17IsoAsp-Gal with and without heparin was added to 3 µL of a 1 mM Thio T solution for the Thio T kinetic assay. To probe the effect of PIMT on D17IsoAsp-Gal fibrillization kinetics. PIMT was added to a freshly prepared 10 µM D17IsoAsp-Gal and heparin solution at a 1/0.1 molar ratio of peptide/PIMT. The normalized fluorescence intensity was plotted against the incubation time and fitted to a sigmoidal growth curve shown in Eq. 1. Normalization was performed by dividing each fluorescence intensity by the maximum fluorescence intensity measured. The lag time was calculated as previously described⁶⁷.

$$y={y}_{0}+\frac{\left({y}_{\max }-{y}_{0}\right)}{1+{{{\rm{e}}}}^{-k\left(t-t{{\rm{\_}}}1/2\right)}}$$

(1)

y is the fluorescence at a particular time, y is the initial Thio T fluorescence, y_max is the maximum Thio T fluorescence, and k is the exponential growth constant.

$${t}_{{lag}}={t}_{1/2}-2/k$$

(2)

Transmission electron microscopy analysis

The side blotting method was employed to prepare samples for TEM analysis. Briefly, 3–5 µL of 50 µM amyloid fibrils formed using Gal and D17IsoAsp-Gal incubated under several conditions discussed above were spotted onto a carbon-coated grid. The samples were absorbed onto the grid surface for about 3 min; then, excess liquid was removed by capillary action. Samples on the grid were stained with a 1% (w/v) uranyl acetate solution. FEI Tecnai G2 F20 S-TWIN STEM (Thermo Fisher) electron microscope was used to acquire images. Images were acquired at 160 kV with an AMT BIOSPR16 camera at 4000–45000 direct magnification.

Statistical analysis

Statistical analyses were conducted using OriginPro software. Welch’s one-way ANOVA was employed to assess differences among more than two groups, followed by the Games-Howell pairwise comparison to account for unequal variances. A two-sample t-test and Welch’s correction were used to evaluate the statistical significance between the two groups. All statistical analyses were performed at a 95% confidence interval.

Bioinformatics data analysis

Peaks Studio X (Bioinformatic Solution Inc) was used for peptide Identification. Mass spectrometric peak integration was performed using Skyline and Origin Pro (OriginLab). Origin Pro was also used for sigmoidal curve fit and statistical analysis. Chromatograms and mobilograms were prepared using DataAnalysis 5.0 from Bruker and Origin Pro.

Data availability

The mass spectrometry peptidomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD053254 and https://doi.org/10.6019/PXD053254. Source data for the Thio T amyloid fibril assay and statistical analysis is included as Supplementary Data. All other data included in this manuscript are available upon request.

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Acknowledgements

Research reported in this publication was supported by the National Institutes of Health, National Institute of Neurological Disorders and Stroke by Award No. R01NS031609, the National Institute on Aging under award No. 1R01AG078797 and the National Institute on Drug Abuse by Award No. P30DA018310. S.O. was supported through the NSF NRT-UtB (DGE 1735252). The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

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Department of Chemistry, University of Illinois, Urbana-Champaign, Urbana, IL, 61801, USA
Samuel Okyem, David H. Mast, Elena V. Romanova, Stanislav S. Rubakhin & Jonathan V. Sweedler
Beckman Institute, University of Illinois, Urbana-Champaign, Urbana, IL, 61801, USA
Samuel Okyem, David H. Mast, Elena V. Romanova, Stanislav S. Rubakhin & Jonathan V. Sweedler

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Samuel Okyem
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David H. Mast
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Elena V. Romanova
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S.O. and J.V.S conceptualized and designed the experiment. S.O, D.H.M., E.R.V., and S.R. contributed to data acquisition and analysis. S.O wrote original draft. Funding and resources for this project were acquired by J.V.S. All authors contributed the manuscript revision and interpretation of data.

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Correspondence to Jonathan V. Sweedler.

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Okyem, S., Mast, D.H., Romanova, E.V. et al. Isoaspartate-containing galanin in rat hypothalamus. Commun Chem 8, 72 (2025). https://doi.org/10.1038/s42004-025-01475-5

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Received: 05 October 2024
Accepted: 27 February 2025
Published: 08 March 2025
Version of record: 08 March 2025
DOI: https://doi.org/10.1038/s42004-025-01475-5