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. 2025 Aug 7;97(32):17570–17579. doi: 10.1021/acs.analchem.5c02612

Untargeted Discovery and Localization of Isomerized Residues in Neuropeptides

Samuel Okyem †,‡, Stanislav S Rubakhin †,‡, Satirtha Saha Protya †, Caden Gunnarson †, Jonathan V Sweedler †,‡,*
PMCID: PMC12368835  PMID: 40774382

Abstract

A reliable and effective analytical method for discovering and characterizing isomerized residues in physiologically active peptides is essential for their comprehensive characterization. Complete structural detail facilitates the determination of a peptide’s biological roles and meets the increasingly stringent demands for peptide-based therapeutics. Here, a comprehensive untargeted analytical workflow predicts possible peptide isomers from peptidomics data and then localizes the isomerized residues by using collision-induced dissociation-trapped ion mobility spectrometry (CID-TIMS) and protein isoaspartyl methyltransferase (PIMT) activity. The approach allows for the discovery and characterization of isomerized isoaspartate (isoAsp) residues and D-amino acids within the peptide. Potential isomeric peptide candidates are first identified from peptide-spectrum matches (PSMs) obtained after a database search by applying a defined retention-time window and comparing the differential ion-mobility values of precursor ions corresponding to nominally identical peptides. CID-TIMS is then utilized to locate the isomerized amino acid(s) within the predicted isomers by comparing the mobilities of fragment ions detected at those retention times. Finally, PIMT is used to label isoAsp residues for confirmation. This integrated workflow enabled the localization of isoAsp residues in eight peptides from the rat hypothalamus. These peptides are from six prohormones, including proenkephalin, promelanin-concentrating hormone, secretogranin II, pituitary adenylate cyclase-activating polypeptide, and prosomatostatin. To show its broad utility, our workflow successfully identified a D-amino acid-containing form of small cardioactive peptide B, FMRFamide, and another D-amino acid-containing peptide from an uncharacterized protein in the sea slug . The presented discovery workflow effectively discovered novel isomerized residues in endogenous peptides directly from complex biological samples.

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Introduction

Isomerization of amino acids in signaling peptides, such as the formation of D-amino acids and isoaspartate (isoAsp) residues, alters peptide three-dimensional structures (3-D), affecting their receptor-binding properties, stability, storage, and capability to aggregate. − For example, the Aplysia neuropeptide GdFFD, containing a d-phenylalanine residue, degrades in the presence of specific enzymes much more slowly than its all-l counterpart. The isomerized peptide acts as an extrinsic modulator of the feeding circuit. In contrast, the nonisomerized form does not affect neuronal activity. IsoAsp presence in proteins affects bacterial biofilm formation, extracellular matrix activation, and integrin binding, , and can even serve as a molecular clock of aging. − Elevated levels of isoAsp have been observed in proteins such as alpha-crystallin in the eye lenses of older individuals, and recent studies have directly linked isoAsp accumulation to Alzheimer’s disease, for instance, through its presence in Aβ42/Aβ40 peptides in patients with cognitive decline. ,− Although isoAsp accumulation in proteins is traditionally viewed as a slow process, high levels have been reported in mice and zebrafish lacking the isoAsp repair enzyme protein isoaspartyl methyltransferase (PIMT), − suggesting that the formation in vivo of this PTM may occur more rapidly than previously reported. Despite these pronounced physiological implications, little attention has been paid to isoAsp formation in short-lived peptide hormones and neuropeptides.

Mass spectrometry (MS) is widely employed for peptide analysis; − however, because isomerization does not change the analyte’s mass, distinguishing peptide isomers by MS alone remains challenging. Fragmentation techniquessuch as electron capture/transfer dissociation, collision-induced dissociation, and radical ion fragmentationcan produce diagnostic peaks for isoAsp and may yield differential fragment ion intensities that facilitate isomer identification when standards are available, − but often require supplemental ion sources or instrument modifications. ,

The integration of ion mobility into MS platforms enables the separation of peptide isomers. ,− Yet localizing isomerized residues in a peptide remains difficult. The Li group utilized traveling wave ion mobility spectrometry (TWIMS) in the analysis of isomerized residues in crustacean hyperglycemic hormone (CHH), which was isolated from , while the Julian group combined tandem mass spectrometry with statistical analyses to localize isomerized residues. , Both methods require specialized dual ion trap configurations that are not widely available across mass spectrometry platforms and may suffer from insufficient mobility resolution and complex data processing. Our group developed a D-amino acid-containing peptide (DAACP) discovery funnel and other approaches that leverage the difference in proteolytic enzymatic degradation of unmodified peptides and DAACPs for such PTM detection and characterization; however, it is limited to N-terminal DAACPs and requires laborious peptide isolation and chiral analysis.

We recently applied PIMT coupled with liquid chromatography-trapped ion mobility mass spectrometry (LC-TIMS-MS) to identify and characterize isoAsp residues in galanin from the rat hypothalamus. Building on this, we now introduce an untargeted bioanalytical and computational workflow that addresses key challenges in detecting and localizing D-amino acid and isoAsp residues in endogenous peptides. First, it eliminates the need for specialized instrumentation by working with a commercial TIMS platform (Bruker timsTOF series). Second, it bypasses labor-intensive peptide isolation and chiral analysis, offering enhanced analyte resolution via TIMS and enabling the localization of isomerized residues throughout peptide sequences.

The workflow integrates LC-TIMS-MS, a custom isomer prediction Python module, PIMT treatment, and collision-induced dissociation with trapped ion mobility (CID-TIM) MS. This strategy significantly improves the speed and accuracy of identifying and characterizing isomerized residues in endogenous therapeutic peptides.

We demonstrate the effectiveness of our approach by successfully identifying isoAsp residues in several endogenous peptides from the rat hypothalamus. Also, this approach enabled the discovery of three novel DAACPs in the cerebral and pleural ganglia of .

Experimental Section

The important details of the experiments are outlined below with detailed protocols presented in the Supporting Information.

PIMT Reactions

The PIMT assay was performed following a previously published protocol with minor modifications. Briefly, the reaction mixture consisted of a 1:10 mass ratio of recombinant PIMT enzyme (Abcam, Cambridge, UK) to peptide and 10 μL of 1 mM S-adenosylmethionine (SAM), prepared in a final reaction volume of 50 μL by using 100 mM Tris-HCl buffer (pH 7.5). The reaction mixture was incubated at 37 °C for 40 min. The reaction was quenched by adding 10 μL of 50 mM potassium phosphate, followed by centrifugation at 1000g for 5 min. Control reactions were performed by substituting an equal volume of water in place of the PIMT enzyme solution. After quenching, the reaction mixtures were acidified to 0.5% (v/v) formic acid and desalted using C18 ZipTips (Millipore Sigma, Burlington, MA).

LC-IMS-MS Analysis

Peptides were separated on a Bruker nanoElute nanoflow LC using a PepSep C18 column (25 cm × 75 μm i.d., 1.9 μm, 100 Å) with a 90 min gradient of 0.1% formic acid in water to 0.1% formic acid in acetonitrile at 600 nL min–1 (∼0.45% B min–1). The eluate was introduced via captive-spray into a Bruker timsTOF Pro 2 in positive-ion mode (m/z 100–1700; 1/k 0 0.6–1.6 Vs cm–2; 100 ms accumulation and ramp). DDA-PASEF was performed at 1.9 Hz (10 MS/MS scans per cycle). Standard TIMS voltages: Δ1 (capillary exit) = −20 V; Δ2 (deflection/discard) = −70 V; Δ3 (Funnel 1 transfer) = 110 V; Δ4 (accumulation trap): 100 V; Δ5 (transfer) = 0 V; and Δ6 (ramp start) = 55 V were employed.

CID-TIMS MS Analysis

The TIMS parameters were tuned to generate fragment ions before mobility separation in the second TIMS cartridge to analyze the mobilities of the peptide fragment ions. This was achieved by employing a tunnel-in pressure of 1.75 mbar and the following TIMS setting, Δ1 = −20 V, Δ2 = −160 V, Δ3 = 110 V, Δ4 = 110 V, Δ5 = 0.0 V, Δ6 = 150 V. The accumulation and ramp times were set as 150 and 400 ms, respectively. Data were acquired in DDA-PASEF mode, 10 PASEF MS/MS per cycle, using custom TIMS settings at a mobility range of 0.3–1.3 Vs cm–2.

Results and Discussion

Figure outlines the approach for the untargeted discovery and site-specific localization of isomerized residues. The workflow begins with peptide extraction from tissues and data acquisition using a standard LC-IMS-MS method. Our custom Python module (github.com/sokyem/PepSIFinder) processes the resulting peptide spectrum matches (PSMs) after peptide identification by database search (Peaks Bioinformatics Solutions) by predicting candidate peptides likely to contain isomerized residues based on the detection of the same peptide eluting at distinct retention times; in other words, it looks for characteristic retention times and mobilities. There are multiple reasons for peptides to have split eluting peaks, but here, the goal is to discover isoAsp and D-amino acid modifications. To distinguish them, we employed PIMT, an enzyme that catalyzes the methylation of the isoAsp residues. The disappearance of multiple eluting peaks and the appearance of methylated peptide forms serve as specific indicators of isoAsp residues. The isomerization site for both D-amino acid and isoAsp can be localized using a CID-TIMS method. The CID-TIMS method was adopted from previous work and optimized for our application on a Bruker timsTOF Pro 2 instrument. In our approach, peptide isomers are separated by liquid chromatography. Then, by adjusting the accumulation exit voltage, ramp start voltage, and tunnel-in pressure, we induce collisional activation of the peptides before mobility analysis. The underlying idea is that fragment ions derived from isomers without an isomerized residue should exhibit similar mobilities, whereas fragment ions containing an isomerized residue will display distinct mobilities. This difference enables localization of the isomerization site. Moreover, we developed a data analysis tool, LCTIMSpy (github.com/sokyem/LCTIMSpy), to streamline the processing and interpretation of CID-TIMS data.

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Schematic workflow for untargeted discovery of isomerized residues in peptides. Peptide Extraction, Processing, and Analysis: Peptides extracted from biological tissues are processed in untreated and PIMT-treated conditions and analyzed by LC-TIMS-MS, followed by database searching to identify peptides. Peptide spectrum matches (PSMs) from database search results are exported and used to identify potential peptide isomers. PepSIFinder: Our custom Python package PepSIFinder identifies potential peptides containing isomerized residues by analyzing retention time (RT) and ion mobility (1/K 0) profiles from the PSMs. To differentiate isoAsp from other isomers, we specifically look for methylated aspartate residues in the PIMT-treated extract and monitor the disappearance of isomeric peaks in the PIMT-treated samples. CID-TIMS: The extract is also analyzed using the CID-TIMS method. We first identify coeluting fragment ions and then compare the mobilities of these coeluting fragment ions at the retention times of isomeric.

Predicting of Neuropeptide Candidates Containing Isomerized Residues

A common hallmark of peptide isomers is the difference in their 3-D structure, which gives them different chromatographic and gas-phase properties. , As a result, peptide isomers can often be separated by reverse-phase chromatography and by ion mobility techniques. ,,− These properties can be leveraged to detect and localize isomerized residues in peptides. In biological samples, peptides without isomerized residues often coexist with those that contain them. Therefore, by analyzing LC-IMS-MS data and looking for peptides with the same m/z that elute at multiple retention times and exhibit different ion mobilities, we can identify the presence of isomerized residues. ,,− However, over 1000 peptides are characterized in most LC-MS experiments, making it time-consuming to manually identify those eluting at multiple retention times. Therefore, we developed a Python module that automates the detection of peptides with the same m/z values but different elution times and ion mobilities.

Here, peptide spectrum matching (PSMs) results obtained from the analysis of LC-IMS-MS data serve as the input to identify peptides that elute at more than one retention time. We first evaluated our Python model using peptides extracted from the pleural and cerebral ganglia of . In this study, peptide extracts were analyzed by liquid chromatography-ion mobility-mass spectrometry (using a Bruker timsTOF Pro2), and the data were processed using the PEAKS bioinformatics platform. We then applied our custom model to predict isomeric peptide candidates by setting a retention time threshold of 1.0 min and an ion mobility variation threshold of 2%; thus, peptides detected after a minute window are considered to have multiple peaks. The retention time and mobility variation threshold above were based on manual analysis of the extracted ion chromatogram and mobilogram of known DAACPs in .

This strategy allowed us to successfully find all known DAACPs in that had more than five amino acid residues (Table S1). Bioinformatics peptide-identification tools lack confidence in sequence determination of peptides containing less than five amino acid residues; hence, the five-residue minimum. We further evaluated our model using neuroendocrine peptides extracted from the hypothalamus of .

Out of approximately 2000 peptides and about 6400 PSMs identified at a 1% false discovery rate using a rat cell–cell signaling peptide database, our analysis identified 45 peptides as having an isomerized residue (Table S2) based on a retention threshold of 1.0 min and a mobility variation of 4%. Although technical issues like column overload, leaking connections, or poor separation can cause split elution peaks, the appearance of multiple peaks for the same peptide may also be due to the presence of diastereomers, positional isomers, or labile modifications such as sulfation. Since the formation of D-amino acid and isoAsp residues impacts their stability, storage, and propensity to form fibrils, we specifically tailored our approach to detect these modifications in endogenous peptides.

CID-TIMS for Site-Specific Localization of Isomerized Residues in Peptide Isomers

A CID-TIMS method was developed to localize the isomerized residues. In a TIMS device with a dual cell, ions accumulate in the first TIMS cell and are separated in the second cell. The internal energy of the ions depends on the applied accumulation transfer (delta 3) and exit (delta 6) DC potentials, as well as the tunnel-in pressure. , Thus, ions build more internal energy at a high DC potential and low tunnel-in pressure. Low-pressure fragmentation can occur in the TIMS tunnel at a specific DC threshold. Borotto and coworkers employed this approach to fragment melittin and ubiquitin before mobility separation. Using this approach and tuning the instrument to fragment a wide m/z range of peptides, we obtained mobility-separated fragment ions for several peptides at a delta 6 potential of 150 V and a tunnel-in pressure of 1.75 mbar. We first demonstrated this approach using peptide standards with known isomerized residues. Figure S1a,b shows the fragment ions and the mobility-separated fragments of alpha-melanocyte-stimulating hormone (alpha-MSH) acquired using this method. Manipulating the ion energy in the accumulation cell allows us to generate high-quality CID fragment ions before mobility separation. If fragment ions can be mobility separated, then the site of isomerization in peptide isomers can be determined by comparing the mobilities of fragment ions of peptide isomers separated by online liquid chromatography. The assumption is that there is enough resolution so that fragment ions with no isomerized residues should have the same mobility. In contrast, a mobility shift will be observed for a fragment ion containing an isomerized residue compared to the nonisomerized counterpart. A D-amino acid-containing alpha-MSH, Met-enkephalin-Arg-Phe, and their all-L forms were used to test our hypothesis. Alpha-MSH had isomerization at the methionine residue, whereas Met-enkephalin-Arg-Phe had isomerization at the phenylalanine and methionine residues. Alpha-MSH, as well as Met-enkephalin-Arg-Phe diastereomers, was chromatographically resolved (Figure S1a, f).

By comparing the mobilities of the fragments at the retention times of both isomers, we identified the site of isomerization in the peptide standards. From Figure S1d, although no substantial mobility difference is observed for the precursors, y12, y9, and y8 fragments of the peptide isomers. However, a substantial shift in ion mobility was observed for the y11 and y10 fragment ions, which shows the presence of an isomer at the y10 residues. Multiple mobility measurements with pairwise t tests (p < 0.05) revealed significant mobility differences for y10, y11, and y12 fragment ions but not for the y7–y9 ions. This result indicates a significant mobility difference for all fragment ions of alpha-MSH isomers containing isomerized residues compared with those without isomers (Figure S1e). Also, the site of isomerization in Met-enkephalin-Arg-Phe standards was localized using this approach. As shown in Figure S1g, we observed a substantial shift in mobilities for the all-L precursor and y6–y3 fragment ions when compared to the isomer with the d-methionine residue; however, no observable mobility difference was recorded for the precursor and the y6 for the all-l and the d-phenylalanine-containing form. However, pairwise t tests confirmed that only fragments bearing isomerized residues differ significantly in mobility, enabling precise localization of isomerization sites (Figure S1h,i). Here, we have demonstrated that we can confidently identify the sites of isomerization in peptide isomers by comparing the mobilities of their fragment ions.

IsoAsp Containing Peptides in the Rat Hypothalamus

Our next objective was to predict and characterize isoAsp residues in endogenous peptides, thereby demonstrating the applicability of our workflow in a complex biological mixture. To validate the presence of isoAsp, we employed protein-l-isoaspartyl methyltransferase (PIMT), an enzyme that selectively methylates isoaspartyl sites and catalyzes their conversion back to aspartyl residues via a targeted methylation pathway. ,, This process involves the methylation of the side chain carboxyl group of the isoAsp residue, generating a succinimide intermediate that subsequently undergoes hydrolysis to yield aspartate. While the succinimide and methyl intermediates typically exhibit half-lives of a few hours , , under optimized reaction conditions, these methylated intermediates can be characterized using high-sensitivity analytical instrumentation.

To identify if any of the predicted peptides with aspartate residues contain an isoAsp, the rat hypothalamic extract was treated with PIMT. We anticipate that one of the multiple eluting peaks of these isomeric peptide candidates will decrease in intensity over time if the peptide contains an isoAsp residue. Furthermore, the postreaction detection of the methylated peptide derivative would provide confirmatory evidence for isoAsp residues. Since isoAsp formation can occur during sample preparation, an isotopically labeled synthetic standard of SPQLEDEAKELQ [penk-198-209] (+14 Da) was spiked into the extraction solution before extraction. As shown in Figure a, no isomerization was observed for the synthetic standard; meanwhile, the endogenous isoAsp form of SPQLEDEAKELQ [penk-198-209] was observed, which indicates that our sample preparation conditions did not induce isomerization.

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Identification of isoAsp residues in neuroendocrine peptides by PIMT treatment. (a) EIC comparison of isotopically labeled synthetic standard SPQ­(13CL)­EDEAKE­(13CL)­Q (m/z 700.85, z = 2) versus endogenous form (m/z 693.84, z = 2). (b) EIC of SPQLEDEAKELQ [penk-198-209] from rat hypothalamic extract before (red) and after (blue) PIMT treatment. (c) EIM showing mobility differences between SPQLEDEAKELQ isomers at RT = 17.8 min (blue) and RT = 22.5 min (red). (d) Pairwise t test of mobility differences for SPQLEDEAKELQ isomers. (e–j) EICs before (red) and after (blue) PIMT treatment for: (e) MDELYPVEPEEEANGGEILA [penk-114-133]; (f) VGRPEWWMDYQ [penk-219-229]; (g) melanin-concentrating hormone; (h) VQLAGTQESVDSAKPRVY [copeptin fragment]; (i) LLDEGHDPVHESPVDTA [scg1-435-451]; (j) GMGENLAAAAVDDRAPLT [paca-109-126] and LPQAAEQDEMRLELQ [sms-73-78]. Arrows indicate isoAsp-containing peptides (peaks disappearing after PIMT). Statistics: pairwise t test with Holmes–Benferroni correction; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Error bars: SEM, n = 3.

Using this analytical strategy, we successfully identified isoAsp residues in peptides from five distinct prohormones: three from the Proenkephalin-A prohormone (MDELYPVEPEEEANGGEILA [penk-114-133]), (SPQLEDEAKELQ [penk-198-209]), and (VGRPEWWMDYQ [penk-219-229]); from Melanin-concentrating hormone (EIGDEENSAKFPI-NH2 [neuropeptide glutamic acid isoleucine]) and its nonamidated form containing glycine at the C-terminus; one from Vasopressin-neurophysin 2-copeptin (VQLAGTQESVDSAKPRVY [copeptin fragment]); one from Secretogranin 1 (LLDEGHDPVHESPVDTA [scg1-435-451]); one from Pituitary adenylate cyclase-activating polypeptide (GMGEN­LAAA­AVDD­RAPLT [paca-109-126]); and (LPQA­AEQD­EMRL­ELQ­[sms-73–87]) from prosomatostatin. Several of these peptides are cleaved at dibasic or monobasic sites, suggesting that they are endogenously processed by prohormone convertases (Figure S4).

Chromatographic analysis of the untreated extract revealed multiple peaks for each peptide, with selective degradation of specific peaks following PIMT treatment (Figure b,e–j), which indicates that these represented isoAsp-containing forms. Tandem MS analysis of the extract confirmed the presence of methylated forms of these peptides post-PIMT treatment (Figure S2). Comparative MS/MS analysis of methylated versus nonmethylated peptide forms enabled precise localization of methylation sites (Figure S3), confirming isoAsp residues at positions 2, 6, 9, 4, 11, 3, 12, and 8 for [penk-114-133], [penk-198-209], [penk-219-229], neuropeptide glutamic acid isoleucine, copeptin, [scg1-435-451], [paca-109-126], and [sms-73-87], respectively.

Additionally, the CID-TIMS method effectively localizes the isoAsp residues. As depicted in Figure c,d, there are significant mobility differences among the y8–y10 fragments of [penk-109-129] isomers. Interestingly, no significant change in mobility was observed for the y7 residue, which contains the isoAsp modification. Typically, one would expect the y7 fragment to exhibit a mobility difference compared to its nonisomerized counterpart. However, our data do not show this, indicating a unique behavior of fragments resulting from cleavage at the isoAsp residue. This finding suggests that cleavage at the methylene insertion site (y7 residue) may alleviate the structural constraints introduced by isoAsp residues into peptide.

Our analysis revealed a noteworthy structural pattern; the methylated aspartate residues were consistently followed by glutamate, aspartate, serine, or tyrosine at position n+1 (with n representing the methylated aspartate). Molecular dynamics simulations conducted by Akifumi Oda and colleagues on three elastin peptides (GVADAAAA, REGDPSSS, AGADEGVR) demonstrated that aspartate residues adjacent to glutamate at the n+1 position undergo isomerization at a higher rate. This increased isomerization was attributed to the overall hydrophilicity of the peptide and the solvent accessibility of both the amide nitrogen and the carboxyl side chain undergoing the cyclization reaction that leads to isoAsp formation. We can infer from this analysis that the presence of a hydrophilic residue may increase the solvent accessibility of the carboxyl side chain and the amide nitrogen, enhancing the isomerization rate.

Identifying isoAsp residues in short-lived peptides challenges the conventional understanding that isoAsp accumulation in proteins and peptides is exclusively a slow process associated with aged proteins. We previously demonstrated that 7–18% of isoAsp can form in Galanin within 48 h at pH 5.8. Observations of elevated isoAsp levels in PIMT-knockout mice and zebrafish models further support this rapid accumulation. ,, Our results suggest that isoAsp accumulation can occur during peptide storage in dense core vesicles. Meanwhile, cytosolic isoAsp levels likely remain minimal due to PIMT activity (as our work confirms that endogenous peptides are PIMT substrates). The detection of isoAsp at lower concentrations has been historically challenging. This limitation stems primarily from inadequacies in analytical methodology, as conventional mass spectrometry workflows and data analysis pipelines typically do not incorporate isomer identification protocols. Our newly developed method addresses this gap by enabling the identification and precise localization of isoAsp residues in short-lived neuroendocrine peptides, even at relatively low concentrations.

Recent research has revealed that neuropeptides and peptide hormones are commonly stored as amyloid fibrils, , and importantly, isoAsp modifications in peptides can initiate or enhance amyloid fibril formation. , Therefore, isoAsp in various endogenous neuropeptides suggests a potential physiological mechanism for peptide storage, wherein the isoAsp-modified forms may promote fibrillation and improve peptide storage and packaging.

Localization of Isomerized Residue in Endogenous Neuropeptides

Our group has previously identified several peptides containing D-amino acids in using the discovery funnel and related approaches. ,,,, To further assess our method’s ability to identify isomerized residues in complex samples, we analyzed peptides extracted from the ganglia of using our LC-CID-TIMS technique and targeting previously identified DAACPs. The resulting spectra are notably complex because all coeluting peptides are fragmented in the TIMS tunnel before mobility separation.

To address this complexity, we developed a custom data analysis tool to analyze the CID-TIMS data. This workflow first identifies coeluting fragment ions corresponding to peptide isomers and then extracts their mobility values. Full details of the workflow are provided in Supporting protocol 1. By comparing the mobility profiles of the fragment ions from the peptide isomers, we can accurately determine the site of the isomerization.

Among the DAACPs detected in Aplysia are the pleurin peptides. For pleurin 1, isomerization occurs at the second residue, while for pleurin 2, isomerization is found at both the second and third residues. Our LC-CID-TIMS approach successfully localized these isomerization sites (Figure b ,e). Pleurin peptide isomers are well resolved by reverse-phase chromatography (Figure a,d), and all-L forms elute earlier than their DAACP counterparts, with pleurin 1 isomers eluting at approximately 18 and 27 min (Figure a). By comparing the mobility of coeluting fragment ions at the two retention times, we confirmed that isomerization in pleurin 1 occurs at the second residue (Figure b,c). Specifically, the y12–y6 fragment ions show no observable mobility differences, while the y13 fragments show different mobilities (Figure b). Similarly, for pleurin 2, the observation of differential mobilities for y13 and y12 fragment ions and similar mobility values in the y11–y9 fragments indicates isomerization at the second and third residues (Figure e).

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Structural characterization of isomerized peptides using CID-TIMS and ion mobility fragment ion analysis (a) EIC of pleurin 1 fragment ions showing chromatographic separation of isomeric forms. (b) EIM of pleurin 1 fragments at RT = 21.5 min (blue) and RT = 28.0 min (red). (c) Pairwise mobility comparison of pleurin 1 fragment ions. (d) EIC of pleurin 2 fragment ions. (e) EIM of pleurin 2 fragments at RT = 18.7 min (blue) and RT = 21.5 min (red). (f) Pairwise mobility comparison of pleurin 2 fragment ion. (g) EIC of FMRGFamide fragment ions. (h) EIM of FMRFamide fragments at RT = 15.8 min (blue) and RT = 21.3 min (red). (i) Pairwise mobility comparison of FMRGFamide fragment ions: pairwise t test with Holmes–Benferroni correction; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Error bars: SEM, n = 3.

Moreover, we confirmed the presence of a d-methionine residue in FMRGFamide by comparing the mobilities of its fragment ions at retention times of 16 and 21 min (Figure h,i). In this case, the precursor ions and y4 fragments showed a clear mobility shift between the isomeric forms.

Statistical analysis using pairwise t tests further supported these findings. In pleurin 1, a significant difference was observed between the mobility difference of the y13 fragment ion pair and that of the y12–y10 fragments (Figure c), which confirms isomerization at the second phenylalanine residue. In pleurin 2, the mobility differences among the y9–y11 fragments were insignificant; the differences between the y13 and y12 fragments were highly significant (Figure f), indicating that the y12 and y13 fragments contained isomerized residues. Additionally, for FMRGF-amide, the precursors and y4 fragment displayed significant mobility differences, confirming isomerization at the methionine residue.

Overall, our results demonstrate that the LC-CID-TIMS method enables reliable localization of isomerization sites in peptide extracts without the need for prior isolation and purification of the peptide isomers.

Discovery of New DAACPS from Aplysia Central Nervous System

Analysis of the cerebral and pleural ganglia predicted several peptides to contain isomerized residues using our peptide isomer finder model (Table S1). Notable examples include the peptides FMRFamide, small cardioactive peptide B (sequence: MNYLAFPRM-NH2), and the peptide FLESIAGHII from the uncharacterized prohormone XP_035829589.1. From this same prohormone, we have previously confirmed two DAACPs, YdLDHLGSSLV and YdLDGIASSLI. These new DAACP candidates exhibited at least two distinct and resolved chromatographic peaks (Figure a,d,g), indicative of isomeric variants. Extracts from the pleural and cerebral ganglia were analyzed using our integrated workflow to identify the precise site of isomerization in these candidate peptides. The extracted ion chromatogram of fragment ions derived from the FLESIAGHII peptide displayed two well-separated fragment ion peaks at retention times of 34.5 and 36.8 min (Figure a). A comparative analysis of the mobility profiles for fragment ions across these two retention times revealed a noticeable difference, specifically for the y9 fragment ions (Figure b). To statistically validate these observations, pairwise t tests were conducted on the mobility differences of fragment ions at the two retention times (Figure c). This analysis indicated a significant difference exclusively for the y9 fragment, suggesting isomerization at the second leucine residue. No statistically significant differences were observed for the other fragment ions or the precursor.

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Discovery of new DAACPs in the cerebral ganglion of . (a) EIC of [FLESIAGHII] derived from the uncharacterized prohormone XP_035829589.1, showing coeluting fragment ions at RT = 34.5 and 36.8 min. (b) EIMs of [FLESIAGHII] fragment ions at RT = 34.5 min (blue traces) and RT = 36.8 min (red traces). (c) Pairwise t test comparison of mobility differences between fragment ions of [FLESIAGHII] at the two RTs. (d) EIC of SCPb fragment ions. (e) EIMs of SCPb fragment ions at RT = 31.8 min (blue traces) and RT = 33.2 min (red traces). (f) Pairwise t test comparison of mobility differences for SCPb fragment ions at the two RTs. (g) EIC of FMRFamide fragment ions. (h) EIMs of FMRFamide fragment ions at RT = 17.6 min (blue traces) and RT = 22.0 min (red traces). (i) Pairwise comparison of mobility differences between FMRFamide fragment ions at RT = 17.6 min and RT = 22.0 min. A pairwise t test comparison with Holmes–Benferroni correction was performed for (c, f, and i), where * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Error bars: SEM, n = 4.

Similarly, analysis of the fragment ion mobilities for small cardioactive peptide B (SCPb) at retention times of 31.8 and 33.3 min (Figure d) revealed a significant mobility difference for the y8 fragment ions and the precursor ions (Figure e). This observation indicates isomerization at the second asparagine residue. Other fragment ions did not show significant mobility differences. Historically, isomerization in peptides leading to DAACPs has been predominantly documented for hydrophobic residues. However, in this study, we observed the isomerization of an asparagine residue in SCPb. To validate this observation, we employed synthetic standards of SCPb for the all-l- and d-asparagine isomers. As demonstrated in Figure S5, the retention times of these synthetic standards closely matched those observed in biological extracts (Figure d). Particularly, the DAACP form of SCPb eluted before its all-L counterpartan elution profile that diverges from previously reported DAACPs. ,, This result indicates that isomerization at polar residues can affect distinct physicochemical properties of peptides, in contrast to the behavior of hydrophobic-residue DAACPs. In the case of FMRFamide, fragment ion mobility analysis at retention times of 17.5 and 22 min revealed a significant shift in the precursor and the y3 fragment ion mobilities, indicating isomerization at the methionine residue (Figure e–h).

Here, we have demonstrated the utility of these methods for the untargeted discovery of three novel DAACPs from the cerebral and pleural ganglia of .

Conclusions

We developed a comprehensive analytical workflow for identifying peptide isomers that includes a computational method for predicting isomerized residues from LC-IMS-MS data, a PIMT method for isoAsp identification, a CID-TIMS method for localizing isomerized residues, and a dedicated data analysis package for interpreting CID-TIMS results. By integrating these complementary approaches, we have established an effective, untargeted workflow for peptide isomer identification that bypasses the conventional requirements for peptide isolation and chiral chromatographic analyses, thus significantly streamlining the characterization process. Furthermore, the ability to identify and localize specific isomerization sites substantially reduces the need for synthetic standards when peptide isomer identities are confirmed via liquid chromatography. By applying these methods, we successfully identified eight isoaspartyl-containing peptides from rat hypothalamus and discovered three previously uncharacterized D-amino acid-containing peptides from . These findings expand our understanding of post-translational modifications in neuropeptides, which are important for the evaluation of the functional significance of peptide isomerization in diverse biological contexts. Our next efforts involve determining the extent of isomerization to determine how DAACP occupancy changes based on the physiological state. Our integrated workflow represents a significant advancement in peptidomics, offering new opportunities to explore the role of isomerized peptides in neuronal signaling pathways and other physiological processes.

Supplementary Material

ac5c02612_si_001.pdf (616.9KB, pdf)

Acknowledgments

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). This work is subject to the NIH Public Access Policy. Through acceptance of this federal funding, NIH has been given a right to make this manuscript publicly available in PubMed Central upon the Official Date of Publication, as defined by NIH. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.5c02612.

  • Additional experimental details, methods, and protocols for data analysis, additional results, including the localization of isomerized residues in the synthetic standard by CID-TIMS (Figure S1), EIC, and tandem MS spectra of a methylated sequence of prohormones of identified isoAsp-containing peptides (Figure S2–S4), and EIC of SCPB standards. Potential peptides with isomerized residues are also shown (Table S1 & S2) (PDF)

S.O. and J.V.S. conceptualized the experiments. S.R. dissected animals and provided several ganglion and rat hypothalamic tissues for the experiment, and S.O. and C.G. wrote code for peptide isomer prediction. S.O. and S.P. performed data analysis. S.O. handled data curation and prepared the original draft. J.V.S. acquired funding and resources for this project. All authors approved the final version of the manuscript.

The authors declare no competing financial interest.

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