Rapid and Efficient Radiolabeling of Short Peptides

Abstract

The use of peptides labeled with radioactive iodine isotopes enables rapid and highly sensitive assessment of their dynamic distribution in the body, as well as effective visualization and quantification of their metabolites in biological fluids and tissues down to femtomolar concentrations. In this work, we present the laboratory protocol for radioiodination of peptides containing oxidation-labile amino acids. Products of iodination of the histidine- and tyrosine-containing peptides were analyzed by NMR and high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) spectra. The proposed protocol describes a simple laboratory method for monitoring the extent of iodine isotope incorporation and a technique for isolating labeled peptides (3–10 amino acids) of high chemical and radiochemical purity without using HPLC. Using HPLC with fluorescence detection, we demonstrate the absence of significant oxidation of labile amino acids (Met, Trp, Tyr) during the radiolabeling process. The peptides labeled according to the protocol are obtained as lyophilizates with minimal inorganic salt content, allowing their direct use in cellular and animal model studies.

1. Introduction

Radiolabeling with iodine isotopes (¹²³I, ¹²⁴I, ¹²⁵I, or ¹³¹I) provides a convenient and widely used approach for imaging biologically active peptides, while such labeling is less frequently applied to full-length proteins, especially those with enzymatic activity. , Since 1948, scientists have employed iodine-labeled peptides in diverse biochemical and pharmacological studies. Commercially available radioactive iodine isotopes span a broad range of half-livesfrom a few hours (¹²³I) to several days (¹²⁴I and ¹³¹I) or even months (¹²⁵I)enabling researchers to select the optimal isotope for their experimental needs. γ-Emitting iodine isotopes outperform β-emitting natural or synthetic isotopes in detection efficiency because γ radiation interacts weakly with biological media and tissues. γ-Emitting compounds can be directly detected without the need for sample homogenization, liquid scintillation cocktails, or adjustments for sample-induced radiation quenching.

Most currently published protein radioiodination techniques provide a convenient and universal method for incorporating an activated iodine atom into the meta-position of tyrosine’s aromatic ring. , Since nearly all protein molecules contain at least one tyrosine residue and the tyrosine iodination reaction proceeds rapidly under physiological conditions (room temperature, pH 7–8), researchers widely use this approach for protein labeling.

The main challenges arise when introducing a radioactive label into a polypeptide chain lacking tyrosine. In our study, we needed to detect short peptides (3–10 amino acid residues) in biological models, where the only available site for radioiodination was the histidine side chain. To address this, we developed an efficient method for labeling such peptides with ¹²⁵I. Due to the chemical identity of iodine isotopes, researchers can apply this method without modifications to any other radioactive iodine isotope (¹²³I, ¹²⁴I, and ¹³¹I). The protocol also works for tyrosine-containing peptides.

2. Results and Discussion

2.1. Strategy for Radioiodination Method Development

Analysis of radiolabeled compounds in cells or tissues can follow two strategies: trace labeling, which incorporates ∼1% of the radioactive isotope, or near-stoichiometric labeling, which incorporates over 30% of the isotope. The trace labeling approach enables researchers to obtain biologically relevant data, reflecting the behavior of the native peptide equivalent. In contrast, while stoichiometric labeling produces biochemically more homogeneous samples, it necessitates careful evaluation of potential metabolic perturbations induced by a high concentration of iodinated compounds in animal or cellular systems. For most biological studies, trace labeling of peptides represents a more rational and physiologically appropriate strategy.

Recent publications predominantly describe reversed-phase high-performance liquid chromatography (RP-HPLC) as the standard method for isolating and purifying radiolabeled peptides. , Although RP-HPLC demonstrates exceptional efficiency for purifying high-molecular-weight radiolabeled compounds, this technique presents several practical limitations: significant operational costs, extended instrument downtime following radioactive work, and a requirement for thorough decontamination before subsequent nonradioactive applications. To address these challenges, our methodology development focused on eliminating HPLC from the purification workflow, designing a simplified chromatographic approach using disposable materials, maintaining high specific activity of the final product, and substantially reducing purification costs. The resulting protocol provides an efficient and cost-effective alternative to conventional RP-HPLC purification while preserving the radiochemical purity and biological activity of the labeled peptides.

Commercial radioactive iodine isotopes are typically supplied as alkaline aqueous sodium iodide solutions. To facilitate electrophilic aromatic substitution into target molecules, the iodide anion (I^–) requires oxidative activation to form reactive species such as molecular iodine (I₂), hypoiodous acid (HOI), or iodine monochloride (ICl). Numerous oxidizing agents for iodine activation have been reported in the literature, with the most commonly employed including hydrogen peroxide (H₂O₂), peroxyacids (e.g., peracetic acid), iodine monochloride (ICl), N-halosuccinimides (e.g., Suc-Cl), and N-chloroamides. ,, The activity of these initiators varies, and the outcome of the reaction may depend on the nature of the reactive species. For example, molecular iodine will form two additional equilibria (I₂ + I^– = I₃ ^– and I₂ + H₂O = HOI + H⁺ + I^–), and the newly gained reactive iodine species (I₃ ^–, HOI, and excess of I^–) may react concurrently with the main reaction. At the same time, adding more nonradioactive molecular iodine will dilute the overall percentage of the labeled product. In the cases of polarized initiators like I^δ+–Cl^δ− and Suc^δ+–Cl^δ−, the reaction mixture is less prone to form multiple reactive iodine species; however, the surface charge distribution and sterical factors may drive mono- or bis- aromatic substitution reactions, depending on the coordinative capabilities of the amino acid. Protected and nonprotected amino acids also differ in the outcome; that is why choosing the right iodination strategy needs careful consideration.

Among commercially available oxidants, N-chloro-p-toluenesulfonamide (chloramine-T, CAT) offers distinct advantages for peptide radiolabeling in aqueous solutions: solid, water-soluble formulation, exceptional storage stability with minimal loss of oxidative capacity, and rapid and efficient conversion of I^– to reactive ICl species. Despite its valuable properties, CAT is a potent oxidizing agent for amino acids. Consequently, when iodinating oxidation-sensitive substrates, researchers must carefully optimize both the reaction conditions and the reaction time to achieve high efficiency and avoid oxidative damage to the peptides.

There are two approaches to protecting labile amino acid side chains (Cys, Met, Trp, and Tyr) from oxidative damage. The first method employs solid heterogeneous oxidants in aqueous media. Phase separation minimizes contact between water-soluble labile compounds and water-insoluble oxidants. Heterogeneous radioiodination activators include Pierce Iodination Beads and iodogen-coated filters and tubes. While heterogeneous oxidizers are less aggressive, they markedly slow down the iodination reaction and necessitate monitoring the reaction’s completeness. Moreover, they often cause substantial peptide adsorption onto oxidant crystals or coated matrices. This undesirable adsorption becomes particularly pronounced for peptides rich in hydrophobic amino acids.

The second approach to mitigate the destructive oxidative effects of N-chloroamides on labile peptide components involves the in situ generation of N-chloro derivatives of secondary amines. These secondary amines exhibit reduced oxidative potential while maintaining sufficient activity to convert iodide anions into reactive HOI species. , Although using intermediate N-chloro derivatives of secondary amines decreases unwanted oxidative modification of peptide and protein side chains, introducing this additional oxidant complicates reaction mixture separation. The final mixture contains various components of both primary and secondary oxidizers. Since each organic byproduct of iodide oxidative activation may possess biological activity, precise purification of the resulting peptide becomes necessary. This requires reversed-phase or ion-exchange HPLC with radioactive detection. For most macromolecules (proteins), size-exclusion chromatography cartridges (Sephadex-G25, G50, etc.) provide rapid and efficient purification of protein preparations from low-molecular-weight radioiodination byproducts. However, this approach has a drawback: sample dilution. For short peptides and hydrophobic macromolecules, size-exclusion chromatography using cartridges or minicolumns proves largely impractical due to the limited capacity and resolution of such sorbents. Increasing the sorbent volume inevitably leads to labeled product loss through irreversible nonspecific adsorption. Therefore, we consider optimization of conventional electrophilic aromatic iodination conditions, with careful consideration of the reaction mechanism’s known features, to be a promising approach.

It is known that the rate of electrophilic aromatic substitution with I⁺ increases with pH and is catalyzed by HPO₄ ^2– ions, while the destructive oxidative effects of both CAT and I₂ (ICl) remain pH-insensitive. CAT exhibits direct chlorinating activity toward amino acids at neutral pH, but this process slows dramatically at elevated pH. Based on these findings, we adopted the following principles for our method development: conducting the reaction under alkaline conditions (pH 8.8–9.5), using minimal temperature to prevent crystallization of inorganic buffer components, and minimizing reaction duration. To determine the optimal time for electrophilic peptide iodination, we monitored reaction progress by thin-layer chromatography (TLC) and stopped the reaction by oxidant neutralization upon reaching 92–95% conversion of radioactive I^– (Figure ). The reaction may be quenched with a reductant, usually by the addition of sodium sulfite or sodium metabisulfite. Section contains a detailed description of the radioiodination protocol we have developed.

1.

Steps of the peptide radioiodination and purification: (A) Chemical synthesis on an ice bath; (B) TLC control of the radioiodination full completion; (C) chromatography fractionalization of the reaction mixture on a cellulose microcolumn; (D) TLC control of the pure product fractions; and (E) aliquoting for experiments with cell cultures and animals.

2.2. Iodide Incorporation in the Amino Acids

There are two amino acids involved in electrophilic aromatic substitution: histidine and tyrosine. Tyrosine is prone to bis-iodination via the S_EAr mechanism in the ortho-position to the phenolic OH, which acts as a directing group for the incoming nucleophile. Histidine captures iodine in the imidazole cycle, with the varying outcome of the iodination products depending on conditions. It was established in the late 1960s in the reactions with methylated histidines that the iodination process required initial ICl coordination via nitrogen of the imidazole to form the monoiodination product, which was assigned to be 5-iodohistidine. Later, the structure of the histidine product was misattributed in several papers as 2-iodohistidine and further frequently cited the wrong way in other publications. At the same time, the 2-iodo derivative can be obtained in acidic conditions via regiospecific dehalogenation: the excess of an iodinating agent may result in 2,5-diiodohistidine formation, which in acidic conditions undergoes exchange of iodide at the fourth position to the proton. Since most protocols recommend using alkaline buffer solutions for nonprotected amino acids, and in this reaction, the possibility of H⁺ assistance is ruled out.

Rates of incorporation of iodine significantly increase in alkaline pH buffer solutions due to deprotonation of the nitrogen atom in the histidine residue or the phenolic OH in tyrosine. It is known that pH 9 facilitates the reaction of a protein to achieve a maximum conversion (between pH values 7 and 9) of His into monoiodohistidine. We have performed preliminary screening using ¹H NMR integration of the signals for approximate conversion rate and yields of the products in the pH range between 4.4 and 10.3 (Figures S1–S3). Iodination at higher pH values (pH 10.3) proceeds with the same yields of 5-iodohistidine; only the 2,5-diiodo-product yield is increased. This phenomenon might complicate the workup in the radioactive synthesis: since the radioactive label is twice as intense in the double-substituted product, it needs to be separated and uniformly counted in the biological studies; multiple His residues in one protein may be affected to result in a row of labeled products. In terms of short peptide transformations, high pH values above 9.5 result in more oxidation products and risks of peptide decomposition.

We decided to validate the outcome of the iodination reaction of the iodide + CAT system based on the model short peptides N-acetyl HAEE amide (or shortly, HAEE below in the text) and GYE and elucidate their structure using HPLC-HRMS and one-dimensional (1D) and two-dimensional (2D) NMR spectroscopies. It appears that both histidine and tyrosine follow the expected patterns described in the literature, which allows us to conclude their mechanism of electrophilic aromatic substitution (Scheme ).

1. Iodination Mechanism of Histidine- and Tyrosine-Containing Peptides HAEE and GYE.

We confirmed by NMR spectra that iodination of l-histidine (both in HAEE sequence and pure amino acid) using the iodide + CAT system leads to 5-iodohistidine isomer (Figure ) with traces of the bis-iodinated product. This was done using a reference sample with comprehensive ¹H, ¹³C, and ¹H–¹³C correlation spectra analysis (Figures S9–S13). For iodinated HAEE tetrapeptide, a direct comparison shows a product chemical shift difference of only about 0.01 ppm. Using HPLC-HRMS, we have verified the position of the iodinated amino acid in HAEE peptide and studied the fragmentation of the product and traces of the starting HAEE and bis-iodinated HAEE (Figure ). Additionally, HPLC-HRMS data for tyrosine-containing GYE tripeptide iodination concluded the formation of neat bis-substituted GYE. Detailed fragmentation analysis of each compound is found in the Supporting Information (Figure S8).

2.

¹H NMR spectra of the aromatic region of iodinated l-histidine and the HAEE tetrapeptide. Color of the signals indicates assignment to the protons in the structure; CAT – chloramine-T; (A) spectrum of l-His iodinated using CAT in the reaction mixture; (B) differential spectrum showing l-His and 5-iodo-l-His; (C) differential spectrum l-His; (D) ¹H NMR spectrum of HAEE tetrapeptide iodinated using chloramine-T; (E) reference ¹H NMR spectrum of the iodinated HAEE tetrapeptide reaction mixture; and (F) HPLC chromatogram characterizing HAEE iodination. Monoiodinated product prevails (EIC C₂₁H₃₀IN₇O₉ with the retention time 2.5 min), traces of the starting HAEE (EIC C₂₁H₃₁N₇O₉ with the retention time of 3.1 min) and bis-iodinated product (EIC C₂₁H₂₉I₂N₇O₉ with the retention time of 2.3 min), and (G–I) HRMS profiles with fragmentation ions of the starting HAEE, monoiodinated product, and bis-iodinated product.

3.

Sample peptides subjected to iodination. The arrows indicate the predominant insertion sites of ¹²⁵I.

2.3. Validation of the Short Peptide Radioiodination Method

To demonstrate the methodology for labeling with iodine-125, we used several histidine-containing peptides in the reaction: N-Ac-His-Ala-Glu-Glu-NH₂ (HAEE), Ac-His-Ala-Glu-Glu-Pro-Gly-Pro-NH₂ (HAEEPGP), amyloid β-protein (1–10) (Asp-Ala-Glu-Phe-Arg-His-Asp-pSer-Gly-Tyr), and RGDREEAH (Arg-Gly-Asp-Arg-Glu-Glu-Ala-His) (Figure ). Peptides containing HAEE sequence are of particular importance for ¹²⁵I-radiolabeling: N-Ac-His-Ala-Glu-Glu-NH₂ is a potential therapeutic agent for the treatment of Alzheimer’s disease (AD). HAEEPGP is regarded as a more HAEE prodrug with the prolonged release time; the hydrolyzable Pro-Gly-Pro motif coherently functions as a regulatory peptide for neuroregeneration. Thus, preparing such ¹²⁵I-labeled peptides is valuable for monitoring drug candidates’ permeability through the blood-brain barrier (BBB) and distribution in tissues.

Briefly, these four peptides were subjected to the following steps. A total of 50 nmol of each peptide reacted with radioactive iodine activated by CAT. The reaction progress was monitored by using TLC (Figure ). Once isotope incorporation into the peptide reached 95%, the reaction was quenched, and the labeled peptides were purified by microcolumn chromatography on microcrystalline cellulose. Fractions collected during chromatography were analyzed by TLC (Figure ). Fractions containing pure iodinated peptides were pooled and lyophilized. Radiochemical purity was confirmed by scanning the radioautograph of the TLC plate. As a result, 50 nmol of the peptides was successfully radiolabeled. The radioactive purity of the labeled peptides exceeded 98%, with a molar radioactivity of 10.8 Ci/mmol.

4.

(A) Monitoring the progress of the reaction of peptides with radioautograph on a silica gel TLC plate. One – HAEE and 2 – HAEEPGP, elution in 2-butanol/water/acetic acid (3:2:1). (B) Monitoring the progress of the reaction with a radioautograph on a cellulose TLC plate. Three – amyloid protein β (1–10) and 4 – RGDREEAH, elution in n-butanol/water/acetic acid (3:1:1).

5.

Silica gel TLC control of the cellulose microcolumn chromatographic purification of radioiodinated HAEE and HAEEPGP. (A) Radioautograph of silica gel TLC plates (the first symbol refers to peptide 4 or 7 amino acids, the second symbol refers to the fraction number). (B, C) Radioautograph scan of 4″8 and 7′′7 lines, correspondingly.

Thus, the versatility of the proposed protocol has been established for His- and Tyr-containing proteins, and the progress of incorporation of iodide-125 has been detected by TLC detection of ¹²⁵I in a continuous and rapid manner, allowing for minimizing oxidative damage of other labile amino acids in the proteins and eliminating byproduct isomers. Furthermore, we have tested the iodination of other lengthy peptides of β-amyloid origin: Aβ16 (DAEFRHDSGYEVHHQK), Aβ42 (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA), and isoAβ42 – the Asp7-isomerized Aβ42 (Figure S15).

2.4. Validation of Radiolabeling Conditions: Confirming the Absence of Oxidative Amino Acid Modifications

The issue of oxidative modifications of amino acid side chains during peptide radiolabeling is critically important for drawing accurate conclusions about peptide distribution in cells and organisms, as well as for studying peptide metabolites. Previous studies have shown that CAT effectively oxidizes the side chains of Met, Tyr, Cys, and Trp in acidic media, whereas under alkaline conditions, only Cys and Met are oxidized, with the latter forming methionine sulfoxide. Some authors report that methionine sulfoxide can be reduced back to methionine using DTT at elevated temperatures, while another study suggests using 2-mercaptoethanol for this purpose.

The peptides used in our study do not contain oxidation-labile amino acids such as Cys, Met, Pro, or Trp. To confirm that our optimized reaction conditions do not cause significant oxidative modifications of susceptible amino acids, we performed a control experiment using a mixture of Met, Tyr, Trp, and Ala (as a reference) instead of a peptide. The reaction products were analyzed by HPLC with precolumn derivatization using dansyl chloride, following the previously published protocol. HPLC analysis revealed no changes in the chromatographic behavior of the amino acids, indicating the absence of significant oxidation (Figures and ). Another control experiment with HPLC detection revealed that the selected temperature of the reaction (0 °C) is optimal for methionine intactness under these conditions (Figure S14). Unfortunately, the DnsCl derivatization method followed by RP-HPLC analysis is not applicable to cysteine because its sulfhydryl group reduces dansyl chloride. It is highly likely that even under the mild conditions of the proposed protocol, cysteine, being a strong reducing agent, undergoes oxidative modification.

6.

RP-HPLC analysis of methionine oxidation with precolumn dansylation. (A) Oxidation under current protocol conditions (1 equiv. CAT, 0 °C, pH 9.0, 5 min), 1 – methionine; (B) oxidation after 5 min in 2% H₂O₂ at pH 9.0, 2 – methionine sulfoxide; and (C) sample compounds, 1 – methionine and 3 – dansylamide.

7.

RP-HPLC analysis of oxidation of the amino acid mixture. 1 – dansylamide, 2 – Gly, 3 – Pro, 4 – Trp, 5 – Tyr, 4ox – oxidized tryptophan; (A) after 5 min in 2% H₂O₂ at pH 9.0; (B) one of the popular radioiodination conditions (1 equiv. CAT, 22 °C, pH 7.0, 2 min); (C) at current protocol conditions (1 equiv. CAT, 0 °C, pH 9.0, 5 min); and (D) a mixture of references (Gly, Pro, Trp, Tyr).

3. Conclusions

A simple, easily scalable, and adaptable laboratory protocol was developed for the radiolabeling of short peptides with varying compositions. This method is particularly suitable for labeling peptides containing oxidation-labile amino acids. The developed protocol includes a rapid method for monitoring iodine isotope incorporation and a fast and cost-effective purification technique for labeled peptides (3–10 amino acids long) without the need for HPLC. The resulting peptides exhibit high chemical and radiochemical purity and the absence of significant oxidative modifications in oxidation-sensitive amino acids. The radiolabeled peptides are obtained as lyophilizates with minimal inorganic salt content, allowing for their direct use in both cell-based experiments and animal studies involving intravenous administration.

4. Materials and Methods

4.1. Cautionary Notes

Methods of radioiodination involve the use of relatively high amounts of ¹²⁵I or ¹³¹I in the form of sodium iodide solutions, which are known to be volatile in air upon acidification and oxidation and readily absorbed by the thyroid gland if inhaled or absorbed internally. So your laboratory must be equipped with a shielded fume hood rated for iodination, a sufficient number of lead blocks, a lead cave, and radiometric and dosimetry equipment. All manipulations with radioactive materials and radioactive waste should be coordinated with a radiation safety department (officer).

4.2. Equipment

Reagents and solvents were purchased from Sigma-Aldrich, Thermo Fischer/Acros, J&K Scientific, and TCI Europe (Belgium) in Molecular biology grade and HPLC-HRMS grade and used without further purification. Storage Phosphor Screen BAS-IP SR 2025 E Super Resolution, 20 cm × 25 cm (Cytiva), screen imager Typhoon FLA 9500 (GE Healthcare). Chromatograms were registered on an HPLC system with the fluorometric detector RF-20A (Shimadzu Scientific; (λ_ex = 340 nm, λ_em = 530 nm)), paired with the DAT controller AD-24 (Ampersand, Russia) and analyzed using Multichrom v.3.4 software (Ampersand, Russia). Compounds were applied onto a Cosmosil C18-MS-II column (Akvilon, Russia; 4.0 mm × 250 mm column, particle size 5 μm; other brands with similar properties are suitable) and eluted at 40 °C with a flow rate of 1.0 mL/min in a following gradient of HPLC buffer A (79.5% H₂O + 10% CH₃CN + 10% MeOH + 0.5% propionic acid) in HPLC buffer B (79.5% CH₃CN + 20% THF + 0.5% propionic acid): 0–3 min 0% B, 3–85 min0–85% B, 85–90 min85–100% B, and 90–95 min100–0%. Fritted glass or polyethylene column 10 mm × 100 mm or polyethylene pipet tips V = 1 mL equipped with a glass fiber pad were used for filtration.

4.3. Materials

Microgranular (microcrystalline) cellulose powder for column chromatography (CC31 from Whatman or analogue), silica gel TLC plates (TLC Silica gel 60 F₂₅₄ from Merck or analogue), 2-butanol, glacial acetic acid, chloramine-T all chemicals were reactive grade from Sigma-Aldrich. ¹²⁵I radioiodine was 1 MBq/μL (80 MBq/nmol), 98% radiochemical purity, and was supplied as a 50 mM NaOH solution.

4.4. Buffers and Solutions

The following buffers and elution systems were prepared for the radioiodination and purification procedures. Buffer A: 200 mM aqueous NaH₂PO₄ buffer, pH 9.0. TLC elution buffer B is a mixture of 2-butanol, water, and glacial acetic acid in a 3:2:1 volume ratio. Column elution system C: a mixture of 2-butanol, water, and glacial acetic acid in a 5:1:1 volume ratio. Column elution system D: a mixture of 2-butanol, water, and glacial acetic acid in a 3:1:1 volume ratio.

4.5. Radioiodination Procedure

The peptide (50 nmol) was initially lyophilized to dryness under a vacuum. The dried peptide was then dissolved in 10 μL of buffer A. To this solution, 20 μL of ¹²⁵I–NaI solution (0.25 nmol, 20 MBq) and 2 μL of a cold carrier NaI solution in buffer A (1.25 nmol) were added. The reaction mixture was cooled to 0–4 °C on an ice bath, and the oxidation was initiated by adding 20 nmol of CAT in 2 μL of buffer A (28 mg/mL). After incubation on ice for 5–7 min, a small aliquot (0.1–0.2 μL) was taken out for immediate TLC analysis. The main reaction mixture was then frozen by using liquid nitrogen or dry ice.

Based on the TLC results, the subsequent steps were determined. If the radioiodine incorporation reached 90–95%, the reaction was quenched by adding 5 μL of a 0.3 mM aqueous DTT solution. If incorporation was below 90%, the protocol was repeated with the addition of CAT. After quenching, the mixture was incubated at room temperature for 5 min, lyophilized to complete dryness, and the residue was dissolved in 50–70 μL of the isopropanol/water mixture (4:1, v/v) for subsequent purification.

4.6. Thin-Layer Chromatography Analysis

For TLC analysis, the collected aliquot was mixed with 100 μL of a 1% aqueous DTT solution to quiet the reaction. Approximately 0.5 μL of this mixture was spotted on a silica gel TLC plate. The plate was developed in a chamber presaturated with TLC elution buffer B. After development, the plate was dried in a ventilated hood for 5–7 min without heating. Radioactive components were visualized and quantified using a phosphor imager with a typical exposure time of 1–2 min for ¹²⁵I.

4.7. Purification by Microcrystalline Cellulose Chromatography

A chromatography column was prepared by swelling 10 g of microgranular cellulose powder in 40 mL of 50% aqueous isopropanol for 12 h at room temperature (20–25 °C). The slurry was packed into a glass column (8–10 mm inner diameter) or a 1 mL pipet tip fitted with a glass fiber pad. After sedimentation, a cellulose bed volume of 450–500 μL was formed. The column was prewashed sequentially with 2 mL of isopropanol, followed by 2 mL of 2-butanol.

The dissolved reaction mixture was then applied to the preconditioned column. The column was washed with 100 μL of a 1:1 (v/v) isopropanol/2-butanol mixture. The labeled peptide was subsequently eluted using a step-gradient: first with 500 μL of elution system C, followed by 500 μL of elution system D. Fractions of approximately 200 μL were collected and analyzed by TLC (as described above; see also Figure ). Fractions containing the pure radiolabeled product were pooled and lyophilized for final storage.

4.8. HPLC Analysis of Oxidation

The lyophilized sample was dissolved in 20 μL of water. Then, 80 μL of a saturated aqueous sodium carbonate (Na₂CO₃) solution and 20 μL of a dansyl chloride solution in dioxane (20 mg/mL) were added for derivatization. The mixture was sonicated in an ultrasonic bath at 40 °C for 10 min. The reaction was quenched by adding 2 μL of dimethylamine. The dansyl-derivatized peptides were extracted twice with 200 μL of n-butanol. Extracts were analyzed by HPLC with fluorescence detection (excitation λ = 340 nm, emission λ = 530 nm).

Glossary

Abbreviations

HPLC

high-performance liquid chromatography

TLC

thin-layer chromatography

dansyl

5-(dimethylamino)­naphthalene-1-sulfonyl

CAT

chloramine-T, sodium N-chloro-4-methylbenzenesulphonylamide

DTT

dithiotreitol, 1,4-dimercapto-2,3-butanediol

RP-HPLC

reverse phase HPLC

DnsCl

5-(dimethylamino)­naphthalene-1-sulfonyl chloride

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c04908.

• Contains preliminary screening of the iodination conditions using ¹H NMR; HPLC, HRMS, and NMR analysis of the iodination products; radioautographs of the sample peptides (PDF)

The manuscript was written through contributions of all authors. Method development: D.V.Y., O.I.K., V.A.M., P.N.S., Y.V.T. Conceptualization: D.V.Y., V.A.M. Funding acquisition: A.A.M. Resources: A.A.M., S.N.K. Drafting and editing: D.V.Y., O.I.K., A.A.M., P.N.S., Y.V.T., V.A.M. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.