The preparation of specifically iodine-125 (125I)-labeled peptides of high purity and specific activity represents a key tool for the detailed characterization of their binding properties in interaction with their binding partners. Early synthetic methods for the incorporation of iodine faced challenges such as harsh reaction conditions, the use of strong oxidants and low reproducibility. Herein, we review well-established radiolabeling strategies available to incorporate radionuclide into a protein of interest, and our long-term experience with a mild, simple and generally applicable technique of 125I late-stage-labeling of biomolecules using the Pierce iodination reagent for the direct solid-phase oxidation of radioactive iodide. General recommendations, tips, and details of optimized chromatographic conditions to isolate pure, specifically 125I-mono-labeled biomolecules are illustrated on a diverse series of (poly)peptides, ranging up to 7.6 kDa and 67 amino acids (aa). These series include peptides that contain at least one tyrosine or histidine residue, along with those featuring disulfide crosslinking or lipophilic derivatization. This mild and straightforward late-stage-labeling technique is easily applicable to longer and more sensitive proteins, as demonstrated in the cases of the insulin-like growth factor binding protein-3 (IGF-BP-3) (29 kDa and 264 aa) and the acid-labile subunit (ALS) (93 kDa and 578 aa).

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague 16610 Czech Republic

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Rangger C., Haubner R. Radiolabelled peptides for positron emission tomography and endoradiotherapy in oncology. Pharmaceuticals (Basel) 2020;13 PubMed PMC

Fani M., Maecke H.R. Radiopharmaceutical development of radiolabelled peptides. Eur. J. Nucl. Med. Mol. Imaging. 2012;39:S11–S30. PubMed

Oliveira M.C., Correia J.D.G. Biomedical applications of radioiodinated peptides. Eur. J. Med. Chem. 2019;179:56–77. PubMed

Ambrosini V., Fani M., Fanti S., et al. Radiopeptide imaging and therapy in Europe. J. Nucl. Med. 2011;52:42S–55S. PubMed

Patel A.C., Matthewson S.R. In: Molecular Biomethods Handbook. Rapley R., Walker J.M., editors. Humana Press; Totowa, NJ: 1998. Radiolabeling of peptides and proteins; pp. 401–411.

Sugiura G., Kühn H., Sauter M., et al. Radiolabeling strategies for tumor-targeting proteinaceous drugs. Molecules. 2014;19:2135–2165. PubMed PMC

Krenning E.P., Breeman W.A.P., Kooij P.P.M., et al. Localisation of endocrine-related tumours with radioiodinated analogue of somatostatin. Lancet. 1989;1:242–244. PubMed

Dillman R.O. Radiolabeled anti-CD20 monoclonal antibodies for the treatment of B-cell lymphoma. J. Clin. Oncol. 2002;20:3545–3557. PubMed

Goldsmith S.J. Radioimmunotherapy of lymphoma: Bexxar and Zevalin. Semin. Nucl. Med. 2010;40:122–135. PubMed

Robinson M.K., Doss M., Shaller C., et al. Quantitative immuno-positron emission tomography imaging of HER2-positive tumor xenografts with an iodine-124 labeled anti-HER2 diabody. Cancer Res. 2005;65:1471–1478. PubMed

Pandit-Taskar N., Zanzonico P.B., Kramer K., et al. Biodistribution and dosimetry of intraventricularly administered 124I-Omburtamab in patients with metastatic leptomeningeal tumors. J. Nucl. Med. 2019;60:1794–1801. PubMed PMC

Yerrabelli R.S., He P., Fung E.K., et al. IntraOmmaya compartmental radioimmunotherapy using 131I-omburtamab—pharmacokinetic modeling to optimize therapeutic index. Eur. J. Nucl. Med. Mol. Imag. 2021;48:1166–1177. PubMed PMC

Petrov S.A., Yusubov M.S., Beloglazkina E.K., et al. Synthesis of radioiodinated compounds. Classical approaches and achievements of recent years. Int. J. Mol. Sci. 2022;23 PubMed PMC

Kim E.J., Kim B.S., Choi D.B., et al. Enhanced tumor retention of radioiodinated anti-epidermal growth factor receptor antibody using novel bifunctional iodination linker for radioimmunotherapy. Oncol. Rep. 2016;35:3159–3168. PubMed PMC

Jiráček J., Žáková L., Marek A. Radiolabeled hormones in insulin research, a minireview. J. Label. Compd. Radiopharm. 2020;63:576–581. PubMed

Pražienková V., Marek A., Maletínská L. Iodination of CART(61-102) peptide: Preserved binding and anorexigenic activity in mice. J. Label. Compd. Radiopharm. 2021;64:61–64. PubMed

Chen X., Park R., Shahinian A.H., et al. Pharmacokinetics and tumor retention of 125I-labeled RGD peptide are improved by PEGylation. Nucl. Med. Biol. 2004;31:11–19. PubMed

Doré S., Kar S., Rowe W., et al. Distribution and levels of [125I]IGF-I, [125I]IGF-II and [125I]insulin receptor binding sites in the hippocampus of aged memory-unimpaired and -impaired rats. Neuroscience. 1997;80:1033–1040. PubMed

Wei S., Li C., Li M., et al. Radioactive iodine-125 in tumor therapy: Advances and future directions. Front. Oncol. 2021;11 PubMed PMC

Wang H., Shi H.-B., Qiang W.-G., et al. CT-guided radioactive 125I seed implantation for abdominal incision metastases of colorectal cancer: Safety and efficacy in 17 patients. Clin. Colorectal Cancer. 2023;22:136–142. PubMed

Wall J.S., Paulus M.J., Gleason S., et al. Micro-imaging of amyloid in mice. Methods Enzymol. 2006;412:161–182. PubMed PMC

Coenen H.H., Mertens J., Mazière B. Springer; Dordrecht: 2006. Radioionidation Reactions for Radiopharmaceuticals: Compendium for Effective Synthesis Strategies; pp. 1–101.

Mushtaq S., Jeon J., Shaheen A., et al. Critical analysis of radioiodination techniques for micro and macro organic molecules. J. Radioanal. Nucl. Chem. 2016;309:859–889.

Wynendaele E., Bracke N., Stalmans S., et al. Development of peptide and protein based radiopharmaceuticals. Curr. Pharmaceut. Des. 2014;20:2250–2267. PubMed

Kręcisz P., Czarnecka K., Królicki L., et al. Radiolabeled peptides and antibodies in medicine. Bioconjugate Chem. 2021;32:25–42. PubMed PMC

Li M., Wang S., Kong Q., et al. Advances in macrocyclic chelators for positron emission tomography imaging. View. 2023;4

Tolmachev V., Stone-Elander S. Radiolabelled proteins for positron emission tomography: Pros and cons of labelling methods. Biochim. Biophys. Acta. 2010;1800:487–510. PubMed

Wilbur D.S. Radiohalogenation of proteins: An overview of radionuclides, labeling methods, and reagents for conjugate labeling. Bioconjugate Chem. 1992;3:433–470. PubMed

Lin S.-L., Lin C.-Y., Lee W., et al. Mini review: Molecular interpretation of the IGF/IGF-1R axis in cancer treatment and stem cells-based therapy in regenerative medicine. Int. J. Mol. Sci. 2022;23 PubMed PMC

Belfiore A., Malaguarnera R., Vella V., et al. Insulin receptor isoforms in physiology and disease: An updated view. Endocr. Rev. 2017;38:379–431. PubMed PMC

Satoh F., Smith D.M., Gardiner J.V., et al. Characterization and distribution of prolactin releasing peptide (PrRP) binding sites in the rat – evidence for a novel binding site subtype in cardiac and skeletal muscle. Br. J. Pharmacol. 2000;129:1787–1793. PubMed PMC

Hunter S.J., Boyd A.C., O’Harte F.P.M., et al. Demonstration of glycated insulin in human diabetic plasma and decreased biological activity assessed by euglycemic-hyperinsulinemic clamp technique in humans. Diabetes. 2003;52:492–498. PubMed

Chard T. third ed. Elsevier; Amsterdam: 1987. An Introduction to Radioimmunoassay and Related Techniques. T.S. Work, E. Work, Laboratory Techniques in Biochemistry and Molecular Biology; pp. 291–527.

Lappin G., Temple S. CRC Press; Boca Raton: 2006. Radiotracers in Drug Development; pp. 1–320.

Derdau V., Elmore C.S., Hartung T., et al. The future of (radio)-labeled compounds in research and development within the life science industry. Angew. Chem. Int. Ed. 2023;62 PubMed

Németh J., Oroszi G., Jakab B., et al. 125I-labeling and purification of peptide hormones and bovine serum albumin. J. Radioanal. Nucl. Chem. 2002;251:129–133.

Salacinski P.R.P., McLean C., Sykes J.E.C., et al. Iodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen) Anal. Biochem. 1981;117:136–146. PubMed

Ferris T., Carroll L., Jenner S., et al. Use of radioiodine in nuclear medicine—a brief overview. J. Label. Compd. Radiopharm. 2021;64:92–108. PubMed

De la Vieja A., Riesco-Eizaguirre G. Radio-iodide treatment: From molecular aspects to the clinical view. Cancers. 2021;13 PubMed PMC

Schäffer L., Larsen U.D., Linde S., et al. Characterization of the three 125I-iodination isomers of human insulin-like growth factor I (IGF1) Biochim. Biophys. Acta. 1993;1203:205–209. PubMed

Bailey G.S. In: Walker J.M., editor. vol. 32. Humana Press; Totowa, NJ: 1994. Labeling of peptides and proteins by radioiodination; pp. 441–448. (Basic Protein and Peptide Protocols).

Maletínská L., Tichá A., Nagelová V., et al. Neuropeptide FF analog RF9 is not an antagonist of NPFF receptor and decreases food intake in mice after its central and peripheral administration. Brain Res. 2013;1498:33–40. PubMed

Behr T.M., Gotthardt M., Becker W., et al. Radioiodination of monoclonal antibodies, proteins and peptides for diagnosis and therapy. A review of standardized, reliable and safe procedures for clinical grade levels kBq to GBq in the Göttingen/Marburg experience. Nuklearmedizin. 2002;41:71–79. PubMed

Hermanson G.T. third ed. Academic Press; Boston: 2013. Isotopic Labeling Techniques. Bioconjugate Techniques; pp. 507–534.

Tolomeu H.V., Fraga C.A.M. Imidazole: Synthesis, functionalization and physicochemical properties of a privileged structure in medicinal chemistry. Molecules. 2023;28 PubMed PMC

Berridge M.S., Jiang V.W., Welch M.J. Intramolecular effects of 125I decay in o-iodotyrosine. Radiat. Res. 1980;82:467–477.

Ramachandran L.K. Protein-iodine interaction. Chem. Rev. 1956;56:199–218.

Liu Z., Julian R.R. Deciphering the peptide iodination code: Influence on subsequent gas-phase radical generation with photodissociation ESI-MS. J. Am. Soc. Mass Spectrom. 2009;20:965–971. PubMed

Conlon J.M. In: The Protein Protocols Handbook. Walker J.M., editor. Humana Press; Totowa, NJ: 2009. Preparation of 125I-labeled peptides and proteins with high specific activity using IODO-GEN; pp. 1735–1742.

Mock B., Zheng Q.-H. In: Nuclear Medicine. second ed. Henkin R.E., editor. Elsevier; 2006. Radiopharmaceutical chemistry: Iodination techniques; pp. 397–405.

Contreras M.A., Bale W.F., Spar I.L. In: Langone J.J., Van Vunakis H., editors. vol. 92. Academic Press; 1983. Iodine monochloride (ICl) iodination techniques; pp. 277–292. (Methods in Enzymology). PubMed

Breslav M., McKinney A., Becker J.M., et al. Preparation of radiolabeled peptides via an iodine exchange reaction. Anal. Biochem. 1996;239:213–217. PubMed

Wajchenberg B.L., Pinto H., Torres de Toledo e Souza I., et al. Preparation of iodine-125-labeled insulin for radioimmunoassay: Comparison of lactoperoxidase and chloramine-T iodination. J. Nucl. Med. 1978;19:900–905. PubMed

Morrison M., Bayse G.S. Catalysis of iodination by lactoperoxidase. Biochemistry. 1970;9:2995–3000. PubMed

Al-Shehri S.S., Duley J.A., Bansal N. Xanthine oxidase-lactoperoxidase system and innate immunity: Biochemical actions and physiological roles. Redox Biol. 2020;34 PubMed PMC

Kristensen J.B., Pedersen M.L., Larsen U.D., et al. [125I], [127I]-and [14C]-labelling of the GLP-1-(7-37) derivative NN2211. J. Label. Compd. Radiopharm. 2003;46:499–510.

Marchalonis J.J. An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem. J. 1969;113:299–305. PubMed PMC

Muccioli G., Ghè C., Ghigo M.C., et al. Specific receptors for synthetic GH secretagogues in the human brain and pituitary gland. J. Endocrinol. 1998;157:99–106. PubMed

Thorell J.I., Johansson B.G. Enzymatic iodination of polypeptides with 125I to high specific activity. Biochim. Biophys. Acta. 1971;251:363–369. PubMed

Fraker P.J., Speck J.C., Jr. Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Biochem. Biophys. Res. Commun. 1978;80:849–857. PubMed

Markwell M.A.K., Fox C.F. Surface-specific iodination of membrane proteins of viruses and eucaryotic cells using 1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril. Biochemistry. 1978;17:4807–4817. PubMed

Koppe M.J., Bleichrodt R.P., Soede A.C., et al. Biodistribution and therapeutic efficacy of 125/131I-, 186Re-, 88/90Y-, or 177Lu-labeled monoclonal antibody MN-14 to carcinoembryonic antigen in mice with small peritoneal metastases of colorectal origin. J. Nucl. Med. 2004;45:1224–1232. PubMed

Liu Z., Jin C., Yu Z., et al. Radioimmunotherapy of human colon cancer xenografts with 131I-labeled anti-CEA monoclonal antibody. Bioconjugate Chem. 2010;21:314–318. PubMed

Wang K., Adelstein S.J., Kassis A.I. DMSO increases radioiodination yield of radiopharmaceuticals. Appl. Radiat. Isot. 2008;66:50–59. PubMed PMC

Ünak T., Akgün Z., Yildirim Y., et al. Self-radioiodination of iodogen. Appl. Radiat. Isot. 2001;54:749–752. PubMed

Janssens Y., Verbeke F., Debunne N., et al. Analysis of iodinated quorum sensing peptides by LC–UV/ESI ion trap mass spectrometry. J. Pharm. Anal. 2018;8:69–74. PubMed PMC

Markwell M.A.K. A new solid-state reagent to iodinate proteins. I. Conditions for the efficient labeling of antiserum. Anal. Biochem. 1982;125:427–432. PubMed

Richardson K., Parker C.D. Identification and characterization of Vibrio cholerae surface proteins by radioiodination. Infect. Immun. 1985;48:87–93. PubMed PMC

Cavina L., van der Born D., Klaren P.H.M., et al. Design of radioiodinated pharmaceuticals: Structural features affecting metabolic stability towards in vivo deiodination. Eur. J. Org. Chem. 2017;2017:3387–3414. PubMed PMC

Geissler F., Anderson S.K., Venkatesan P., et al. Intracellular catabolism of radiolabeled anti-μ antibodies by malignant B-cells. Cancer Res. 1992;52:2907–2915. PubMed

Bakker W.H., Krenning E.P., Breeman W.A., et al. In vivo use of a radioiodinated somatostatin analogue: Dynamics, metabolism, and binding to somatostatin receptor-positive tumors in man. J. Nucl. Med. 1991;32:1184–1189. PubMed

Foulon C.F., Reist C.J., Bigner D.D., et al. Radioiodination via D-amino acid peptide enhances cellular retention and tumor xenograft targeting of an internalizing anti-epidermal growth factor receptor variant III monoclonal antibody. Cancer Res. 2000;60:4453–4460. PubMed

Chaturvedi R., Heimburg J., Yan J., et al. Tumor immunolocalization using 124I-iodine-labeled JAA-F11 antibody to Thomsen–Friedenreich alpha-linked antigen. Appl. Radiat. Isot. 2008;66:278–287. PubMed PMC

Russell J., O'Donoghue J.A., Finn R., et al. Iodination of annexin V for imaging apoptosis. J. Nucl. Med. 2002;43:671–677. PubMed

Vaidyanathan G., Zalutsky M.R. Synthesis of N-succinimidyl 4-guanidinomethyl-3-[∗I]iodobenzoate: A radio-iodination agent for labeling internalizing proteins and peptides. Nat. Protoc. 2007;2:282–286. PubMed

Wilbur D.S., Hylarides M.D. Radiolabeling of a monoclonal antibody with N-succinimidyl para-[77Br]bromobenzoate. Int. J. Radiat. Appl. Instrum. B. 1991;18:363–365. PubMed

Garg P.K., Garg S., Zalutsky M.R. N-succinimidyl 4-methyl-3-(tri-n-butylstannyl)benzoate: Synthesis and potential utility for the radioiodination of monoclonal antibodies. Nucl. Med. Biol. 1993;20:379–387. PubMed

Zalutsky M.R., Narula A.S. A method for the radiohalogenation of proteins resulting in decreased thyroid uptake of radioiodine. Int. J. Radiat. Appl. Instrum. A. 1987;38:1051–1055. PubMed

Reist C.J., Garg P.K., Alston K.L., et al. Radioiodination of internalizing monoclonal antibodies using N-succinimidyl 5-iodo-3-pyridinecarboxylate. Cancer Res. 1996;56:4970–4977. PubMed

Garg S., Garg P.K., Zalutsky M.R. N-Succinimidyl 5-(trialkylstannyl)-3-pyridinecarboxylates: A new class of reagents for protein radioiodination. Bioconjugate Chem. 1991;2:50–56. PubMed

Yang Y., Liu N., Zan L., et al. Radioiodination of insulin using N-succinimidyl 5-(tributylstannyl)-3-pyridine-carboxylate (SPC) as a bi-functional linker: Synthesis and biodistribution in mice. J. Radioanal. Nucl. Chem. 2006;268:205–210.

Ali H., van Lier J.E. Synthesis of radiopharmaceuticals via organotin intermediates. Synthesis. 1996;1996:423–445.

Yamamoto T., Toyota K., Morita N. An efficient and regioselective iodination of electron-rich aromatic compounds using N-chlorosuccinimide and sodium iodide. Tetrahedron Lett. 2010;51:1364–1366.

Vaidyanathan G., Affleck D.J., Li J., et al. A polar substituent-containing acylation agent for the radioiodination of internalizing monoclonal antibodies: N-succinimidyl 4-guanidinomethyl-3-[131I]iodobenzoate ([131I]SGMIB) Bioconjugate Chem. 2001;12:428–438. PubMed

Vaidyanathan G., Affleck D.J., Bigner D.D., et al. N-succinimidyl 3-[211At]astato-4-guanidinomethylbenzoate: An acylation agent for labeling internalizing antibodies with α-particle emitting 211At. Nucl. Med. Biol. 2003;30:351–359. PubMed

Wood F.T. Radioactive labeling of proteins with an iodinated amidination reagent. J. Dent. Res. 1975;54:C86–C92. PubMed

Praissman M., Praissman L., Kent S.B.H., et al. Preparation and characterization of a biologically active gastrin derivative modified with an 125I-labeled imidoester. Anal. Biochem. 1981;115:287–296. PubMed

Wall K.A., Fitch F.W. Cell-surface modification with an iodinatible imidoester to enhance radiolabeling. J. Immunol. Methods. 1985;77:1–8. PubMed

Ram S., Buchsbaum D.J. Radioiodination of monoclonal antibodies D612 and 17-1A with 3-iodophenylisothiocyanate and their biodistribution in tumor-bearing nude mice. Cancer. 1994;73:808–815. PubMed

Rana T.M., Meares C.F. N-terminal modification of immunoglobulin polypeptide chains tagged with isothiocyanato chelates. Bioconjugate Chem. 1990;1:357–362. PubMed

Orlova A., Bruskin A., Sivaev I., et al. Radio-iodination of monoclonal antibody using potassium [125I]-(4-isothiocyanatobenzylammonio)-iodo-decahydro-closo-dodecaborate (Iodo-DABI) Anticancer Res. 2006;26:1217–1223. PubMed

Knoth W.H., Miller H.C., Sauer J.C., et al. Chemistry of boranes. IX. Halogenation of B10H10-2 and B12H12-2. Inorg. Chem. 1964;3:159–167.

Khawli L.A., van den Abbeele A.D., Kassis A.I. N-(m-[125I]iodophenyl)maleimide: An agent for high yield radiolabeling of antibodies. Int. J. Radiat. Appl. Instrum. B. 1992;19:289–295. PubMed

Srivastava P.C., Buchsbaum D.J., Allred J.F., et al. A new conjugating agent for radioiodination of proteins: Low in vivo deiodination of a radiolabeled antibody in a tumor model. Biotechniques. 1990;8:536–545. PubMed

Bhojani M.S., Ranga R., Luker G.D., et al. Synthesis and investigation of a radioiodinated F3 peptide analog as a SPECT tumor imaging radioligand. PLoS One. 2011;6 PubMed PMC

Wilbur D.S., Chyan M.-K., Hamlin D.K., et al. Reagents for astatination of biomolecules. 5. Evaluation of hydrazone linkers in 211At- and 125I-labeled closo-decaborate(2-) conjugates of Fab' as a means of decreasing kidney retention. Bioconjugate Chem. 2011;22:1089–1102. PubMed PMC

Mushtaq S., Nam Y.R., Kang J.A., et al. Efficient and site-specific 125I-radioiodination of bioactive molecules using oxidative condensation reaction. ACS Omega. 2018;3:6903–6911. PubMed PMC

Chizzonite R., Truitt T., Podlaski F.J., et al. IL-12: Monoclonal antibodies specific for the 40-kDa subunit block receptor binding and biologic activity on activated human lymphoblasts. J. Immunol. 1991;147:1548–1556. PubMed

Asai S., Žáková L., Selicharová I., et al. A radioligand receptor binding assay for measuring of insulin secreted by MIN6 cells after stimulation with glucose, arginine, ornithine, dopamine, and serotonin. Anal. Bioanal. Chem. 2021;413:4531–4543. PubMed

Potalitsyn P., Selicharová I., Sršeň K., et al. A radioligand binding assay for the insulin-like growth factor 2 receptor. PLoS One. 2020;15 PubMed PMC

Honetschlägerová Z., Hejnová L., Novotný J., et al. Effects of renal denervation on the enhanced renal vascular responsiveness to angiotensin II in high-output heart failure: Angiotensin II receptor binding assessment and functional studies in Ren-2 transgenic hypertensive rats. Biomedicines. 2021;9 PubMed PMC

Mráziková L., Neprašová B., Mengr A., et al. Lipidized prolactin-releasing peptide as a new potential tool to treat obesity and type 2 diabetes mellitus: Preclinical studies in rodent models. Front. Pharmacol. 2021;12 PubMed PMC

Maletínská L., Maixnerová J., Matyšková R., et al. Cocaine- and amphetamine-regulated transcript (CART) peptide specific binding in pheochromocytoma cells PC12. Eur. J. Pharmacol. 2007;559:109–114. PubMed

Maixnerová J., Hlaváček J., Blokešová D., et al. Structure-activity relationship of CART (cocaine- and amphetamine-regulated transcript) peptide fragments. Peptides. 2007;28:1945–1953. PubMed

Behr T.M., Gotthardt M., Barth A., et al. Imaging tumors with peptide-based radioligands. Q. J. Nucl. Med. 2001;45:189–200. PubMed

Conlon J.M. Purification of naturally occurring peptides by reversed-phase HPLC. Nat. Protoc. 2007;2:191–197. PubMed

Gairin J.E., Jomary C., Pradayrol L., et al. 125I-DPDYN, monoiodo[D-Pro10]dynorphin(1–11): A highly radioactive and selective probe for the study of kappa opioid receptors. Biochem. Biophys. Res. Commun. 1986;134:1142–1150. PubMed

Frank B.H., Peavy D.E., Hooker C.S., et al. Receptor binding properties of monoiodotyrosyl insulin isomers purified by high performance liquid chromatography. Diabetes. 1983;32:705–711. PubMed

Conlon J.M. In: Irvine G.B., Williams C.H., editors. vol. 73. Humana Press; Totowa, NJ: 1997. The use of IODO-GEN for preparing 125I-labeled peptides and their purification by reversed-phase high performance liquid chromatography; pp. 231–237. (Neuropeptide Protocols). PubMed

Potalitsyn P., Mrázková L., Selicharová I., et al. Non-glycosylated IGF2 prohormones are more mitogenic than native IGF2. Commun. Biol. 2023;6 PubMed PMC

Catt K.J., Baukal A. Prolonged retention of high specific activity by 125I-labeled angiotensin II – a consequence of ‘decay catastrophe’. Biochim. Biophys. Acta. 1973;313:221–225. PubMed

Jiang V.W., Krohn K.A., Welch M.J. Intramolecular effects of radioiodine decay in o-iodophenol, a model for radioiodinated proteins. J. Am. Chem. Soc. 1975;97:6551–6556. PubMed

Li W.B. Calculation of DNA strand breakage by neutralisation effect after 125I decays in a synthetic oligodeoxynucleotide using charge transfer theory. Radiat. Protect. Dosim. 2006;122:89–94. PubMed

Eichler D.C., Solomonson L.P., Barber M.J., et al. Radiation inactivation analysis of enzymes. Effect of free radical scavengers on apparent target sizes. J. Biol. Chem. 1987;262:9433–9436. PubMed

Ayene I.S., Koch C.J., Krisch R.E. Role of scavenger-derived radicals in the induction of double-strand and single-strand breaks in irradiated DNA. Radiat. Res. 1995;142:133–143. PubMed

Murray J., Garman E. Investigation of possible free-radical scavengers and metrics for radiation damage in protein cryocrystallography. J. Synchrotron Radiat. 2002;9:347–354. PubMed

Nomura S., Tsuchida H., Furuya R., et al. Effects of radical scavengers on aqueous solutions exposed to heavy-ion irradiation using the liquid microjet technique. Nucl. Instrum. Methods Phys. Res. B. 2015;365:611–615.

Hofer K.G. Biophysical aspects of auger processes. Acta Oncol. 2000;39:651–657. PubMed

Howell R.W. Auger processes in the 21st century. Int. J. Radiat. Biol. 2008;84:959–975. PubMed PMC

Berson S.A., Yalow R.S. Radioimmunoassay of ACTH in plasma. J. Clin. Invest. 1968;47:2725–2751. PubMed PMC

Doran A.C., Wan Y.-P., Kopin A.S., et al. Established theory of radiation-induced decay is not generalizable to Bolton–Hunter labeled peptides. Biochem. Pharmacol. 2003;65:1515–1520. PubMed

Linde S., Hansen B., Sonne O., et al. Tyrosine A14 [125I]monoiodoinsulin: Preparation, biologic properties, and long-term stability. Diabetes. 1981;30:1–8. PubMed

Li C.H. Iodination of tyrosine groups in serum albumin and pepsin. J. Am. Chem. Soc. 1945;67:1065–1069.

Schambye H.T., Hjorth S.A., Bergsma D.J., et al. Differentiation between binding sites for angiotensin II and nonpeptide antagonists on the angiotensin II type 1 receptors. Proc. Natl. Acad. Sci. USA. 1994;91:7046–7050. PubMed PMC

Strnadová V., Pačesová A., Charvát V., et al. Anorexigenic neuropeptides as anti-obesity and neuroprotective agents. Biosci. Rep. 2024;44 PubMed PMC

Karnošová A., Strnadová V., Holá L., et al. Palmitoylation of prolactin-releasing peptide increased affinity for and activation of the GPR10, NPFF-R2 and NPFF-R1 receptors: In vitro study. Int. J. Mol. Sci. 2021;22 PubMed PMC

Maixnerová J., Špolcová A., Pýchová M., et al. Characterization of prolactin-releasing peptide: Binding, signaling and hormone secretion in rodent pituitary cell lines endogenously expressing its receptor. Peptides. 2011;32:811–817. PubMed

Lin Y., Hall R.A., Kuhar M.J. CART peptide stimulation of G protein-mediated signaling in differentiated PC12 cells: Identification of PACAP 6-38 as a CART receptor antagonist. Neuropeptides. 2011;45:351–358. PubMed PMC

Freitas-Lima L.C., Pačesová A., Staňurová J., et al. GPR160 is not a receptor of anorexigenic cocaine- and amphetamine-regulated transcript peptide. Eur. J. Pharmacol. 2023;949 PubMed

Stanley S.A., Murphy K.G., Bewick G.A., et al. Regulation of rat pituitary cocaine- and amphetamine-regulated transcript (CART) by CRH and glucocorticoids. Am. J. Physiol. Endocrinol. Metab. 2004;287:E583–E590. PubMed

Charvát V., Strnadová A., Myšková A., et al. Lipidized analogues of the anorexigenic CART (cocaine- and amphetamine-regulated transcript) neuropeptide show anorexigenic and neuroprotective potential in mouse model of monosodium-glutamate induced obesity. Eur. J. Pharmacol. 2024;980 PubMed

Sheikh S.P., O'Hare M.M.T., Tortora O., et al. Binding of monoiodinated neuropeptide Y to hippocampal membranes and human neuroblastoma cell lines. J. Biol. Chem. 1989;264:6648–6654. PubMed

Holubová M., Blechová M., Kákonová A., et al. In vitro and in vivo characterization of novel stable peptidic ghrelin analogs: Beneficial effects in the settings of lipopolysaccharide-induced anorexia in mice. J. Pharmacol. Exp. Therapeut. 2018;366:422–432. PubMed

Zemenova J., Sykora D., Adamkova H., et al. Novel approach to determine ghrelin analogs by liquid chromatography with mass spectrometry using a monolithic column. J. Separ. Sci. 2017;40:1032–1039. PubMed

Maletínská L., Pýchová M., Holubová M., et al. Characterization of new stable ghrelin analogs with prolonged orexigenic potency. J. Pharmacol. Exp. Therapeut. 2012;340:781–786. PubMed

Murphy C.T., Hu P.J. Wormbook; 2013. Insulin/insulin-like Growth Factor Signaling in C. elegans. The C. elegans Research Community; pp. 1–43.http://www.wormbook.org/chapters/www_insulingrowthsignal/insulingrowthsignal.html PubMed PMC

Ullrich A., Gray A., Tam A.W., et al. Insulin-like growth factor I receptor primary structure: Comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J. 1986;5:2503–2512. PubMed PMC

Alberini C.M. IGF2 in memory, neurodevelopmental disorders, and neurodegenerative diseases. Trends Neurosci. 2023;46:488–502. PubMed PMC

Clemmons D.R. Role of IGF-binding proteins in regulating IGF responses to changes in metabolism. J. Mol. Endocrinol. 2018;61:T139–T169. PubMed

Huhtala T., Rytkönen J., Jalanko A., et al. Native and complexed IGF-1: Biodistribution and pharmacokinetics in infantile neuronal ceroid lipofuscinosis. J. Drug Deliv. 2012;2012 PubMed PMC

Kertisová A., Žáková L., Macháčková K., et al. Insulin receptor Arg717 and IGF-1 receptor Arg704 play a key role in ligand binding and in receptor activation. Open Biol. 2023;13 PubMed PMC

Hamlin J.L., Arquilla E.R. Monoiodoinsulin: Preparation, purification, and characterization of a biologically active derivative substituted predominantly on tyrosine A14. J. Biol. Chem. 1974;249:21–32. PubMed

Chrudinová M., Žáková L., Marek A., et al. A versatile insulin analog with high potency for both insulin and insulin-like growth factor 1 receptors: Structural implications for receptor binding. J. Biol. Chem. 2018;293:16818–16829. PubMed PMC

Jiráček J., Žáková L. Structural perspectives of insulin receptor isoform-selective insulin analogs. Front. Endocrinol. 2017;8 PubMed PMC

Kim H.-S. Role of insulin-like growth factor binding protein-3 in glucose and lipid metabolism, Ann. Pediatr. Endocrinol. Metab. 2013;18:9–12. PubMed PMC