摘要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-la-beling 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).