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. 2023 Jan 4;8(2):2367-2376.
doi: 10.1021/acsomega.2c06758. eCollection 2023 Jan 17.

Tritium Labeling of Neuromedin S by Conjugation with [3H] N-Succinimidyl Propionate

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Tritium Labeling of Neuromedin S by Conjugation with [3H] N-Succinimidyl Propionate

Martin R Edelmann et al. ACS Omega. .

Abstract

The human neuropeptide neuromedin S (NMS) consists of 33 amino acids. The introduction of tritium atoms into NMS has not been described so far. This represents a gap for using [3H]NMS in radioreceptor binding assays or in tracking and monitoring their metabolic pathway. Two approaches for the incorporation of tritium into NMS were explored in this study: (1) halogenation at the His-18 residue followed by catalyzed iodine-127/tritium exchange and (2) conjugation of tritiated N-succinimidyl-[2,3-3H3]propionate ([3H]NSP) to at least one of the three available primary amines of amino acids Ile-1, Lys-15, and Lys-16 in the peptide sequence. Although iodination of histidine was achieved, subsequent iodine-127/deuterium exchange was unsuccessful. Derivatization at the three possible amino positions in the peptide using nonradioactive NSP resulted in a mixture of unconjugated NSM and 1- to 3-conjugations at different amino acids in the peptide sequence. Each labeling position in the mixture was assigned following detailed LC-MS/MS analysis. After separating the mixture, it was shown in an in vitro fluorometric imaging plate reader (FLIPR) and in a competitive binding assay that the propionyl-modified NMS derivatives were comparable to the unlabeled NMS, regardless of the degree of labeling and the labeling position(s). A molecular simulation with NMS in the binding pocket of the protein neuromedin U receptor 2 (NMUR2) confirmed that the possible labeling positions are located outside the binding region of NMUR2. Tritium labeling was achieved at the N-terminal Ile-1 using [3H]NSP in 7% yield with a radiochemical purity of >95% and a molar activity of 90 Ci/mmol. This approach provides access to tritiated NMS and enables new investigations to characterize NMS or corresponding NMS ligands.

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Conflict of interest statement

The authors declare the following competing financial interest(s): All authors are in paid employment by the company F. Hoffmann-La Roche AG during the completion of the study.

Figures

Figure 1
Figure 1
Considerations to tritium-labeled Neuromedin S (NMS). A: Derivatization of peptide using [3H]N-succinimidyl propionate [3H]NSP; B: catalytic hydrogen/tritium exchange starting from the native peptide; C: tritiation of the NMS precursor; D: peptide synthesis using tritiated amino acid.
Figure 2
Figure 2
HPLC analysis of the crude solution on NMS treated with 1 equiv of NSP showed seven peaks with retention times between 14 and 17 min. Assignment of a–e refers to the isolated fractions, where b and d consist of two nonseparable products.
Figure 3
Figure 3
LC/MS chromatograms (MS trace; TIC: 100–2000 Da) of the fractions a–e.
Figure 4
Figure 4
Deconvoluted low-energy mass ([M + H]+) spectra of the main peaks of the fractions a–e.
Figure 5
Figure 5
y ion pattern of high-energy MS spectra of fractions a–e. y ions marked with “prop” indicate the labeling positions. Zoom left column: 2050–2550 Da; zoom right column: 3480–4000 Da.
Figure 6
Figure 6
Concentration–response curves (CRCs) for fractions a–e in in vitro FLIPR assay. The y-axis represents the maximum minus baseline fluorescence signal for each sample. The x-axis represents the log of the molar concentration. Data were fitted by the Hill equation, and data points represent the mean ± standard deviation of two individual experiments.
Figure 7
Figure 7
Concentration–response curves (CRCs) for fractions a–e in in vitro affinity receptor binding assay. The y-axis represents the percentage of inhibition of specific binding for the radiolabeled ligand [125I]NMU-8 for each sample. The x-axis represents the log of the molar concentration. Data were fitted by the Hill equation, and data points represent the mean ± standard deviation of two individual experiments.
Figure 8
Figure 8
NMS in the binding pocket of NMUR2 (PDB code 7W57). Phe-26 to Asn-33 are engaged in intermolecular interactions with the receptor.
Figure 9
Figure 9
Analytical HPLC shows the normalized chromatograms of (A) crude solution after dialysis, (B) isolated fraction from peak B, and (C) isolated fraction C. Green lines: absorbances at the wavelength of 280 nm. Blue lines: time-shifted radio detection in counts per min (cpm).
Figure 10
Figure 10
HPLC analysis of the crude solution on NMS treated with 3 equiv of NSP (for assignments of c–e, see the text).
Figure 11
Figure 11
HPLC analysis of the crude solution on NMS:NSP (3:1) (for assignments of a–d, see the text).

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