Ji Soo An (Inje University)

Young Beom Kwak (Inje University)

Jundong Yu (Korea Racing Authority)

Ji Soo An (Inje University)

Young Beom Kwak (Inje University)

Jundong Yu (Korea Racing Authority)

Jang Mi Han (Inje University)

Won Suk Jung (Inje University)

In hyeok Jang (Korea Racing Authority)

Jong Min Park (Hallym University)

IVF-C is a single-ingredient pharmaceutical formulation containing human chorionic gonadotropin (hCG), a placental glycoprotein hormone. hCG contains β-subunit variants encoded by multiple genes, which may differ in structural characteristics. Therefore, accurate characterization of the molecular forms present in the formulation is essential. In this study, intact protein mass analysis and a bottom-up proteomic approach using UHPLC-Q-Exactive were performed to characterize the structural composition of IVF-C. Intact protein mass analysis revealed a mass signal corresponding to whole hCG at approximately 37 kDa and a putative α-subunit dimer species at approximately 27 kDa. For bottom-up analysis, hCG was reduced, alkylated, and digested with trypsin prior to UHPLC-Q-Exactive analysis. The acquired data were processed using MaxQuant, and hCG-derived peptide candidates were selected based on chromatographic characteristics and MS/MS spectral evaluation. PCA and PLS-DA demonstrated separation between experimental groups, and peptides derived from both α- and β-subunits were identified. β-subunit-derived peptides showed high sequence homology with CGB1 and CGB2 genes, supporting their specificity as potential analytical targets. The integrated application of intact protein mass analysis and bottom-up proteomics enabled structural characterization of IVF-C at both protein and peptide levels. These findings provide fundamental information for the development of mass spectrometry-based analytical methods for hCG characterization and future quantitative analysis.

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