Screening Differential Protein Pathways in Insulin Resistance Complicated with PCOS Using iTRAQ Combined with LC-MS/MS Technology

XIE Fang; LI Ping; CHEN Yang

Abstract：Objective: To screen potential serum differential proteins in insulin resistance (IR) complicated with polycystic ovary syndrome (PCOS) using isobaric tags for relative and absolute quantitation (iTRAQ) combined with ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Methods: A total of 20 patients diagnosed with PCOS in our hospital were selected, including 10 cases of simple PCOS and 10 cases of PCOS+IR. iTRAQ combined with LC-MS/MS was used for proteomic analysis to identify serum differential proteins. Bioinformatics analyses, including gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and protein-protein interaction (PPI) analysis, were conducted to understand the biological processes, cellular components, and molecular functions of the differentially expressed proteins detected by the two methods. Results: A total of 454,675 secondary spectra were detected by iTRAQ, with 14,376 matched spectra. Combined with LC-MS/MS, 74,386 peptides, 47,542 unique peptide sequences, and 54,675 proteins were identified. A total of 249 differentially expressed proteins with a fold change ≥1.30 or ≤0.83 were found, among which 9 had a P-value <0.05. There were 5 up-regulated and 4 down-regulated proteins, namely RPAP3, ALDH6A1, COX20, RASSF3, ALPK2, NANOS1, FAM210A, CHGA, and CGA. These differential proteins were imported into the STRING database for PPI network analysis, which revealed 21 nodes and 69 edges with a PPI enrichment P-value <0.001. KEGG enrichment results included hsa04913 (Ovarian steroidogenesis), hsa04024 (cAMP signaling pathway), and hsa04080 (Neuroactive ligand-receptor interaction). Conclusion: The combination of iTRAQ and LC-MS/MS technologies identified nine differential proteins including CHGA and CGA, which are mainly enriched in pathways related to ovarian steroidogenesis, cAMP signaling, and neuroactive ligand-receptor interaction. This combination provides a powerful platform for exploring the pathogenesis of IR complicated with PCOS and discovering new biomarkers.