利用SWATH-MS技術分析小鼠脾臟中塑膠微粒毒性與乳癌藥物抗藥性之差異蛋白質體學研究

Abstract

蛋白質體學是生物體內蛋白質組成、結構、功能及交互作用的研究，透過質譜與生物資訊分析，可了解不同生理與病理狀態下的蛋白質變化，廣泛應用於疾病標記物發掘及毒理機制研究。塑膠微粒因其耐用且不易降解，對環境與生態造成威脅，並可能對哺乳類產生器官損傷及免疫毒性等潛在威脅。乳癌作為全球女性好發癌症，雖有多元治療策略，但荷爾蒙受體陽性乳癌在長期內分泌治療後常面臨抗藥性挑戰，腫瘤細胞會透過改變受體訊號或啟動替代路徑來逃避免疫與藥物壓力。本研究分為兩部分，第一部分以被餵食塑膠微粒的小鼠為模型，應用SWATH-MS技術分析脾臟中的蛋白質，總共鑑定4701個蛋白質，其中2,823個蛋白質透過 SWATH 定量分析被檢測到，Metascape分析結果顯示，蛋白質主要參與嗜中性球去顆粒化、Rho GTPases訊號傳導與細胞骨架調控等免疫相關過程，顯示塑膠微粒可能降低免疫細胞遷移能力並引發組織損傷。第二部分我們針對不同抗藥性的乳癌細胞進行分析，共鑑定 4,423 個蛋白質，並定量到3,479個蛋白質，其中包含806個磷酸化蛋白質。途徑分析結果顯示，抗藥性乳癌細胞中顯著富集多條與治療耐受相關的生物過程，包括 RNA 剪接異常、Rho GTPases訊號、自噬(Autophagy)及 ErbB訊號等，顯示腫瘤細胞可能透過基因轉錄後調控、細胞骨架重塑及能量回收機制來維持存活並抵禦藥物壓力。我們應用脂肪族羥基酸修飾金屬氧化物層析法(HAMMOC)進行磷酸化胜肽富集，深入探討塑膠微粒毒性與乳癌抗藥性的分子影響機制，期望為環境毒理與乳癌臨床治療策略提供科學依據。Proteomics offers a comprehensive view of protein composition, function, and interactions, providing key insights into how cells and tissues respond to health and disease. By combining mass spectrometry with bioinformatics, it has become a vital tool for biomarker discovery and understanding mechanisms of toxicity and disease. Microplastic pollution is a growing global concern, with particles under 5 mm accumulating in mammalian tissues and linked to immune dysfunction, metabolic disruption, and organ damage. Breast cancer, especially the hormone receptor-positive (HR⁺) subtype, remains difficult to treat long-term due to acquired resistance, as tumor cells reprogram signaling pathways to evade endocrine therapy. In this study, we employed a mouse model of microplastic exposure and applied SWATH-MS-based proteomic analysis to investigate spleen tissue alterations. We identified 4,701 proteins, with 2,823 quantified across samples. Pathway enrichment analysis revealed significant involvement of immune-related processes, including neutrophil degranulation, Rho GTPase signaling, and cytoskeletal organization, suggesting that microplastic exposure impairs immune cell migration and promotes tissue injury. In addition, proteomic analysis of breast cancer cell lines with different drug resistance profiles identified 4,423 proteins, with 3,479 quantified, including 806 phosphoproteins. Pathway enrichment analysis revealed significant enrichment of several processes associated with therapeutic resistance, such as abnormal RNA splicing, Rho GTPase signaling, autophagy, and ErbB signaling, indicating that resistant cancer cells may maintain survival under drug pressure through post-transcriptional regulation, cytoskeletal remodeling, and energy recycling mechanisms. Furthermore, phosphopeptide enrichment was performed using hydroxy acid–modified metal oxide chromatography (HAMMOC) to further investigate the molecular mechanisms underlying microplastic toxicity and breast cancer drug resistance. These findings provide important insights for future toxicological studies and the development of clinical therapeutic strategies.