Intracellular proteins localize and translocate to perform specific functions essential for maintaining physiological activities. Dysfunctional subcellular protein localization is implicated in various diseases, including heart failure, neurodegenerative diseases, and cancer. Traditionally, the study of differential protein translocation has relied on low-throughput methods such as immunofluorescence for screening and verification.
Here, we established and optimized a high-throughput approach based on mass spectrometry to investigate the differential localization of subcellular proteins under varying conditions. Specifically, we utilized differential centrifugation to separate organelles, followed by label-free quantification through mass spectrometry and data-independent acquisition (DIA) to obtain systematic subcellular protein profiles. To identify differentially localized proteins, we employed the Bayesian ANalysis of Differential Localization Experiments (BANDLE), a Bayesian algorithm model designed to calculate the probability of protein localization changes under various experimental conditions. This model analyzes protein abundance across different cellular components, providing a robust method for identifying proteins with significant localization changes.
Our experiments revealed that treatment with paclitaxel resulted in the subcellular differential localization of 198 proteins in triple-negative breast cancer (TNBC) cell line MDA-MB-231. Notably, proteins in the Golgi apparatus, mitochondria, and endoplasmic reticulum exhibited significant translocation. In addition,we found that the position of JAK-STAT signaling pathway related proteins in the cell was significantly changed under PTX, with O60674 protein transferred from an unknown location to the cell membrane and P40763 protein transferred to the cytoplasm. This result is consistent with previously reported results of changes in the signaling pathway under PTX drug treatment.
This high-throughput method provides a novel perspective on the mechanisms underlying chemotherapy resistance in cancer and offers potential therapeutic strategies for targeting TNBC based on organelle protein translocation. Future applications of this method will explore the differential localization of proteins in cardiomyocytes under hypoxic conditions. Additionally, validating the function of key translocated proteins through in vivo and in vitro models using cellular and molecular biology techniques are also part of our next steps.