Proteomic Analysis of Lysosomal Membrane Proteins in Bovine Mammary Epithelial Cells Illuminates Potential Novel Lysosome Functions in Lactation.

Lactation of bovine mammary epithelial cells (BMEC) is a complex biological process that involves in various organelles. Studies have shown that lysosome and lysosomal membrane proteins (LMP) plays an important role in lactation of BMEC. But the LMP of BMEC remains poorly understood. To obtain a global view of the LMP of BMEC and the affect of lysosome on lactation, the LMP of BMEC was identified using sequential windowed acquisition of all theoretical mass spectra (LC-SWATH/MS). 1214 LMP were identified and 559 were reported to be localized on lysosomal membrane for the first time in BMEC. Gene ontology annotation of these identified proteins showed that both previously reported casein synthesis-related LMP, such as LAMTOR1, 2, 3, and rRagC, and newly identified casein and milk fat synthesis-related LMP, such as EIF4E and ACAA1, were found. KEGG pathway analysis of these identified proteins showed that some pathways involved in lactation, such as PI3K-Akt, mTOR, insulin, PPAR, and JAK-STAT pathway, were found. The lysosomal location of five proteins (PRKCA, EIF4E, ACAA1, HRAS, and THBS1) was analyzed by laser confocal microscopy, and all five were associated with the lysosomal membrane. These findings help to elucidate lysosome functions in the regulation of lactation. The results implicate lysosomes as important organelles in regulation of lactation of BMEC that have been previously undervalued.

Pull-down Assay on Streptavidin Beads and Surface Plasmon Resonance Chips for SWATH-MS-based Interactomics.

BACKGROUND/AIM: Pul-down assay is a popular in vitro method for identification of physical interactors of selected proteins. Here, for the first time, we compared three conventional variants of pull-down assay with the streptavidin-modified surface plasmon resonance (SPR) chips for the detection of PDZ and LIM domain protein 2 (PDLIM2) interaction partners. MATERIALS AND METHODS: PDLIM2 protein-protein interactions were analysed by three variants of pull-down assay on streptavidin beads using LC-MS/MS in "Sequential Window Acquisition of all Theoretical fragment ion spectra (SWATH)" mode and compared with LC-SWATH-MS/MS data from SPR chips. RESULTS: The results showed that (i) the use of SPR chip led to comparable data compared to on-column streptavidin beads, (ii) gravity flow and microflow in wash and elution steps provided better results than centrifugation, and (iii) type and concentration of detergent did not significantly affect the interactome data of cancer-associated PDLIM2. CONCLUSION: Our study supports further application of SPR-based affinity purification with SWATH mass spectrometry for reproducible and controlled characterization of cancer-associated interactomes.