Extracellular vesicles (EVs) are actively secreted by mammalian cells. They are increasingly recognized as promising circulating
biomarkers of
disease progression. Matrix-assisted
laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is currently one of the most powerful techniques for the rapid analysis of
biological samples, especially for discovering
biomarkers for disease diagnosis and prognosis. It is unclear what cell culture medium components and EV isolation methods are suitable for MALDI-TOF MS analysis. Using a human lung
carcinoma cell line (A549), we investigated and optimized the critical experimental conditions for EVs'
protein profiling by combining differential ultracentrifugation and MALDI-TOF MS. The results demonstrated that medium components and ultracentrifugation procedures to extract EVs played important roles in MS detection. Compared with EV-depleted serum and normal serum
medium, conditioned medium with 2%
fetal bovine serum in this study maintained cell proliferation and displayed significant
protein profiling of EVs. RPS27A (
ribosomal protein), which plays an essential role in mRNA translation and ribosome assembly for the differentiation of
cancer cells, was detected from the EVs of
lung cancer cells associated with
cancer cell migration and invasion. We also found the known
tumor diagnosis marker, which is S100A10_
S100 calcium-binding protein A10. Therefore, MALDI-TOF MS-based EV analysis with optimized experimental protocols can contribute to future development of rapid screening techniques of
protein biomarkers associated with early
cancer diagnosis.