Systematic Profiling of Exosomal Small RNA Epigenetic Modifications by High-Performance Liquid Chromatography-Mass Spectrometry.

Exosomes are nanosized extracellular vesicles that have a critical role in intercellular communication and tumor microenvironment regulation. Extensive research has shown that exosomal small RNAs contribute to metastasis in multiple tumor types and that abnormal epigenetic modifications in nucleic acids also have an association with diverse diseases. However, the content of modified nucleosides on exosomal small RNAs has not been quantitatively reported. Because of the trace amounts of exosomes and matrix complexity, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a powerful tool for label-free sensitive and simultaneous determinations of six important modified nucleosides on small RNAs inside exosomes. This system performed well using only approximately 10-10 particles of exosomes to obtain modified nucleoside levels between 0.001 and 0.03, and the most striking result was that the content of mA in exosomal small RNAs was continuously higher than that in the cells being analyzed. We hope that this conclusion helps establish a greater degree of deciphering accuracy on exosomes, which has considerable application potential in the diagnosis and prognosis of diseases.