On-line trapping/capillary hydrophilic-interaction liquid chromatography/mass spectrometry for sensitive determination of RNA modifications from human blood

RNA modification has recently been proposed to play important roles in biological regulation. The detection and quantification of RNA modifications generally are challenging tasks since most of the modifications exist in low abundance in vivo. Here we developed an on-line trapping/capillary hydrophilic-interaction liquid chromatography/electrospray ionization-mass spectrometry (on-line trapping/cHILIC/MS) method for sensitive and simultaneous quantification of RNA modifications of N-6-methyladenosine (m(6)A) and 5-methylcytosine (5-mC) from human blood. The hydrophilic organic-silica hybrid monolith was prepared using sol-gel combined with "thiol-ene" click reaction for the separation of nucleosides. A poly(MAA-co-EGDMA) monolithic capillary was used as the on-line trapping column. With the developed on-line trapping/cHILIC/MS analytical platform, the detection limits of m(6)A and 5-mC can reach to 0.06 fmol and 0.10 fmol. We then investigated the contents of m(6)A and 5-mC in human blood RNA from healthy persons at the age of 6-14 and 60-68 years. Our results showed that both m(6)A and 5-mC contents were significantly decreased in elder persons, suggesting the RNA modifications of m(6)A and 5-mC are correlated to aging. (C) 2018 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.