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 N⁶-methyladenosine (m⁶A) 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 m⁶A and 5-mC can reach to 0.06 fmol and 0.10 fmol. We then investigated the contents of m⁶A 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 m⁶A and 5-mC contents were significantly decreased in elder persons, suggesting the RNA modifications of m⁶A and 5-mC are correlated to aging.