Quantification of mRNA cap-modifications by means of LC-QqQ-MS

Enzymatic modification of the 5′-cap is a versatile approach to modulate the properties of mRNAs. Transfer of methyl groups from S-adenosyl-l-methionine (AdoMet) or functional moieties from non-natural analogs by methyltransferases (MTases) allows for site-specific modifications at the cap. These modifications have been used to tune translation or control it in a temporal manner and even influence immunogenicity of mRNA. For quantification of the MTase-mediated cap modification, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) provides the required sensitivity and accuracy. Here, we describe the complete workflow starting from in vitro transcription to produce mRNAs, via their enzymatic modification at the cap with natural or non-natural moieties to the quantification of these cap-modifications by LC-QqQ-MS.