The CAP-MAP is a method that allows detection and quantifying messenger RNA (mRNA) cap structures present in cells and tissues. CAP-MAP is a quantitative method for detecting mRNA cap dinucleotides. The technique allows the analysis of the methylation status on the N-7 position of the terminal guanosine as well as the methylation status of the 2'-oxygen (2'-O) of the first nucleotide ribose and the N-6 methylation status of the first nucleotide adenosine in mRNA caps. The method was developed by Galloway et al. and published in 2020.
1) Enrichment of mRNA from total cellular RNA using oligo dT affinity beads.
2) Digest mRNA with the non-specific nuclease P1 to release nucleotide 5’-monophosphates and cap dinucleotides, m7GpppNm.
3) Add synthetic cap standard, ARCA.
4) Separate the released caps and nucleotide 5’-monophosphate using liquid chromatography and a porous graphite (PGC) carbon column.
5) Identify cap structures using negative ion electrospray mass spectrometry in multiple reaction monitoring modes (MRM).
6) Analyze and quantify observed caps.
The research group establishing this method used a TSQ Quantiva mass spectrometer interfaced with an Ultimate 3000 Liquid Chromatography system (ThermoScientific), equipped with a PGC HyperCarb column (See Galloway et al. 2020 for more detail).
Oligo dT-conjugated beads allow the purification of cellular RNA. Digestion of captured RNA molecules using P1 nuclease releases cap dinucleotides and nucleotide monophosphates. The use of the synthetic ARCA cap allows spiking of the digested sample. Sample solutions are separated using a TSQ LC-MS/MS system and a PGC column. Operating the mass spectrometer in negative and MRM mode enables detection and identification of cap dinucleotides.
Alison Galloway, Abdelmadjid Atrih, Renata Grzela, Edward Darzynkiewicz, Michael A. J. Ferguson and Victoria H. Cowling; CAP-MAP: cap analysis protocol with minimal analyte processing, a rapid and sensitive approach to analysing mRNA cap structures. Open BiologyVolume 10, Issue 2. Published:26 February 2020. [Open Biology]