Targeted Quantitative Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Using Stable Isotope-Labeled Peptides.

-Methyladenosine (mA) and its reader, writer, and eraser (RWE) proteins assume crucial roles in regulating the splicing, stability, and translation of mRNA. Aside from mA, RNA is known to carry many other types of chemical modifications; no systematic investigations, however, have been conducted about the crosstalk between mA and other modified nucleosides in RNA. Here, we modified our recently established liquid chromatography-parallel-reaction monitoring (LC-PRM) method by incorporating stable isotope-labeled (SIL) peptides as internal or surrogate standards for profiling epitranscriptomic RWE proteins. We were able to detect reproducibly a total of 114 RWE proteins in HEK293T cells with the genes encoding mA eraser proteins (i.e., , ) and the catalytic subunit of the major mA writer complex (i.e., ) being individually ablated. Notably, eight proteins, including writer proteins for 5-methylcytidine and pseudouridine, were altered by more than 1.5-fold in the opposite directions in HEK293T cells depleted of and . Analysis of previously published mA mapping results revealed the presence of mA in the corresponding mRNAs for four of these proteins. Together, we integrated SIL peptides into our LC-PRM method for quantifying epitranscriptomic RWE proteins, and our work revealed potential crosstalks between mA and other epitranscriptomic modifications. Our modified LC-PRM method with the use of SIL peptides should be applicable for high-throughput profiling of epitranscriptomic RWE proteins in other cell types and in tissues.