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

N 6 -Methyladenosine (m 6 A) and its reader, writer, and eraser (RWE) proteins assume crucial roles in regulating the splicing, stability, and translation of mRNA. Aside from m 6 A, RNA is known to carry many other types of chemical modifications; no systematic investigations, however, have been conducted about the crosstalk between m 6 A 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 m 6 A eraser proteins (i.e., ALKBH5 , FTO ) and the catalytic subunit of the major m 6 A writer complex (i.e., METTL3 ) 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 METTL3 and ALKBH5 . Analysis of previously published m 6 A mapping results revealed the presence of m 6 A 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 m 6 A 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.