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

N6-Methyladenosine
(m6A)
and its reader, writer, and eraser (RWE) proteins assume crucial roles
in regulating the splicing, stability, and translation of mRNA. Aside
from m6A, RNA is known to carry many other types of chemical
modifications; no systematic investigations, however, have been conducted
about the crosstalk between m6A 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 m6A eraser
proteins (i.e., ALKBH5, FTO) and
the catalytic subunit of the major m6A 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 m6A mapping results
revealed the presence of m6A 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 m6A
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.