biostudies-literatureQi TFNational Institute of Environmental Health SciencesNIEHS NIH HHS12559-12564https://www.ebi.ac.uk/biostudies/studies/S-EPMC9867940biostudies-literatureUnknown94(37)<i>N</i>⁶-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., <i>ALKBH5</i>, <i>FTO</i>) and the catalytic subunit of the major m⁶A writer complex (i.e., <i>METTL3</i>) 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 <i>METTL3</i> and <i>ALKBH5</i>. 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.Analytical chemistryTargeted Quantitative Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Using Stable Isotope-Labeled Peptides.PMC9867940R35 ES031707Liu XWang YTang FYu KQi TFYin JfalseTargeted Quantitative Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Using Stable Isotope-Labeled Peptides.<i>N</i>⁶-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., <i>ALKBH5</i>, <i>FTO</i>) and the catalytic subunit of the major m⁶A writer complex (i.e., <i>METTL3</i>) 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 <i>METTL3</i> and <i>ALKBH5</i>. 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.2022-01-01T00:00:00Z2022 Sep2025-04-04T21:13:34.399Z2025-04-04T21:13:34.399ZS-EPMC98679403608428110.1021/acs.analchem.2c03549