{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Qi TF"],"funding":["National Institute of Environmental Health Sciences","NIEHS NIH HHS"],"pagination":["12559-12564"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC9867940"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["94(37)"],"pubmed_abstract":["<i>N</i><sup>6</sup>-Methyladenosine (m<sup>6</sup>A) and its reader, writer, and eraser (RWE) proteins assume crucial roles in regulating the splicing, stability, and translation of mRNA. Aside from m<sup>6</sup>A, RNA is known to carry many other types of chemical modifications; no systematic investigations, however, have been conducted about the crosstalk between m<sup>6</sup>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<sup>6</sup>A eraser proteins (i.e., <i>ALKBH5</i>, <i>FTO</i>) and the catalytic subunit of the major m<sup>6</sup>A 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 m<sup>6</sup>A mapping results revealed the presence of m<sup>6</sup>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<sup>6</sup>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."],"journal":["Analytical chemistry"],"pubmed_title":["Targeted Quantitative Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Using Stable Isotope-Labeled Peptides."],"pmcid":["PMC9867940"],"funding_grant_id":["R35 ES031707"],"pubmed_authors":["Liu X","Wang Y","Tang F","Yu K","Qi TF","Yin J"],"additional_accession":[]},"is_claimable":false,"name":"Targeted Quantitative Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Using Stable Isotope-Labeled Peptides.","description":"<i>N</i><sup>6</sup>-Methyladenosine (m<sup>6</sup>A) and its reader, writer, and eraser (RWE) proteins assume crucial roles in regulating the splicing, stability, and translation of mRNA. Aside from m<sup>6</sup>A, RNA is known to carry many other types of chemical modifications; no systematic investigations, however, have been conducted about the crosstalk between m<sup>6</sup>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<sup>6</sup>A eraser proteins (i.e., <i>ALKBH5</i>, <i>FTO</i>) and the catalytic subunit of the major m<sup>6</sup>A 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 m<sup>6</sup>A mapping results revealed the presence of m<sup>6</sup>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<sup>6</sup>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.","dates":{"release":"2022-01-01T00:00:00Z","publication":"2022 Sep","modification":"2025-04-04T21:13:34.399Z","creation":"2025-04-04T21:13:34.399Z"},"accession":"S-EPMC9867940","cross_references":{"pubmed":["36084281"],"doi":["10.1021/acs.analchem.2c03549"]}}