Updated project metadata. Epitransciptomic reader, writer and eraser (RWE) proteins recognize, install, and remove modified nucleosides in RNA, which are known to play essential roles in RNA processing, splicing, and stability. Here, we established a liquid chromatography–parallel reaction monitoring (LC-PRM) method for high-throughput profiling of a total of 152 epitransciptomic RWE proteins, including 68 human epitranscriptomic RWE proteins deposited in the Modomics database. To our knowledge, this is the first report of quantifying a comprehensive list of epitranscriptomic RWE proteins using LC-PRM. To access the roles of epitranscriptomic RWE proteins in breast cancer radioresistance, we applied the LC-PRM method, in conjunction with stable isotope labelling by amino acids in cell culture (SILAC), to quantify these proteins in two pairs of matched parental/radioresistant breast cancer cells (i.e., MDA-MB-231 and MCF-7 cells, and their radioresistant counterparts, i.e., C5 and C6 cells). We found that eight epitranscriptomic RWE proteins were commonly altered by over 1.5-fold in the two pairs of matched breast cancer cells, from which TRMT1 (an m2,2G writer) may play a role in promoting breast cancer radioresistance due to its clinical relevance and correlation with DNA repair gene sets. Furthermore, we envision that the LC-PRM method is applicable for studying the roles of epitranscriptomic RWE proteins in the metastatic transformation of cancer and therapeutic resistance of other types of cancer.