Updated project metadata. Acid and heat lability greatly hindered the study of N-phosphorylation (pArg, pHis and pLys), and limited progresses were focus on prokaryotes since the O-phosphorylation is of high abundance in eukaryotes. To meet the challenge, this paper mainly described the identification of phosphoarginine (pArg) proteins and sites in human Jurkat cells by LC-MS/MS under an optimized proteomic experimental procedure. The optimization included cell stimulation conditions, protein extraction methods and MS/MS fragmentation modes. Under this best procedureļ¼Œ hundreds of pArg proteins and sites were unambiguously identified in Jurkat cells from two biological replicates. GO analysis revealed that the pArg proteins may play an important role in nucleic acid-related functions. Besides, this resource also contains the identification information of pHis and pLys.