We provide comprehensive proteomic profiling of hUBE2O expressed and purified in HEK293T cells, including: (1) hUBE2O-catalyzed polyubiquitin chain linkages and its self-ubiquitination sites via in-gel MS; (2) NEM-modified sites on hUBE2O before and after binding with zinc ions. Dataset includes raw files, and processed identification files. Raw data were analyzed using MaxQuant (version 2.4.2.0) searched against hUBE2O FASTA (Q9C0C9) alone or together with human Ubiquitin FASTA (P62987, P62979, P0CG48 and P0CG47), with the GlyGly or NEM remnants search enabled.