We used shotgun proteomics to analyze three subcellular fractions isolated from mesenchymal stem cells and from differentiated adipocytes and myocytes: nuclear envelopes (NEs), cytoplasmic membranes and nuclear contents. The proteomics data were analyzed by a NSAF-based scoring system to calculate relative protein enrichment in the NE fraction. High NE enrichment scores were obtained for most well-characterized NE proteins. High scores also were seen for a number of new proteins not known to be concentrated at the NE, and we validated that five of these are substantially concentrated at the NE of multiple cell types. Our datasets can be useful for identifying additional NE-concentrated proteins in the future.