Updated project metadata. Updated publication reference for PubMed record(s): 31763404. This article describes a mass spectrometry data set generated from osteogenic differentiated bone marrow stromal cells (BMSCs) and adipose tissue derived stromal cells (ASCs) of a 24-year old healthy donor spiked with iRT peptides. Cells have been identified via FACS-Analysis positive for CD90 and CD105 and negative for CD14, CD34, CD45 and CD11b and tri-lineage differentiation. Obtaining a sufficient amount of high-quality tissue is the key limiting factor for establishing a region-specific spectral library. Hence, combining existing spectral libraries for data-independent acquisition analysis (DIA) can overcome this major limitation. Moreover, these data can be used to map region-specific proteins and to model region-specific pathways. Both can improve our understanding of the functioning in greater depth. In addition, these data can also be used to determine the optimal settings for measuring proteins and peptides of interest. To create the specific spectral library, the tissue was first homogenized and then fractionated via different types of SDS gel electrophoresis, resulting in 11 fractions. These fractions were analysed by nanoHPLC-ESI-MS/MS, resulting in 24 data files.