Fig. 3.

Pericyte-specific protein purification and LC-MS/MS proteomics.A, streptavidin beads were made protease resistant (PrS) through chemical modification of lysine and arginine residues, inhibiting tryptic digestion. B, experimental design to identify pericyte-specific proteins in spheroid cultures in response to hypoxia using PrS-biotin affinity purification and mass spectrometry. C, filtering scheme for mass spectrometric data. Raw LC-MS/MS data was transformed, and global protein abundance was normalized. Biotinylated proteins were filtered for with a cut-off at log2 FC >0.5 compared to WT-pericytes with omitted biotin, and results were contrasted between the conditions of interest. D, principal component analysis of PrS-purified secreted proteins after 24 h of normoxic or hypoxic incubation both in mono- and spheroid-cultures, n = 3. HM, hypoxia–monoculture; HS, hypoxia–spheroids; NM, normoxia–monoculture; NS, normoxia–spheroids.