Ting and colleagues describe a technique that allows spatio-temporal information to be captured by mass spectrometry (MS). The authors used engineered ascorbate peroxidase as a targetable genetic tag to label proteins in live cells. Ascorbate peroxidase biotinylates nearby proteins when biotin-phenol and H2O2 are added to tagged cells, allowing them to be captured for MS with streptavidin beads. The authors fused ascorbate peroxidase to a peptide that targeted it to the mitochondrial matrix. This enabled them to identify 495 proteins within the human mitochondrial matrix, 94% of which had previously been associated with mitochondria, and they performed various experiments to verify their results. As well as presenting a technique that can be used to map the proteome of whole organelles from living cells, this paper identifies 31 proteins with a novel mitochondrial matrix association.