Transient I-DIRT technology for identifying stable and less stable protein–protein interactions. One culture containing an affinity-tagged protein is grown in isotopically light media and cross-linked with formaldehyde (FA). A second culture of wild-type cells is grown in isotopically heavy media (e.g., ¹³C₆-arginine) and also cross-linked with formaldehyde. Following independent harvesting and freezing, the cultures are mixed 1:1 and cryogenically lysed under liquid nitrogen temperature. The affinity-tagged protein is purified with associating proteins and resolved by SDS-PAGE. The gel lane is sliced into 2 mm sections and proteins are identified with mass spectrometry. Proteins interacting stably with the affinity-tagged protein complex have ~100% isotopically light tryptic peptides (i.e., these protein interactions have been preserved from the original isotopically light culture), while nonspecific proteins co-purifying with the affinity-tagged protein complex will have ~50% isotopically light tryptic peptides (i.e., contamination occurs during the purification procedure and these nonspecific protein associations have an equal probability to be isotopically light or heavy). Transiently interacting proteins will have an intermediate level of isotopically light tryptic peptides. Examples are shown of each interaction from a purification of the NuA3 histone acetyltrans-ferase. Following mass spectrometric analysis, the role of the specific protein interactions are explored with functional analyses.