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Although enzyme catalyzed ¹⁸O labeling has been used as a tool in quantitative proteomics, this type of labeling has not yielded the same impact yet as alternative techniques for quantitation like SILAC or labeling with chemical mass tags. The practical difficulties involved in ¹⁸O labeling, most importantly the occurrence of incomplete labeling and, as a result, the difficulties in data analysis and interpretation have hampered its implementation in high-throughput comparative proteomics protocols. In this paper, we have optimized the ¹⁸O labeling procedure to such an extent that complete labeling can be achieved in a routine manner. We have implemented this approach into a protein−protein interaction analysis pipeline to differentiate between bona fide interaction partners of the low-level expressing cell cycle regulator cyclin-dependent kinase 9 (Cdk9) and nonspecifically binding or background proteins …