The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network around mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and analyzed in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation. Additional contact details: <a href="mailto:k.thedieck@umcg.nl">Prof. Dr. Kathrin Thedieck</a> Department of Pediatrics, University of Groningen, University Medical Center Groningen (UMCG), 9713 AV Groningen, The Netherlands. Faculty VI - School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, 26111 Oldenburg, Germany. Prof. Dr. Bettina Warscheid Faculty of Biology, Institute of Biology II, University of Freiburg, 79104 Freiburg, Germany. BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany.