Tris-buffered saline plus 0.05% tween-20.Stem cells have long-term self-renewing activity and can commit to multiple celltypes upon differentiation signals. Liquid chromatography-tandem mass spectrometry LDS 1Īffinity purification mass spectrometry BSA This article contains supplemental material.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. were supported by the Training Program in Environmental Toxicology T32ES007028. This work was supported by National Institutes of Health Grant R01ES022936 (to D.

contributed new reagents or analytic tools J.D.F., A.M.P., C.M., and B.M. designed research J.D.F., S.G.C., A.M.P., A.J.W., and C.M.

This study further demonstrates the value of combining global and targeted MS approaches to interrogate multiprotein complex composition and dynamics.Īuthor contributions: J.D.F., C.M., B.M., and D.C.L. These data support an ASK signalosome comprising a multimeric core complex of ASK1, ASK2, and 14–3-3 proteins, which dynamically engages other binding partners needed to mediate diverse stress-response signaling events. Several other proteins, including ASK3, PARK7, PRDX1, and USP9X were detected with stoichiometries of 0.1:1 or less. We used precise stable isotope dilution assays to quantify protein stoichiometry in the ASK signalosome complex and identified ASK2 at a 1:1 stoichiometric ratio with ASK1 and 14–3-3 proteins (YWHAQ, YWHAB, YWHAH, and YWHAE) collectively at a 0.5:1 ratio with ASK1 as the main components. Using both epitope-tagged overexpression and endogenous expression cell systems, we verified most of the previously reported ASK1 protein-protein interactions and identified 14 proteins that exhibited dynamic shifts in association with ASK1 in response to HNE stress. We employed both shotgun and targeted mass spectrometry assays to catalogue the ASK1 protein-protein interactions in HEK-293 cells treated with the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE). ASK1 is regulated by a large, dynamic multiprotein signalosome complex, potentially including over 90 reported ASK1-interacting proteins. Apoptosis signal-regulating kinase 1 (ASK1) is a key sensor kinase in the mitogen-activated protein kinase pathway that transduces cellular responses to oxidants and electrophiles.

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