Label-free proteomic identification of endogenous, insulin-stimulated interaction partners of insulin receptor substrate-1

Thangiah Geetha, Paul Langlais, Moulun Luo, Rebekka Mapes, Natalie Lefort, Shu Chuan Chen, Lawrence J. Mandarino, Zhengping Yi

    Research output: Contribution to journalArticle

    27 Scopus citations

    Abstract

    Protein-protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein-protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein-protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLCESI- MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.

    Original languageEnglish (US)
    Pages (from-to)457-466
    Number of pages10
    JournalJournal of the American Society for Mass Spectrometry
    Volume22
    Issue number3
    DOIs
    StatePublished - Mar 2011

    Keywords

    • HPLC-ESI-MS/MS
    • Insulin receptor substrate-1
    • Insulin signaling complexes
    • Label-free
    • Protein-protein interactions

    ASJC Scopus subject areas

    • Structural Biology
    • Spectroscopy

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