title: Label-free proteomic identification of endogenous, insulin-stimulated interaction partners of insulin receptor substrate-1.
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. HPLC-ESI-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.
authors.affiliation: Center for Metabolic and Vascular Biology, Arizona State University, Tempe, AZ, USA.
authors.forename: Thangiah
authors.initials: T
authors.lastname: Geetha
authors.forename: Paul
authors.initials: P
authors.lastname: Langlais
authors.forename: Moulun
authors.initials: M
authors.lastname: Luo
authors.forename: Rebekka
authors.initials: R
authors.lastname: Mapes
authors.forename: Natalie
authors.initials: N
authors.lastname: Lefort
authors.forename: Shu-Chuan
authors.initials: SC
authors.lastname: Chen
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Mandarino
authors.forename: Zhengping
authors.initials: Z
authors.lastname: Yi
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D055504:Insulin Receptor Substrate Proteins / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D018709:Statistics, Nonparametric; D053719:Tandem Mass Spectrometry
chemical_list: D055504:Insulin Receptor Substrate Proteins; D010446:Peptide Fragments