Biochemical and Biophysical Research Communications
Proteomic identification of proteins conjugated to ISG15 in mouse and human cells
Section snippets
Materials and methods
Cell culture and antibodies. Ubp43−/− MEFs [30] were cultured in DMEM (Cellgro, Mediatech, Herndon, VA) supplemented with 10% low-endotoxin FBS (HyClone, Logan, UT), 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM Hepes, 1 mM sodium pyruvate, and 2 mM l-glutamine (Biosource, Camarillo, CA). U937 cells were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS and 2 mM l-glutamine (Invitrogen). BMMs were prepared [31] from groups of 2 to 3 C57/BL6 (wild-type) or Ifnar1−/− mice [32]
Identification of candidate ISGylated targets by mass spectrometry
As a first step towards understanding the biological role of ISGylation in mediating the pleiotropic effects of IFN, it is important to elucidate the cellular components targeted by this protein modification system. To identify ISGylation targets by mass spectrometry, we enriched conjugates by ISG15 immunoaffinity purification. Due to the sensitivity of mass spectrometry, trace contaminants could potentially be identified along with true ISGylation targets, making it difficult to distinguish
Discussion
Using immunoprecipitation and mass spectrometry, we identified 76 potential targets of ISGylation. In order to minimize false positives, we chose to purify and identify ISGylated proteins from both mouse and human IFN-treated cells. By using different monoclonal antibodies and washing six of eight samples with 3–4 M NaCl, we believed that we could enrich candidate ISGylated proteins and reduce background contaminants. Supplementary Table 1 contains a listing of all proteins identified in either
Acknowledgments
We thank Dr. Reid Townsend at the Siteman Cancer Center Proteomics Core (DDRCC Grant 52574, Washington University School of Medicine) and Dr. Mark Crankshaw at Protein and Nucleic Acid Chemistry Laboratories (PNACL; Washington University School of Medicine) for their invaluable expertise and assistance. We thank Dr. Keun-Il Kim at the Sookmyung Women’s University for his expert advice and suggestions throughout the course of this work. Ifnar1−/− mice back-crossed to C57/BL6 were kindly provided
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These two authors contributed equally to this work.