title: Capillary Isoelectric Focusing Immunoassay for Fat Cell Differentiation Proteomics.
abstract: Profiling cellular proteome is critical to understanding signal integration during cell fate determination. In this study, the capability of capillary isoelectric focusing (cIEF) immunoassays to detect post-translational modifications (PTM) of protein isoforms is demonstrated. cIEF immunoassays exhibit protein detection sensitivity at up to 5 orders of magnitude higher than traditional methods. This detection ultra-sensitivity permits proteomic profiling of several nanograms of tissue samples. cIEF immunoassays are employed to simultaneously profile three protein kinases during fat cell differentiation: cGMP-dependent protein kinase type I (PKG-I) of the nitric oxide (NO) signaling pathway, protein kinase B (Akt) of the insulin signaling pathway, and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway. Interestingly, a switch in the expression level of PKG- isoforms is observed during fat cell differentiation. While both PKG-Iα and PKG-Iβ isoforms are present in preadipocytes, only PKG-Iβ isoform is expressed in adipocytes. On the other hand, the phosphorylation level increases for Akt while decreases for ERK1 and ERK2 following the maturation of preadipocytes into adipocytes. Taken together, cIEF immunoassay provides a highly sensitive means to study fat cell differentiation proteomics. cIEF immunoassay should be a powerful proteomics tool to study complex protein signal integration in biological systems.
authors.affiliation: Department of Biomedical Sciences, Center for Diabetes and Obesity Research, College of Medicine, Roseman University of Health Sciences, 10530 Discovery Drive, Las Vegas, Nevada, 89135, United States of America.
authors.forename: Mary G
authors.initials: MG
authors.lastname: Johlfs
authors.affiliation: Department of Biomedical Sciences, Center for Diabetes and Obesity Research, College of Medicine, Roseman University of Health Sciences, 10530 Discovery Drive, Las Vegas, Nevada, 89135, United States of America.
authors.forename: Priyatham
authors.initials: P
authors.lastname: Gorjala
authors.affiliation: Department of Biomedical Sciences, Center for Diabetes and Obesity Research, College of Medicine, Roseman University of Health Sciences, 10530 Discovery Drive, Las Vegas, Nevada, 89135, United States of America.
authors.forename: Yasuyo
authors.initials: Y
authors.lastname: Urasaki
authors.affiliation: Department of Biomedical Sciences, Center for Diabetes and Obesity Research, College of Medicine, Roseman University of Health Sciences, 10530 Discovery Drive, Las Vegas, Nevada, 89135, United States of America.
authors.forename: Thuc T
authors.initials: TT
authors.lastname: Le
authors.affiliation: Department of Biomedical Sciences, Center for Diabetes and Obesity Research, College of Medicine, Roseman University of Health Sciences, 10530 Discovery Drive, Las Vegas, Nevada, 89135, United States of America.
authors.forename: Ronald R
authors.initials: RR
authors.lastname: Fiscus
mesh_terms: D017667:Adipocytes / Q000166:cytology / Q000201:enzymology*; D050156:Adipogenesis; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000032:analysis / Q000096:biosynthesis; D019075:Electrophoresis, Capillary / Q000379:methods*; D006367:HeLa Cells / Q000737:chemistry; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D008853:Microscopy / Q000379:methods; D019950:Mitogen-Activated Protein Kinase 1 / Q000032:analysis; D048052:Mitogen-Activated Protein Kinase 3 / Q000032:analysis; D009852:Omentum / Q000166:cytology; D010766:Phosphorylation; D020033:Protein Isoforms / Q000032:analysis; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D051057:Proto-Oncogene Proteins c-akt / Q000032:analysis; D015398:Signal Transduction; D013059:Spectrum Analysis, Raman
chemical_list: D020033:Protein Isoforms; D051057:Proto-Oncogene Proteins c-akt; D062965:Cyclic GMP-Dependent Protein Kinase Type I; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3