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.

Potency Assessment of CBD Oils by Their Effects on Cell Signaling Pathways

“…Assignment of pI values to protein phosphoisoforms was based on the literature and our own data [15][16][17][18][19][20][21][22][23][24][25][26][27]. Quantitation of protein expression and phosphorylation levels were performed using Compass software from Protein Simple.…”

Quantitative Assessment of Liver Steatosis and Affected Pathways with Molecular Imaging and Proteomic Profiling

“…On the other hand, nanofluidic proteomics quantitatively measured perturbations to the PTM profiles of selective proteins to identify abnormalities in associated cellular processes. Significantly, nanofluidic proteomics allowed analysis of protein species using less than 40 ng of total cellular protein per assay 28 , 52 , 53 . By comparison, Western blot analysis required approximately 10 µg of total cellular protein per assay 52 .…”

“…Significantly, nanofluidic proteomics allowed analysis of protein species using less than 40 ng of total cellular protein per assay 28 , 52 , 53 . By comparison, Western blot analysis required approximately 10 µg of total cellular protein per assay 52 . Using less than the amount of total cellular protein required for a single Western blot, up to 96 cIEF immunoassays could be performed in a single run with the timescale of several hours 54 .…”

Detection of the Cell Cycle-Regulated Negative Feedback Phosphorylation of Mitogen-Activated Protein Kinases in Breast Carcinoma using Nanofluidic Proteomics

“…The isoelectric points corresponding to the protein isoforms are summarized in Table 1 . The pI range assignments for protein isoforms were experimentally determined by this study as well as by several previous studies from multiple independent research groups 17 – 20 , 23 , 31 , 32 . Compared with size separation by Western blots, charge separation by the cIEF immunoassay was highly sensitive to the analysis of protein isoforms of the MAPK pathway.…”