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O-GlcNAc (2-acetamino-2-deoxy-β-D-glucopyranose), an important modification for cellular processes, is catalyzed by O-GlcNAc transferase and O-GlcNAcase. O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc) is a nonselective inhibitor of O-GlcNAcase, which increases the level of protein O-GlcNAcylation and is known to induce insulin-resistance in adipose cells due to uncharacterized targets of this inhibitor. In this study, using ATP affinity chromatography, we applied a targeted proteomic approach for identification of proteins induced by treatment with PUGNAc. For optimization of proteomic methods using ATP affinity chromatography, comparison of two cell lines (3T3-L1 adipocytes and C2C12 myotubes) and two different digestion steps was performed using four different structures of immobilized ATP-bound resins. Using this approach, based on DNA sequence homologies, we found that the identified proteins covered almost half of ATP-binding protein families classified by PROSITE. The optimized ATP affinity chromatography approach was applied for identification of proteins that were differentially expressed in 3T3-L1 adipocytes following treatment with PUGNAc. For label-free quantitation, a gel-assisted method was used for digestion of the eluted proteins, and analysis was performed using two different MS modes, data-independent (671 proteins identified) and data-dependent (533 proteins identified) analyses. Among identified proteins, 261 proteins belong to nucleotide-binding proteins and we focused on some nucleotide-binding proteins, ubiquitin-activation enzyme 1 (E1), Hsp70, vasolin-containing protein (Vcp), and Hsp90, involved in ubiquitin-proteasome degradation and insulin signaling pathways. In addition, we found that treatment with PUGNAc resulted in increased ubiquitination of proteins in a time-dependent manner, and a decrease in both the amount of Akt and the level of phosphorylation of Akt, a key component in insulin signaling, through downregulation of Hsp90. In this study, based on a targeted proteomic approach using ATP affinity chromatography, we found four proteins related to ubiquitination and insulin signaling pathways that were induced by treatment with PUGNAc. This result would provide insight into understanding functions of PUGNAc in 3T3-L1 cells., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hepatocytes are used widely as a cell model for investigation of xenobiotic metabolism and the toxic mechanism of drugs. Simvastatin is the first statin drug used extensively in clinical practice for control of elevated cholesterol or hypercholesterolemia. However, it has also been reported to cause adverse effects in liver due to cellular damage. In this study, for proteomic and transcriptomic analysis, rat primary hepatocytes were exposed to simvastatin at IC20 concentration for 24 h. Among a total of 607 differentially expressed proteins, 61 upregulated and 29 downregulated proteins have been identified in the simvastatin-treated group. At the mRNA level, results of transcriptomic analysis revealed 206 upregulated and 41 downregulated genes in the simvastatin-treated group. Based on results of transcriptomic and proteomic analysis, NRF2-mediated oxidative stress response, xenobiotics by metabolism of cytochrome P450, fatty acid metabolism, bile metabolism, and urea cycle and inflammation metabolism pathways were focused using IPA software. Genes (FASN, UGT2B, ALDH1A1, CYP1A2, GSTA2, HAP90, IL-6, IL-1, FABP4, and ABC11) and proteins (FASN, CYP2D1, UG2TB, ALDH1A1, GSTA2, HSP90, FABP4, and ABCB11) related to several important pathways were confirmed by real-time PCR andWestern blot analysis, respectively. This study will provide new insight into the potential toxic pathways induced by simvastatin., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Reactive glia plays a central role in neuroinflammation associated with secondary damage after brain injury. In order to understand the global effects of therapeutic hypothermia on glial activation and neuroinflammation, we performed proteomic profiling of glial cultures following inflammatory stimulation and hypothermic exposure. Primary mixed glial cultures prepared from mouse brains were stimulated with lipopolysaccharide and interferon-γ under normothermic (37°C) or moderate hypothermic (29°C) conditions, and their proteome profiles were compared by LC-ESI-MS/MS. Differentially expressed proteins were determined by high-throughput label-free quantification. Under hypothermic conditions, 64 and 16 proteins were upregulated (≥1.5-fold) and downregulated (≤ 0.7-fold), respectively, compared to normothermic conditions. More importantly, hypothermia altered the abundance of 143 proteins that were either increased or decreased by inflammatory stimulation. The results were validated for several proteins (ICAM-1, STAT-1, YWHAB, and IFIT-3) by Western blot analysis. Pathway and network analysis indicate that hypothermia influences various biological functions of glia such as molecular transport, cell movement, immune response, cell death, and stress response. In conclusion, moderate hypothermia seems to have a significant effect on the protein expression profiles of brain glia and possibly ensuing neuroinflammation. These proteins may be involved in the protective mechanism of hypothermia against brain injuries., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To identify biomarker candidates associated with early IgA nephropathy (IgAN) and thin basement membrane nephropathy (TBMN), the most common causes presenting isolated hematuria in childhood, a proteomic approach of urinary exosomes from early IgAN and TBMN patients was introduced. The proteomic results from the patients were compared with a normal group to understand the pathophysiological processes associated with these diseases at the protein level. The urinary exosomes, which reflect pathophysiological processes, collected from three groups of young adults (early IgAN, TBMN, and normal) were trypsin-digested using a gel-assisted protocol, and quantified by label-free LC-MS/MS, using an MS(E) mode. A total of 1877 urinary exosome proteins, including cytoplasmic, membrane, and vesicle trafficking proteins, were identified. Among the differentially expressed proteins, four proteins (aminopeptidase N, vasorin precursor, α-1-antitrypsin, and ceruloplasmin) were selected as biomarker candidates to differentiate early IgAN from TBMN. We confirmed the protein levels of the four biomarker candidates by semi-quantitative immunoblot analysis in urinary exosomes independently prepared from other patients, including older adult groups. Further clinical studies are needed to investigate the diagnostic and prognostic value of these urinary markers for early IgAN and TBMN. Taken together, this study showed the possibility of identifying biomarker candidates for human urinary diseases using urinary exosomes and might help to understand the pathophysiology of early IgAN and TBMN at the protein level., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We introduce a new methodology named ligand profiling and identification for effective discovery of bioactive ligands such as peptide hormones. This technology was developed from a new concept of parallel column chromatography and active fraction profiling by nano-LC MS. Traditional methods use sequential column chromatography, and thus are inevitably limited by the low abundance of the peptide of interest and by a low yield due to the many column steps. Using this new technology, insulin was successfully identified and diarginylinsulin, a minor intermediate form of insulin, was unexpectedly also identified simultaneously from 100 mg of porcine pancreatic tissue. This integrative technology could be used to search for various low-abundance peptides (or bioactive molecules) rapidly and simultaneously, by applying this to the later stages of traditional sequential purification.