Dong Gi Mun

A protein profile of visceral adipose tissues linked to early pathogenesis of type 2 diabetes mellitus

Adipose tissue is increasingly recognized as an endocrine organ playing important pathophysiological roles in metabolic abnormalities, such as obesity, cardiovascular disease, and type 2 diabetes mellitus (T2DM). In particular, visceral adipose tissue (VAT), as opposed to subcutaneous adipose tissue, is closely linked to the pathogenesis of insulin resistance and T2DM. Despite the importance of VAT, its molecular signatures related to the pathogenesis of T2DM have not been systematically explored. Here, we present comprehensive proteomic analysis of VATs in drug-naïve early T2DM patients and subjects with normal glucose tolerance. A total of 4,707 proteins were identified in LC-MS/MS experiments. Among them, 444 increased in abundance in T2DM and 328 decreased. They are involved in T2DM-related processes including inflammatory responses, peroxisome proliferator-activated receptor signaling, oxidative phosphorylation, fatty acid oxidation, and glucose metabolism. Of these proteins, we selected 11 VAT proteins that can represent alteration in early T2DM patients. Among them, up-regulation of FABP4, C1QA, S100A8, and SORBS1 and down-regulation of ACADL and PLIN4 were confirmed in VAT samples of independent early T2DM patients using Western blot. In summary, our profiling provided a comprehensive basis for understanding the link of a protein profile of VAT to early pathogenesis of T2DM.

PTRF/cavin-1 is essential for multidrug resistance in cancer cells

Since detergent-resistant lipid rafts play important roles in multidrug resistance (MDR), their comprehensive proteomics could provide new insights to understand the underlying molecular mechanism of MDR in cancer cells. In the present work, lipid rafts were isolated from MCF-7 and adriamycin-resistant MCF-7/ADR cells and their proteomes were analyzed by label-free quantitative proteomics. Polymerase I and transcript release factor (PTRF)/cavin-1 was measured to be upregulated along with multidrug-resistant P-glycoprotein, caveolin-1, and serum deprivation protein response/cavin-2 in the lipid rafts of MCF-7/ADR cells. PTRF knockdown led to reduction in the amount of lipid rafts on the surface of MCF7/ADR cells as determined by cellular staining with lipid raft-specific dyes such as S-laurdan2 and FITC-conjugated cholera toxin B. PTRF knockdown also reduced MDR in MCF-7/ADR cells. These data indicate that PTRF is necessary for MDR in cancer cells via the fortification of lipid rafts.

Fully automated multifunctional ultrahigh pressure liquid chromatography system for advanced proteome analyses.

A multifunctional liquid chromatography system that performs 1-dimensional, 2-dimensional (strong cation exchange/reverse phase liquid chromatography or SCX/RPLC) separations and online phosphopeptide enrichment using a single binary nanoflow pump has been developed. With a simple operation of a function selection valve equipped with a SCX column and a TiO2 (titanium dioxide) column, a fully automated selection of three different experiment modes was achieved. Because the current system uses essentially the same solvent flow paths, the same trap column, and the same separation column for reverse-phase separation of 1D, 2D, and online phosphopeptides enrichment experiments, the elution time information obtained from these experiments is in excellent agreement, which facilitates correlating peptide information from different experiments. The final reverse-phase separation of the three experiments is completely decoupled from all of the function selection processes; thereby salts or acids from SCX or TiO2 column do not affect the efficiency of the reverse-phase separation.

The effect and potential of using a temperature controlled separation column with ultra-high pressure microcapillary liquid chromatography/tandem mass spectrometry on proteomic analysis

The microcapillary liquid chromatography (µLC)/tandem mass spectrometry (MS/MS) system has become a prevailing analytical platform in proteomics. Typical proteomic studies aimed at proteome-wide identification of peptides and proteins rely heavily on producing an accurate and reproducible solvent-composition gradient throughout microcapillary separation columns to improve LC separation. With the recent advent of targeted proteomic approaches utilizing the LC retention time as a physicochemical parameter for peptides, high reproducibility of LC separation additionally becomes an important factor. In this study, column temperature elevation is utilized to improve reproducibility and separation efficiency of the µLC-MS/MS system. The simple incorporation of a semi-rigid gas line heater allowed precise control of the temperature of microcapillary columns longer than 70 cm, up to 60 °C. Tryptic enolase peptides were used as a standard sample to evaluate the effect of the controlled temperature elevation on the peptide separation efficiency and reproducibility. In addition to the increased reproducibility in peptide elution time due to the controlled column temperature, the temperature elevation resulted in a decrease in the column operation pressure, which, in turn, allowed a higher solvent flow-rate to be employed using the same LC pumps, leading to further improvements in the performance of µLC systems.

Compact variant‐rich customized sequence database and a fast and sensitive database search for efficient proteogenomic analyses

In proteogenomic analysis, construction of a compact, customized database from mRNA‐seq data and a sensitive search of both reference and customized databases are essential to accurately determine protein abundances and structural variations at the protein level. However, these tasks have not been systematically explored, but rather performed in an ad‐hoc fashion. Here, we present an effective method for constructing a compact database containing comprehensive sequences of sample‐specific variants—single nucleotide variants, insertions/deletions, and stop‐codon mutations derived from Exome‐seq and RNA‐seq data. It, however, occupies less space by storing variant peptides, not variant proteins. We also present an efficient search method for both customized and reference databases. The separate searches of the two databases increase the search time, and a unified search is less sensitive to identify variant peptides due to the smaller size of the customized database, compared to the reference database, in the target‐decoy setting. Our method searches the unified database once, but performs target‐decoy validations separately. Experimental results show that our approach is as fast as the unified search and as sensitive as the separate searches. Our customized database includes mutation information in the headers of variant peptides, thereby facilitating the inspection of peptide‐spectrum matches.

Integrated analysis of global proteome, phosphoproteome, and glycoproteome enables complementary interpretation of disease-related protein networks

Multi-dimensional proteomic analyses provide different layers of protein information, including protein abundance and post-translational modifications. Here, we report an integrated analysis of protein expression, phosphorylation, and N-glycosylation by serial enrichments of phosphorylation and N-glycosylation (SEPG) from the same tissue samples. On average, the SEPG identified 142,106 unmodified peptides of 8,625 protein groups, 18,846 phosphopeptides (15,647 phosphosites), and 4,019 N-glycopeptides (2,634 N-glycosites) in tumor and adjacent normal tissues from three gastric cancer patients. The combined analysis of these data showed that the integrated analysis additively improved the coverages of gastric cancer-related protein networks; phosphoproteome and N-glycoproteome captured predominantly low abundant signal proteins, and membranous or secreted proteins, respectively, while global proteome provided abundances for general population of the proteome. Therefore, our results demonstrate that the SEPG can serve as an effective approach for multi-dimensional proteome analyses, and the holistic profiles of protein expression and PTMs enabled improved interpretation of disease-related networks by providing complementary information.

Multi-omics analysis identifies pathways and genes involved in diffuse-type gastric carcinogenesis induced by E-cadherin, p53, and Smad4 loss in mice

The molecular mechanisms underlying the pathogenesis of diffuse‐type gastric cancer (DGC) have not been adequately explored due to a scarcity of appropriate animal models. A recently developed tool well suited for this line of investigation is the Pdx‐1‐Cre;Cdh1F/+;Trp53F/F;Smad4F/F (pChePS) mouse model that spontaneously develops metastatic DGC showing nearly complete E‐cadherin loss. Here, we performed a proteogenomic analysis to uncover the molecular changes induced by the concurrent targeting of E‐cadherin, p53, and Smad4 loss. The gene expression profiles of mouse DGCs and in vivo gastric phenotypes from various combinations of gene knockout demonstrated that these mutations collaborate to activate cancer‐associated pathways to generate aggressive DGC. Of note, WNT‐mediated epithelial‐to‐mesenchymal transition (EMT) and extracellular matrix (ECM)‐cytokine receptor interactions were prominently featured. In particular, the WNT target gene osteopontin (OPN) that functions as an ECM cytokine is highly upregulated. In validation experiments, OPN contributed to DGC stemness by promoting cancer stem cell (CSC) survival and chemoresistance. It was further found that Bcl‐xL acts as a targetable downstream effector of OPN in DGC CSC survival. In addition, Zeb2 and thymosin‐β4 (Tβ4) were identified as prime candidates as suppressors of E‐cadherin expression from the remaining Cdh1 allele during DGC development. Specifically, Tβ4 suppressed E‐cadherin expression and anoikis while promoting cancer cell growth and migration. Collectively, these proteogenomic analyses broaden and deepen our understanding of the contribution of key driver mutations in the stepwise carcinogenesis of DGC through novel effectors, namely OPN and Tβ4.