I would like to take this opportunity to introduce the studies performed by our team in cancer proteomics. An increased expression of HSP70 family proteins were identified in hepatoma tissues. Furthermore, increased IgG level of anti-HSP70.1 autoantibodies were identified in hepatoma patients’ sera, and we developed a highly sensitive protein chip which measured anti-HSP70.1 antibodies in sera. In another set of cancer proteomic studies, increased expression of Cofilin-phosphatase slingshot-1L (SSH1L) was identified in pancreatic cancer cells and we elucidated that SSH1L facilitated the motility and invasive ability of such cells. Furthermore, proteomic analysis for GEM-resistant and sensitive pancreatic cancer cells showed up-regulation of HSP27 in GEM-resistant cells and expression levels of HSP27 were significantly related to the survival period. In conclusion, proteomics has been a powerful research tool for the identification of biomarkers and target molecules towards cancer treatment.
We have been developing techniques for monitoring the specified enzymatic activities with the use of small molecular fluorescent substrates (molecules that become fluorescent upon cleavage by specific enzymes) that enable highly sensitive detection of the targeted activities. Based on the techniques, we have developed the novel methodology to search for cell-type-specific and disease-related enzymatic activities by comprehensively evaluating enzymatic activities in bio-samples and identifying the enzymes that metabolize specified substrates. With those techniques, we have successfully characterized several disease-related alternations of enzymatic functions from enzymome (total of active enzymes in living systems).
Silkworm (Bombyx mori) cocoons and silk fibers were partially hydrolyzed by weak acids and the resulting peptide fragments could be analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Acid cleavage was a characteristic phenomenon in silk fibers. Peptide mass fingerprints obtained via MALDI-TOF MS analysis (MALDI biotyping) were compared among local silkworm cocoon varieties. Dendrograms were created based on the peptide mass fingerprints of raw, heat-treated, sodium carbonate-treated, and Marseille soap-treated cocoons. The identified differences suggested that it was possible to distinguish varieties and qualities of silkworm cocoons and silk fibers based on their peptide mass fingerprints.
Protein palmitoylation is the most common, dynamic lipid modification and has become topical, as recent proteomic analyses using acyl-biotinyl exchange chemistry have identified numerous palmitoylated proteins in diverse cells, tissues and species. However, their physiological importance remains poorly understood, as there have been no methods to assess the palmitoylation stoichiometries of proteins. The fields have been long awaiting a quantitative method such as Phos-tag SDS-PAGE to monitor the phosphorylation stoichiometry. Here, we developed a simple, sensitive, and specific method to quantify the palmitoylation states of endogenous proteins, i.e., stoichiometries and site-occupancies. The method was named the acyl-PEGyl exchange gel shift (APEGS) method. The APEGS assay consists of four chemical steps: (1) cleavage of disulfide bonds with tris-(2-carboxyethyl) phosphine (TCEP), (2) blockade of free cysteine thiols with N-ethyl maleimide (NEM), (3) specific cleavage of palmitoylation thioester linkages with hydroxylamine (NH2OH), and (4) labeling of newly exposed cysteinyl thiols with maleimide-conjugated PEG (mPEG), which causes the mobility shift of palmitoylated proteins on SDS-PAGE. Western blotting (WB) with antibodies against proteins of interest would separate palmitoylated bands from non-palmitoylated. Quantifying the relative intensities of bands would provide information about the palmitoylation stoichiometry and palmitoylation states. Advantageous features of the APEGS assay include: (1) versatile application to any biological samples, (2) no need for protein purification, (3) reliably examining whether proteins of interest are palmitoylated if antibodies are available, and (4) investigating the dynamic changes in palmitoylation states. Thus, the APEGS assay contributes to understanding regulatory mechanisms for protein palmitoylation.
Chronic hyperglycemia causes impaired insulin secretion and suppression of insulin gene expression in pancreatic β-cells, referred to as glucotoxicity. However, the molecular mechanisms of glucotoxicity are still not fully understood. We attempted to elucidate signaling pathways for suppression of insulin gene expression in glucotoxicity by techniques using Multi-PK antibody and Microarray analysis. Using Multi-PK antibody, we found that calcium/calmodulin-dependent protein kinase IV (CaMKIV) was decreased in rat pancreatic insulinoma INS-1 cells under glucotoxic conditions. The reduction of CaMKIV was induced by degradation with calpain. Furthermore, CaMKIV was reduced in pancreatic islets of diabetic OLETF (Otsuka Long-Evans Tokushima fatty) rats compared with nondiabetic LETO (Long-Evans Tokushima Otsuka) rats. Next, we performed Microarray and Real-time PCR analysis, resulting that nine protein kinases were affected by glucotoxic condition. Among them, candidate plasticity gene 16 (CPG16) was increased in INS-1 cells under glucotoxic conditions and suppressed insulin promoter activity in a kinase activity-dependent manner. In addition, CPG16 bound and phosphorylated jun dimerization protein 2 (JDP2). JDP2 bound to the G1 element of the insulin promoter and up-regulated insulin promoter activity. Furthermore, CPG16 suppressed the up-regulation of insulin promoter activity by JDP2 in a kinase activity-dependent manner. These results suggest that calpain-CaMKIV and CPG16-JDP2 pathways play important roles in the suppression of insulin gene expression in pancreatic β-cells under glucotoxic conditions.
Phos-tag electrophoresis, which was developed by Kinoshita et al. in 2006, has attracted the attention of many researchers as an epoch-making method that can analyze the phosphorylation state of proteins. The authors were involved in the research and development of Phos-tag diagonal electrophoresis as one of the new techniques for Phos-tag electrophoresis. In this review, the authors describe why research was initiated to develop Phos-tag diagonal electrophoresis, and proteomic significance and the practical protocol of this electrophoresis.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is a promising technique for the high-resolution separation of protein components extracted from crude biological samples and is popular for the use in sample prefractionation processing for mass spectrometry-based proteomics. In order to analyze SDS-PAGE-separated proteins via mass spectrometry, the proteins must first be enzymatically digested in the gel matrix and then recovered from the gel as peptide fragments. Such an in-gel digestion procedure often requires overnight reaction and is more prone to sample loss than in-solution digestion. Here, the focus is on dissolvable polyacrylamide gels prepared using N,N’-bis(acryloyl)cystamine (BAC) as a cross-linker to overcome these problems. The BAC cross-linked polyacrylamide gel is easily dissolved by reductive treatment, allowing lossless protein recovery into the surrounding solution. The suitability of recovered proteins for rapid trypsin digestion under high temperature conditions led to the establishment of a sample preparation workflow for protein mass spectrometry called BAC-DROP (BAC-Gel Dissolution to Digest PAGE-Resolved Objective Proteins). According to this workflow, the entire process from SDS-PAGE separation to the end of trypsin digestion can be completed in approximately 5 h. Combining reduced proteome complexity via high-resolution PAGE fractionation with rapid trypsin digestion provides a unique opportunity for conducting high-throughput analysis of in-depth proteome with a focus on specific molecular weight ranges.
For lipid staining of lipoprotein fractions on electrophoresis, Fat red 7B and Sudan black B are used. These dyes utilize the lipid-soluble characteristic of lipophilic dyes. Accordingly, the value of the fraction may vary due to differences in lipids contained in lipoproteins. We have recently developed a new fully automatic lipoprotein fractionation method, the CH•TG method, which stains total cholesterol (CH) and triglycerides (TG) using enzymes. Compared with the conventional Fat red method, the separation of LDL and VLDL became clearer by the CH•TG method, and full automation was possible. The correlation with the Fat red method was y=1.04x−9.1, r=0.934 for the HDL fraction, y=1.27x−6.6, r=0.893 for the VLDL fraction, and y=1.26x−3.2, r=0.734 for the LDL fraction. In addition, the VLDL and LDL fractions by the CH•TG method better reflected fluctuations in TG and CH, respectively. Based on these results, the CH•TG method may be a new lipoprotein fractionation method that overcomes the problems with the Fat red method.
The characteristics of tumor cells are greatly influenced by the tumor microenvironment, which contains multiple natural extracellular matrices (ECMs). However, only single or specific ECMs are presently used to assess the behavioral characteristics of tumor cells in vitro. Recently, as an alternative to conventional in vitro tumor models, to address the complex challenges in tumor in vitro assays, “decellularization” technology has recently emerged as a new platform for mimicking the tumor microenvironment. Especially, the use of decellularized tissue gels (DTGs) has gained attention as they provide multiple ECM components of the microenvironment of tumor cells. However, as tissues contain unique components of ECMs, it is essential to reproduce the tissue-specific ECM components when evaluating tumor functions in vitro. We hypothesized that DTGs affect the in vitro tumor cell behavior and that the protein composition of DTGs varies between tissues. To test this hypothesis, we created lung and liver DTGs via freeze-thawing and investigated the effects of DTGs on tumor cell behavior such as cell proliferation. We found that DTGs regulated the proliferation of tumor cells when the cells were cultured on DTGs-coated plates. The proteins present in the DTGs were separated via SDS-PAGE, and multiple bands were observed via silver staining. The band pattern varied depending on the tissue from which the DTGs were derived. It was found that certain DTGs modulated the behavioral characteristics of tumor cells, which may have potential applications in cancer studies and the development of therapies. Hence, DTGs are worth investigating as useful tools for tumor cell culture and in vitro assays.