The GeLC-MS workflow, which combines low-cost, easy-to-use sodium dodecyl sulfate (SDS)-polyacryl... more The GeLC-MS workflow, which combines low-cost, easy-to-use sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) with liquid chromatography-mass spectrometry (LC-MS), is very popular in current bottom-up proteomics. However, GeLC-MS requires that PAGE-separated proteins undergo overnight enzymatic digestion in a gel, resulting in more than 20 h of sample preparation for LC-MS. In this study, we overcame the limitations of GeLC-MS by developing a rapid digestion workflow for PAGE separation of proteins using N,N'-bis(acryloyl)cystamine (BAC) cross-linked gels that can be solubilized by reductive treatment. Making use of an established workflow called BAC-DROP (BAC-gel dissolution to digest PAGE-resolved objective proteins), crude proteome samples were fractionated based on molecular weight by BAC cross-linked PAGE. After fractionation, the gel fragments were reductively dissolved in under 5 min, and in-solution trypsin digestion of the protein released from the gel was completed in less than 1 h at 70 °C, equivalent to a 90-95% reduction in time compared to conventional in-gel trypsin digestion. The introduction of the BAC-DROP workflow to the MS assays for inflammatory biomarker CRP and viral marker HBsAg allowed for serum sample preparation to be completed in as little as 5 h, demonstrating successful marker quantification from a 0.5 μL sample of human serum.
Cytomegalovirus (CMV)-induced interstitial pneumonitis is a well-known lethal complication in bon... more Cytomegalovirus (CMV)-induced interstitial pneumonitis is a well-known lethal complication in bone marrowrecipients. CMV is also knownto cause gastroenteritis. Wereport the first case of a bone marrow recipient who developed CMV-induced gastritis which is verified both histologically and virologically. The gastritis preceded the interstitial pneumonitis which was detected in an early stage and was successfully treated. Wepropose that bonemarrowrecipients whoshowsigns and symptoms of gastritis undergoan endoscopic examination, and that biopsied specimens should be scrutinized for CMVby both histological examination and culture investigation.
α2-Macroglobulin is a highly abundant serum protein involved in the development of atherosclerosi... more α2-Macroglobulin is a highly abundant serum protein involved in the development of atherosclerosis and cardiac hypertrophy. However, its circulating molecular form and exact concentrations in human health/diseases are not known. Blue native-polyacrylamide gel electrophoresis of human serum was used to confirm the native conformation of α2-macroglobulin. We created an enzyme-linked immunosorbent assay suitable for quantifying its circulating molecular form and undertook a cross-sectional study to measure its serum levels in 248 patients with diabetes mellitus and 59 healthy volunteers. The predominant circulating molecular form of α2-macroglobulin was the tetramer, whereas its dimer was detectable in patients with high serum levels of α2-macroglobulin. The serum α2-macroglobulin concentration was not associated with glycated hemoglobin or any other glycemic variable as evaluated from 48-h continuous glucose monitoring, but showed close correlation with left ventricular posterior wall...
Early diagnosis of biliary tract cancer (BTC) is important for curative surgical resection. Curre... more Early diagnosis of biliary tract cancer (BTC) is important for curative surgical resection. Current tumor markers of BTC are unsatisfactory in terms of sensitivity and specificity. In a search for novel biomarkers for BTC, serum samples obtained from 62 patients with BTC were compared with those from patients with benign biliary diseases and from healthy controls, using the MALDI-TOF/TOF ClinProt system. Initial screening and further validation identified a peak at 4204 Da with significantly greater intensity in the BTC samples. The 4204 Da peak was partially purified and identified as a fragment of prothrombin by amino acid sequencing. The sensitivity of the 4204 Da peptide for detection of stage I BTC cancer was greater than those for CEA and CA19-9. Also, serum levels of the 4204 Da peptide were above the cut-off level in 15 (79%) of 19 cases in which the CEA and CA19-9 levels were both within their cut-off values. Receiver operating characteristic analysis showed that the combin...
Recent advances in two-dimensional electrophoresis (2-DE) such as fluorescent 2-D differential ge... more Recent advances in two-dimensional electrophoresis (2-DE) such as fluorescent 2-D differential gel electrophoresis (2-D DIGE) has made it possible to detect and quantitate the critical changes involved in disease pathogenesis. We have previously identified novel proteins with altered expression in primary colorectal cancer using agarose 2-DE that has a higher loading capacity than immobilized pH gradient gel. The aim of this study is to identify novel proteins with altered expression in primary esophageal cancer using the powerful method of agarose 2-DE and agarose 2-D DIGE. Excised tissues from 12 patients of primary esophageal cancer were obtained. Proteins with altered expression between cancer and adjacent non-cancer tissues were analyzed by agarose 2-D DIGE and identified by mass spectrometry. Thirty-three proteins out of 74 spots with altered expression in tumors were identified. Among them, a 195-kDa protein, periplakin, was significantly downregulated in esophageal cancer, which was confirmed by immunoblotting. Immunohistochemistry showed that periplakin was mainly localized at cell-cell boundaries in normal epithelium and dysplastic lesions, while it disappeared from cell boundaries, shifted to cytoplasm, in early cancers and scarcely expressed in advanced cancers. These results suggest that periplakin could be a useful marker for detection of early esophageal cancer and evaluation of tumor progression.
Head and neck squamous cell carcinoma (HNSCC) is usually found at a late stage and distant metast... more Head and neck squamous cell carcinoma (HNSCC) is usually found at a late stage and distant metastasis occurs at high frequency; therefore, novel prognostic markers are needed. This study was aimed to identify novel tumor markers in HNSCC. We identified 65 proteins which were significantly increased or decreased in the tumors by 2D-DIGE using 12 HNSCC and adjacent non-cancer tissues. Western blotting and immunohistochemical analysis confirmed that the expression of plectin was significantly increased in most cancer tissues as compared with non-cancer tissues. Strikingly, the suppression of endogenous plectin using siRNA inhibited the proliferation, migration and invasion of HNSCC cells and down-regulated Erk 1/2 kinase. Furthermore, immunohistochemistry using paraffin-embedded tissues from 62 patients showed not only that the frequency of recurrence was correlated with the plectin expression but that the prognosis of patients with a high plectin was extremely poor. Moreover, the survival rate of patients with a high plectin was significantly lower than that of patients with low E-cadherin levels, which is known to correlate with the poor prognosis of HNSCC. Our findings suggest that plectin promotes the migration and invasion of HNSCC cells through activation of Erk 1/2 kinase and is a potential prognostic biomarker of HNSCC.
Although various samples, including tissue, cells, serum, and urine, from patients with renal cel... more Although various samples, including tissue, cells, serum, and urine, from patients with renal cell carcinoma (RCC) have been analyzed, biomarkers with diagnostic value have yet to be identified. We used a proteomics approach to analyze cyst fluid in cases of cyst-associated RCC to identify accessible and abundant proteins that are overexpressed and/or secreted by RCC cells. Proteins in the cyst fluid were separated by reverse-phase highperformance liquid chromatography and agarose twodimensional gel electrophoresis and were identified by tandem mass spectrometry. We conducted a National Center for Biotechnology Information search and a MEDLINE search to predict the function of these identified proteins and to select a tumor-marker candidate protein. Our search resulted in the identification and selection of the differentially regulated protein known as 14-3-3 protein beta/alpha, which was overexpressed in cyst fluid from cyst-associated RCC but has not been previously associated with RCC. We then measured its incidence through Western blotting of various normal and RCC samples (serum, urine, tissue, and cyst fluid). The expression levels of 14-3-3 protein beta/ alpha were higher in urine samples from patients with RCC than in samples from healthy volunteers. Receiver operating characteristic (ROC) curve analyses were performed to assess this potential biomarker; these data (area under the ROC curve value was 0.8813) indicate a high degree of accuracy for this screening method. 14-3-3 Protein beta/ alpha may be a diagnostically useful biomarker for early diagnosis of RCC.
Alcoholism: Clinical and Experimental Research, 2010
Background: The search for biological markers of alcohol abuse is of continual interest in experi... more Background: The search for biological markers of alcohol abuse is of continual interest in experimental and clinical alcohol research. We previously used gel-free proteome analysis methods such as the ProteinChip Ò system and the ClinProtÔ system to search for new serum markers for alcoholism and found several novel marker candidates. As serum contains thousands of proteins and peptides that are present in a large dynamic concentration, depletion of the abundant proteins and further fractionation of the remainder is necessary to get into the deep proteome. We recently described a simple and highly reproducible three-step method for identifying potential disease-marker candidates among the low-abundance serum proteins. Methods: Two serum samples-one on admission and one after 8 weeks of abstinence-were obtained from 8 patients with alcohol dependency. The samples were subjected to a three-step serum proteome analysis. The steps were the following: first, immunodepletion of the 6 most abundant proteins; second, fractionation using reverse-phase high-performance liquid chromatography; and third, separation using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Differences revealed by protein staining were further confirmed by Western blotting and by enzyme-linked immunosorbent assays (ELISA). Results: Three-step serum proteome analysis revealed that the serum levels of 5 proteins, alpha2-HS glycoprotein, apolipoprotein A-I, glutathione peroxidase 3, heparin cofactor II, and pigment epithelial-derived factor (PEDF), were significantly greater on admission than after 8 weeks of abstinence. We focused on PEDF because alterations in its levels in alcoholic subjects are not well known. Western blotting and ELISA confirmed the upregulation of PEDF. Serum PEDF levels were significantly greater in moderate to heavy habitual drinkers (14.2 ± 7.7 lg ⁄ ml) than in healthy subjects without a drinking history (5.5 ± 3.0 lg ⁄ ml) (p < 0.001). The serum PEDF levels in subjects with nonalcoholic chronic liver diseases were comparable to the PEDF levels in healthy subjects. Conclusion: Three-step serum proteome analysis reveals that excessive alcohol drinking increases the PEDF level.
Oxidative stress has been linked to a number of chronic diseases, and this has aroused interest i... more Oxidative stress has been linked to a number of chronic diseases, and this has aroused interest in the identification of clinical biomarkers that can accurately assess its severity. We used liquid chromatography-high resolution mass spectrometry (LC-MS) to show that oxidised and non-oxidised Met residues at position 147 of human serum albumin (Met147) can be accurately and reproducibly quantified with stable isotope-labelled peptides. Met147 oxidation was significantly higher in patients with diabetes than in controls. Least square multivariate analysis revealed that glycated haemoglobin (HbA1c) and glycated albumin (GA) did not significantly influence Met147 oxidation, but the GA/HbA1c ratio, which reflects glycaemic excursions, independently affected Met147 oxidation status. Continuous glucose monitoring revealed that Met147 oxidation strongly correlates with the standard deviation of sensor glucose concentrations and the time spent with hypoglycaemia or hyperglycaemia each day. T...
Serum/plasma contains thousands of different types of proteins and peptides that can provide valu... more Serum/plasma contains thousands of different types of proteins and peptides that can provide valuable information on numerous inherent processes. However, the existence of high-abundance proteins and the large dynamic range of serum proteins/peptides make analysis of low-abundance proteins/peptides challenging. In particular, detailed analysis and identification of native peptides remain extremely difficult due to the tremendous variety of cleavage possibilities and posttranslational modifications. Therefore, widely ranging searches based on peptide identification are difficult. Herein, I describe the highly accurate and sensitive quantitative analysis of over 2,500 peptides. The strategy combined isobaric tag labeling, amine-reactive 6-plex tandem mass tag labeling, and a modified differential solubilization method for high-yield peptide extraction. We quantitatively analyzed six pooled plasma samples (three pre-surgery and three post-surgery) to discover potential candidate biomarker peptides of renal cell carcinoma. The concentrations of about 30 peptides were found to be altered following surgery. A preliminary validation study was conducted using 77 plasma samples to demonstrate the possibility that even unidentified potential candidate biomarker peptides can be verified using the dimethyl labeling method.
Recent mass spectrometry (MS)-based techniques enable deep proteome coverage with relative quanti... more Recent mass spectrometry (MS)-based techniques enable deep proteome coverage with relative quantitative analysis, resulting in increased identification of very weak signals accompanied by increased data size of liquid chromatography (LC)–MS/MS spectra. However, the identification of weak signals using an assignment strategy with poorer performance results in imperfect quantification with misidentification of peaks and ratio distortions. Manually annotating a large number of signals within a very large dataset is not a realistic approach. In this study, therefore, we utilized machine learning algorithms to successfully extract a higher number of peptide peaks with high accuracy and precision. Our strategy evaluated each peak identified using six different algorithms; peptide peaks identified by all six algorithms (i.e., unanimously selected) were subsequently assigned as true peaks, which resulted in a reduction in the false-positive rate. Hence, exact and highly quantitative peptide...
Nihon Rinsho Men'eki Gakkai kaishi = Japanese journal of clinical immunology, 2005
and Life Sciences S2-3 Achievements and improvements in multi-center pilot study of newborn scree... more and Life Sciences S2-3 Achievements and improvements in multi-center pilot study of newborn screening by tandem mass spectrometry
N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions h... more N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that constitutively activates the receptor. However, the molecular basis for this mechanism remains unstudied because no external agonist has been discovered. Here, we describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC). ThC-induced activation persists due to limited receptor internalization. Strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. We show that MPL is subject to sugar-mediated activation and that lectin-mediated activation kinetics differ from cytokine-mediated activation kinetics. Our data demonstrated the potential of lectins to provide deeper insight into human pathogenesis.One-sentence summaryA marine sponge lectin catal...
Uterine carcinosarcoma (UCS) represents a true example of cancer associated with epithelial-mesen... more Uterine carcinosarcoma (UCS) represents a true example of cancer associated with epithelial-mesenchymal transition (EMT), which exhibits cancer stem cell (CSC)-like traits. Although S100A4 is an inducer of EMT, little is known about its involvement in UCS tumorigenesis. Herein, we focused on the functional role of S100A4 during development of UCS. Expression of S100A4 and molecules associated with its function were also examined in 35 UCS cases. In endometrial carcinoma cell lines, S100A4 promoter activity and mRNA levels were significantly increased by the transfection of NF-κB/ p65, independent of a putative κB-binding site in the promoter. Cells stably overexpressing S100A4 showed enhancement of CSC properties, along with decreased cell proliferation and acceleration of cell migration. These phenotypes were abrogated in S100A4-knockdown cells. A combination of S100A4 antibody-mediated co-immunoprecipitation and shotgun proteomics analysis revealed that S100A4 strongly interacted with non-muscle myosin II (NMII) heavy chains, including myosin 9 and myosin 14. Specific inhibition of NMII by blebbistatin phenocopied S100A4 overexpression and induced a fibroblast-like morphology. In clinical samples, S100A4 score was significantly higher in sarcomatous as compared with carcinomatous components of UCS, and was positively correlated with ALDH1, Slug, and vimentin scores, and inversely with Ki-67 labeling indices. These findings suggest that an S100A4/NMII-related signaling cascade may contribute to the establishment and maintenance of EMT/CSC properties, along with changes in cell proliferation and migration capability. These events may be initiated in carcinomatous components in UCS and lead to divergent sarcomatous differentiation.
Cancer types can be classified according to novel antibody-based proteomics using anti-phosphopro... more Cancer types can be classified according to novel antibody-based proteomics using anti-phosphoprotein monoclonal antibodies (PPmAbs) and multiple discriminant analysis. The antibody-based phosphoproteomics using an antibody panel from over 150 uncharacterized PPmAbs combined with multiple discriminant analysis makes it possible to classify cancer cells. To improve the system, new antibody panels need to be developed using characterized PPmAbs for clinical diagnosis. The uncharacterized 154 PPmAbs were tested for reactivity based on immunohistostaining of several cancer tissues. We focused on AKPS288 PPmAb, and the PPmAb-related antigen was localized in the cytoplasm of tumor cells of the colon and stomach but did not react with non-tumor cells in both tissues. Moreover, the AKPS288 PPmAb showed positive staining in the cytoplasm of normal prostate tissue but not cancer tissue. Based on the mass spectrometry (MS), the PPmAb-related antigen was identified as TATA-element modulatory factor 1 (TMF/ARA160), a tumor-associated antigen (TAA). These results indicate that the use of a novel antibody panel consisting of anti-TAA mAbs could have considerably greater utility for cancer classification than the PPmAb panel with unknown specificity identified in our previous study.
Autoimmune mechanisms have been hypothesized to underlie a number of human disorders in which bot... more Autoimmune mechanisms have been hypothesized to underlie a number of human disorders in which both disease pathogenesis and diagnostic biomarkers remain poorly understood. This is partly due to a lack of efficient techniques for identification of plasma autoantibodies associated with specific pathophysiological conditions. We have developed a novel proteomic methodology to comprehensively identify plasma IgG-bound proteins using liquid chromatography tandem mass spectrometry (LC-MS/MS) after denaturing enriched plasma IgG to solubilize and release low molecular weight proteins. In total, we identified 44 proteins using this method that were undetectable in unprocessed plasma, 21 of which were not identified in the Human Plasma Proteome Draft of 2017. Comparison of plasma IgGbound proteins between healthy subjects and patients with isolated adrenocorticotropic hormone deficiency, a rare endocrine disorder speculated to involve autoimmune mechanisms, revealed several distinct IgG-bound proteins specifically detected in patient plasma but not in healthy subjects. Our results suggest that solubilization of low molecular weight proteins bound to enriched plasma IgG and subsequent proteomic analysis by LC-MS/MS could provide a promising strategy for identification of autoantigens in human peripheral blood.
The following versions of software and data (see references i O) were used in the production of t... more The following versions of software and data (see references i O) were used in the production of this report:
The GeLC-MS workflow, which combines low-cost, easy-to-use sodium dodecyl sulfate (SDS)-polyacryl... more The GeLC-MS workflow, which combines low-cost, easy-to-use sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) with liquid chromatography-mass spectrometry (LC-MS), is very popular in current bottom-up proteomics. However, GeLC-MS requires that PAGE-separated proteins undergo overnight enzymatic digestion in a gel, resulting in more than 20 h of sample preparation for LC-MS. In this study, we overcame the limitations of GeLC-MS by developing a rapid digestion workflow for PAGE separation of proteins using N,N'-bis(acryloyl)cystamine (BAC) cross-linked gels that can be solubilized by reductive treatment. Making use of an established workflow called BAC-DROP (BAC-gel dissolution to digest PAGE-resolved objective proteins), crude proteome samples were fractionated based on molecular weight by BAC cross-linked PAGE. After fractionation, the gel fragments were reductively dissolved in under 5 min, and in-solution trypsin digestion of the protein released from the gel was completed in less than 1 h at 70 °C, equivalent to a 90-95% reduction in time compared to conventional in-gel trypsin digestion. The introduction of the BAC-DROP workflow to the MS assays for inflammatory biomarker CRP and viral marker HBsAg allowed for serum sample preparation to be completed in as little as 5 h, demonstrating successful marker quantification from a 0.5 μL sample of human serum.
Cytomegalovirus (CMV)-induced interstitial pneumonitis is a well-known lethal complication in bon... more Cytomegalovirus (CMV)-induced interstitial pneumonitis is a well-known lethal complication in bone marrowrecipients. CMV is also knownto cause gastroenteritis. Wereport the first case of a bone marrow recipient who developed CMV-induced gastritis which is verified both histologically and virologically. The gastritis preceded the interstitial pneumonitis which was detected in an early stage and was successfully treated. Wepropose that bonemarrowrecipients whoshowsigns and symptoms of gastritis undergoan endoscopic examination, and that biopsied specimens should be scrutinized for CMVby both histological examination and culture investigation.
α2-Macroglobulin is a highly abundant serum protein involved in the development of atherosclerosi... more α2-Macroglobulin is a highly abundant serum protein involved in the development of atherosclerosis and cardiac hypertrophy. However, its circulating molecular form and exact concentrations in human health/diseases are not known. Blue native-polyacrylamide gel electrophoresis of human serum was used to confirm the native conformation of α2-macroglobulin. We created an enzyme-linked immunosorbent assay suitable for quantifying its circulating molecular form and undertook a cross-sectional study to measure its serum levels in 248 patients with diabetes mellitus and 59 healthy volunteers. The predominant circulating molecular form of α2-macroglobulin was the tetramer, whereas its dimer was detectable in patients with high serum levels of α2-macroglobulin. The serum α2-macroglobulin concentration was not associated with glycated hemoglobin or any other glycemic variable as evaluated from 48-h continuous glucose monitoring, but showed close correlation with left ventricular posterior wall...
Early diagnosis of biliary tract cancer (BTC) is important for curative surgical resection. Curre... more Early diagnosis of biliary tract cancer (BTC) is important for curative surgical resection. Current tumor markers of BTC are unsatisfactory in terms of sensitivity and specificity. In a search for novel biomarkers for BTC, serum samples obtained from 62 patients with BTC were compared with those from patients with benign biliary diseases and from healthy controls, using the MALDI-TOF/TOF ClinProt system. Initial screening and further validation identified a peak at 4204 Da with significantly greater intensity in the BTC samples. The 4204 Da peak was partially purified and identified as a fragment of prothrombin by amino acid sequencing. The sensitivity of the 4204 Da peptide for detection of stage I BTC cancer was greater than those for CEA and CA19-9. Also, serum levels of the 4204 Da peptide were above the cut-off level in 15 (79%) of 19 cases in which the CEA and CA19-9 levels were both within their cut-off values. Receiver operating characteristic analysis showed that the combin...
Recent advances in two-dimensional electrophoresis (2-DE) such as fluorescent 2-D differential ge... more Recent advances in two-dimensional electrophoresis (2-DE) such as fluorescent 2-D differential gel electrophoresis (2-D DIGE) has made it possible to detect and quantitate the critical changes involved in disease pathogenesis. We have previously identified novel proteins with altered expression in primary colorectal cancer using agarose 2-DE that has a higher loading capacity than immobilized pH gradient gel. The aim of this study is to identify novel proteins with altered expression in primary esophageal cancer using the powerful method of agarose 2-DE and agarose 2-D DIGE. Excised tissues from 12 patients of primary esophageal cancer were obtained. Proteins with altered expression between cancer and adjacent non-cancer tissues were analyzed by agarose 2-D DIGE and identified by mass spectrometry. Thirty-three proteins out of 74 spots with altered expression in tumors were identified. Among them, a 195-kDa protein, periplakin, was significantly downregulated in esophageal cancer, which was confirmed by immunoblotting. Immunohistochemistry showed that periplakin was mainly localized at cell-cell boundaries in normal epithelium and dysplastic lesions, while it disappeared from cell boundaries, shifted to cytoplasm, in early cancers and scarcely expressed in advanced cancers. These results suggest that periplakin could be a useful marker for detection of early esophageal cancer and evaluation of tumor progression.
Head and neck squamous cell carcinoma (HNSCC) is usually found at a late stage and distant metast... more Head and neck squamous cell carcinoma (HNSCC) is usually found at a late stage and distant metastasis occurs at high frequency; therefore, novel prognostic markers are needed. This study was aimed to identify novel tumor markers in HNSCC. We identified 65 proteins which were significantly increased or decreased in the tumors by 2D-DIGE using 12 HNSCC and adjacent non-cancer tissues. Western blotting and immunohistochemical analysis confirmed that the expression of plectin was significantly increased in most cancer tissues as compared with non-cancer tissues. Strikingly, the suppression of endogenous plectin using siRNA inhibited the proliferation, migration and invasion of HNSCC cells and down-regulated Erk 1/2 kinase. Furthermore, immunohistochemistry using paraffin-embedded tissues from 62 patients showed not only that the frequency of recurrence was correlated with the plectin expression but that the prognosis of patients with a high plectin was extremely poor. Moreover, the survival rate of patients with a high plectin was significantly lower than that of patients with low E-cadherin levels, which is known to correlate with the poor prognosis of HNSCC. Our findings suggest that plectin promotes the migration and invasion of HNSCC cells through activation of Erk 1/2 kinase and is a potential prognostic biomarker of HNSCC.
Although various samples, including tissue, cells, serum, and urine, from patients with renal cel... more Although various samples, including tissue, cells, serum, and urine, from patients with renal cell carcinoma (RCC) have been analyzed, biomarkers with diagnostic value have yet to be identified. We used a proteomics approach to analyze cyst fluid in cases of cyst-associated RCC to identify accessible and abundant proteins that are overexpressed and/or secreted by RCC cells. Proteins in the cyst fluid were separated by reverse-phase highperformance liquid chromatography and agarose twodimensional gel electrophoresis and were identified by tandem mass spectrometry. We conducted a National Center for Biotechnology Information search and a MEDLINE search to predict the function of these identified proteins and to select a tumor-marker candidate protein. Our search resulted in the identification and selection of the differentially regulated protein known as 14-3-3 protein beta/alpha, which was overexpressed in cyst fluid from cyst-associated RCC but has not been previously associated with RCC. We then measured its incidence through Western blotting of various normal and RCC samples (serum, urine, tissue, and cyst fluid). The expression levels of 14-3-3 protein beta/ alpha were higher in urine samples from patients with RCC than in samples from healthy volunteers. Receiver operating characteristic (ROC) curve analyses were performed to assess this potential biomarker; these data (area under the ROC curve value was 0.8813) indicate a high degree of accuracy for this screening method. 14-3-3 Protein beta/ alpha may be a diagnostically useful biomarker for early diagnosis of RCC.
Alcoholism: Clinical and Experimental Research, 2010
Background: The search for biological markers of alcohol abuse is of continual interest in experi... more Background: The search for biological markers of alcohol abuse is of continual interest in experimental and clinical alcohol research. We previously used gel-free proteome analysis methods such as the ProteinChip Ò system and the ClinProtÔ system to search for new serum markers for alcoholism and found several novel marker candidates. As serum contains thousands of proteins and peptides that are present in a large dynamic concentration, depletion of the abundant proteins and further fractionation of the remainder is necessary to get into the deep proteome. We recently described a simple and highly reproducible three-step method for identifying potential disease-marker candidates among the low-abundance serum proteins. Methods: Two serum samples-one on admission and one after 8 weeks of abstinence-were obtained from 8 patients with alcohol dependency. The samples were subjected to a three-step serum proteome analysis. The steps were the following: first, immunodepletion of the 6 most abundant proteins; second, fractionation using reverse-phase high-performance liquid chromatography; and third, separation using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Differences revealed by protein staining were further confirmed by Western blotting and by enzyme-linked immunosorbent assays (ELISA). Results: Three-step serum proteome analysis revealed that the serum levels of 5 proteins, alpha2-HS glycoprotein, apolipoprotein A-I, glutathione peroxidase 3, heparin cofactor II, and pigment epithelial-derived factor (PEDF), were significantly greater on admission than after 8 weeks of abstinence. We focused on PEDF because alterations in its levels in alcoholic subjects are not well known. Western blotting and ELISA confirmed the upregulation of PEDF. Serum PEDF levels were significantly greater in moderate to heavy habitual drinkers (14.2 ± 7.7 lg ⁄ ml) than in healthy subjects without a drinking history (5.5 ± 3.0 lg ⁄ ml) (p < 0.001). The serum PEDF levels in subjects with nonalcoholic chronic liver diseases were comparable to the PEDF levels in healthy subjects. Conclusion: Three-step serum proteome analysis reveals that excessive alcohol drinking increases the PEDF level.
Oxidative stress has been linked to a number of chronic diseases, and this has aroused interest i... more Oxidative stress has been linked to a number of chronic diseases, and this has aroused interest in the identification of clinical biomarkers that can accurately assess its severity. We used liquid chromatography-high resolution mass spectrometry (LC-MS) to show that oxidised and non-oxidised Met residues at position 147 of human serum albumin (Met147) can be accurately and reproducibly quantified with stable isotope-labelled peptides. Met147 oxidation was significantly higher in patients with diabetes than in controls. Least square multivariate analysis revealed that glycated haemoglobin (HbA1c) and glycated albumin (GA) did not significantly influence Met147 oxidation, but the GA/HbA1c ratio, which reflects glycaemic excursions, independently affected Met147 oxidation status. Continuous glucose monitoring revealed that Met147 oxidation strongly correlates with the standard deviation of sensor glucose concentrations and the time spent with hypoglycaemia or hyperglycaemia each day. T...
Serum/plasma contains thousands of different types of proteins and peptides that can provide valu... more Serum/plasma contains thousands of different types of proteins and peptides that can provide valuable information on numerous inherent processes. However, the existence of high-abundance proteins and the large dynamic range of serum proteins/peptides make analysis of low-abundance proteins/peptides challenging. In particular, detailed analysis and identification of native peptides remain extremely difficult due to the tremendous variety of cleavage possibilities and posttranslational modifications. Therefore, widely ranging searches based on peptide identification are difficult. Herein, I describe the highly accurate and sensitive quantitative analysis of over 2,500 peptides. The strategy combined isobaric tag labeling, amine-reactive 6-plex tandem mass tag labeling, and a modified differential solubilization method for high-yield peptide extraction. We quantitatively analyzed six pooled plasma samples (three pre-surgery and three post-surgery) to discover potential candidate biomarker peptides of renal cell carcinoma. The concentrations of about 30 peptides were found to be altered following surgery. A preliminary validation study was conducted using 77 plasma samples to demonstrate the possibility that even unidentified potential candidate biomarker peptides can be verified using the dimethyl labeling method.
Recent mass spectrometry (MS)-based techniques enable deep proteome coverage with relative quanti... more Recent mass spectrometry (MS)-based techniques enable deep proteome coverage with relative quantitative analysis, resulting in increased identification of very weak signals accompanied by increased data size of liquid chromatography (LC)–MS/MS spectra. However, the identification of weak signals using an assignment strategy with poorer performance results in imperfect quantification with misidentification of peaks and ratio distortions. Manually annotating a large number of signals within a very large dataset is not a realistic approach. In this study, therefore, we utilized machine learning algorithms to successfully extract a higher number of peptide peaks with high accuracy and precision. Our strategy evaluated each peak identified using six different algorithms; peptide peaks identified by all six algorithms (i.e., unanimously selected) were subsequently assigned as true peaks, which resulted in a reduction in the false-positive rate. Hence, exact and highly quantitative peptide...
Nihon Rinsho Men'eki Gakkai kaishi = Japanese journal of clinical immunology, 2005
and Life Sciences S2-3 Achievements and improvements in multi-center pilot study of newborn scree... more and Life Sciences S2-3 Achievements and improvements in multi-center pilot study of newborn screening by tandem mass spectrometry
N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions h... more N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that constitutively activates the receptor. However, the molecular basis for this mechanism remains unstudied because no external agonist has been discovered. Here, we describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC). ThC-induced activation persists due to limited receptor internalization. Strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. We show that MPL is subject to sugar-mediated activation and that lectin-mediated activation kinetics differ from cytokine-mediated activation kinetics. Our data demonstrated the potential of lectins to provide deeper insight into human pathogenesis.One-sentence summaryA marine sponge lectin catal...
Uterine carcinosarcoma (UCS) represents a true example of cancer associated with epithelial-mesen... more Uterine carcinosarcoma (UCS) represents a true example of cancer associated with epithelial-mesenchymal transition (EMT), which exhibits cancer stem cell (CSC)-like traits. Although S100A4 is an inducer of EMT, little is known about its involvement in UCS tumorigenesis. Herein, we focused on the functional role of S100A4 during development of UCS. Expression of S100A4 and molecules associated with its function were also examined in 35 UCS cases. In endometrial carcinoma cell lines, S100A4 promoter activity and mRNA levels were significantly increased by the transfection of NF-κB/ p65, independent of a putative κB-binding site in the promoter. Cells stably overexpressing S100A4 showed enhancement of CSC properties, along with decreased cell proliferation and acceleration of cell migration. These phenotypes were abrogated in S100A4-knockdown cells. A combination of S100A4 antibody-mediated co-immunoprecipitation and shotgun proteomics analysis revealed that S100A4 strongly interacted with non-muscle myosin II (NMII) heavy chains, including myosin 9 and myosin 14. Specific inhibition of NMII by blebbistatin phenocopied S100A4 overexpression and induced a fibroblast-like morphology. In clinical samples, S100A4 score was significantly higher in sarcomatous as compared with carcinomatous components of UCS, and was positively correlated with ALDH1, Slug, and vimentin scores, and inversely with Ki-67 labeling indices. These findings suggest that an S100A4/NMII-related signaling cascade may contribute to the establishment and maintenance of EMT/CSC properties, along with changes in cell proliferation and migration capability. These events may be initiated in carcinomatous components in UCS and lead to divergent sarcomatous differentiation.
Cancer types can be classified according to novel antibody-based proteomics using anti-phosphopro... more Cancer types can be classified according to novel antibody-based proteomics using anti-phosphoprotein monoclonal antibodies (PPmAbs) and multiple discriminant analysis. The antibody-based phosphoproteomics using an antibody panel from over 150 uncharacterized PPmAbs combined with multiple discriminant analysis makes it possible to classify cancer cells. To improve the system, new antibody panels need to be developed using characterized PPmAbs for clinical diagnosis. The uncharacterized 154 PPmAbs were tested for reactivity based on immunohistostaining of several cancer tissues. We focused on AKPS288 PPmAb, and the PPmAb-related antigen was localized in the cytoplasm of tumor cells of the colon and stomach but did not react with non-tumor cells in both tissues. Moreover, the AKPS288 PPmAb showed positive staining in the cytoplasm of normal prostate tissue but not cancer tissue. Based on the mass spectrometry (MS), the PPmAb-related antigen was identified as TATA-element modulatory factor 1 (TMF/ARA160), a tumor-associated antigen (TAA). These results indicate that the use of a novel antibody panel consisting of anti-TAA mAbs could have considerably greater utility for cancer classification than the PPmAb panel with unknown specificity identified in our previous study.
Autoimmune mechanisms have been hypothesized to underlie a number of human disorders in which bot... more Autoimmune mechanisms have been hypothesized to underlie a number of human disorders in which both disease pathogenesis and diagnostic biomarkers remain poorly understood. This is partly due to a lack of efficient techniques for identification of plasma autoantibodies associated with specific pathophysiological conditions. We have developed a novel proteomic methodology to comprehensively identify plasma IgG-bound proteins using liquid chromatography tandem mass spectrometry (LC-MS/MS) after denaturing enriched plasma IgG to solubilize and release low molecular weight proteins. In total, we identified 44 proteins using this method that were undetectable in unprocessed plasma, 21 of which were not identified in the Human Plasma Proteome Draft of 2017. Comparison of plasma IgGbound proteins between healthy subjects and patients with isolated adrenocorticotropic hormone deficiency, a rare endocrine disorder speculated to involve autoimmune mechanisms, revealed several distinct IgG-bound proteins specifically detected in patient plasma but not in healthy subjects. Our results suggest that solubilization of low molecular weight proteins bound to enriched plasma IgG and subsequent proteomic analysis by LC-MS/MS could provide a promising strategy for identification of autoantigens in human peripheral blood.
The following versions of software and data (see references i O) were used in the production of t... more The following versions of software and data (see references i O) were used in the production of this report:
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Papers by Yoshio Kodera