Pub Date : 2005-12-01DOI: 10.2198/JELECTROPH.49.85
T. Funato, Mayu Takeda
Resistance to chemotherapy is an obstacle to the successful treatment of acute myeloid leukemia (AML). Failure of therapeutic treatment may be due to the development of multidrug resistance (MDR), the mechanisms of which include upregulation of membrane-resident transporters which efflux chemotherapeutic drugs from tumor cells, as well as failure of cancer cells to undergo apoptosis in response to chemotherapy. We developed a quantitative reverse transcription PCR method for MDR1 and multidrug resistance-related protein 1 (MRP1) transcripts to evaluate drug resistance, and applied it to clinical samples. P-glycoprotein encoding MDR1 (P-gp) expression was determined by Western blot analysis, rhodamine 123 was used for functional study of P-gp protein, and sensitization of leukemic cells to drugs was quantified by methyl thiazolyl tetrazolium (MTT) assays. In this review, we cover current findings and suggest that the different methods for determining MDR and, in particular, discuss the efficacy of this quantitative analysis of MDR1 transcripts for the prediction of clinical drug resistance in acute leukemia.
{"title":"Approaches to detect the drug resistance in acute leukemia","authors":"T. Funato, Mayu Takeda","doi":"10.2198/JELECTROPH.49.85","DOIUrl":"https://doi.org/10.2198/JELECTROPH.49.85","url":null,"abstract":"Resistance to chemotherapy is an obstacle to the successful treatment of acute myeloid leukemia (AML). Failure of therapeutic treatment may be due to the development of multidrug resistance (MDR), the mechanisms of which include upregulation of membrane-resident transporters which efflux chemotherapeutic drugs from tumor cells, as well as failure of cancer cells to undergo apoptosis in response to chemotherapy. We developed a quantitative reverse transcription PCR method for MDR1 and multidrug resistance-related protein 1 (MRP1) transcripts to evaluate drug resistance, and applied it to clinical samples. P-glycoprotein encoding MDR1 (P-gp) expression was determined by Western blot analysis, rhodamine 123 was used for functional study of P-gp protein, and sensitization of leukemic cells to drugs was quantified by methyl thiazolyl tetrazolium (MTT) assays. In this review, we cover current findings and suggest that the different methods for determining MDR and, in particular, discuss the efficacy of this quantitative analysis of MDR1 transcripts for the prediction of clinical drug resistance in acute leukemia.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"6 1","pages":"85-93"},"PeriodicalIF":0.0,"publicationDate":"2005-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82675544","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-09-01DOI: 10.2198/JELECTROPH.49.71
K. Kameyama, Tomohiro Nanri, Y. Yamanaka, M. Arima, H. Kawasaki
In-gel digestion is an important technique for proteome analysis using polyacrylamide gel electrophoresis. Although the identification by MSMS ion search is much reliable than PMF, MSMS analysis of peptides separated by nanoLC directly coupled to MS is time consuming and clogging with particles from in-gel digest interrupts separation of peptide by nanoLC. Removal of particles from the digest with filtration is essential for nanoLC analysis, but it is laborious process, when a large number of samples should be processed. Simple and high-throughput method is necessary for in-gel digestion and the sample preparation of nanoLC. The characteristics of filter plates are investigated and a method of in-gel digestion for nanoLC-MSMS systems using inexpensive 96-well filter plate is presented.
{"title":"Assessment of filter plates for multi-well in-gel digestion of proteins separated by polyacrylamide gel electrophoresis to identify them with LC-ESI / MSMS","authors":"K. Kameyama, Tomohiro Nanri, Y. Yamanaka, M. Arima, H. Kawasaki","doi":"10.2198/JELECTROPH.49.71","DOIUrl":"https://doi.org/10.2198/JELECTROPH.49.71","url":null,"abstract":"In-gel digestion is an important technique for proteome analysis using polyacrylamide gel electrophoresis. Although the identification by MSMS ion search is much reliable than PMF, MSMS analysis of peptides separated by nanoLC directly coupled to MS is time consuming and clogging with particles from in-gel digest interrupts separation of peptide by nanoLC. Removal of particles from the digest with filtration is essential for nanoLC analysis, but it is laborious process, when a large number of samples should be processed. Simple and high-throughput method is necessary for in-gel digestion and the sample preparation of nanoLC. The characteristics of filter plates are investigated and a method of in-gel digestion for nanoLC-MSMS systems using inexpensive 96-well filter plate is presented.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"36 4","pages":"71-75"},"PeriodicalIF":0.0,"publicationDate":"2005-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91400465","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-06-01DOI: 10.2198/JELECTROPH.49.61
H. Kajiwara, Yoko Ito, A. Imamaki, Masatoshi Nakamura, K. Mita, M. Ishizaka
Proteomic analysis was performed on the midgut of fifth-instar day-3 female silkworm (Bombyx mori, strain p50) larvae. Though silkworm genome analysis has not yet been completed, the Drosophila genome and silkworm expression sequence tag (EST) data were applied to the analysis of the midgut proteins in the database (DB) for identification. The spots, which were excised manually from two-dimensional electrophoresis (2-DE) gels, were treated with 4-vinylpyridine for alkylation. Each spot was analyzed by capillary high-performance liquid chromatography coupled with ion-trap mass spectrometry (MS) after proteolysis using a trypsin. Nearly 60% of the proteins analyzed by MS were identified. These included many kinds of cytoskeleton proteins, ATPase, and chaperonins.
{"title":"Protein profile of silkworm midgut of fifth-instar day-3 larvae","authors":"H. Kajiwara, Yoko Ito, A. Imamaki, Masatoshi Nakamura, K. Mita, M. Ishizaka","doi":"10.2198/JELECTROPH.49.61","DOIUrl":"https://doi.org/10.2198/JELECTROPH.49.61","url":null,"abstract":"Proteomic analysis was performed on the midgut of fifth-instar day-3 female silkworm (Bombyx mori, strain p50) larvae. Though silkworm genome analysis has not yet been completed, the Drosophila genome and silkworm expression sequence tag (EST) data were applied to the analysis of the midgut proteins in the database (DB) for identification. The spots, which were excised manually from two-dimensional electrophoresis (2-DE) gels, were treated with 4-vinylpyridine for alkylation. Each spot was analyzed by capillary high-performance liquid chromatography coupled with ion-trap mass spectrometry (MS) after proteolysis using a trypsin. Nearly 60% of the proteins analyzed by MS were identified. These included many kinds of cytoskeleton proteins, ATPase, and chaperonins.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"31 1","pages":"61-69"},"PeriodicalIF":0.0,"publicationDate":"2005-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82607002","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-06-01DOI: 10.2198/JELECTROPH.49.45
H. Ihara, Takayuki Matsumoto, Takashi Kakinoki, Y. Aoki, N. Hashizume, Yoshinobu Inada, S. Nanba, G. Yoshino, Mitsutaka Yoshida
We investigated whether the electrophoretic patterns and clinical information derived from the five principal fractions of the Beckman Paragon capillary zone electrophoresis (CZE) 2000 system are comparable to the results obtained by the cellulose acetate electrophoresis (CAE: Olympus AES 320) system. Concordance studies between the results obtained by CAE and CZE were performed on sera from pregnant women and from patients with nephrotic syndrome, acute and chronic inflammatory diseases, cirrhosis, monoclonal and polyclonal gammopathy, and bisalbuminemia. In all subjects except for the pregnant women, the results of CAE and CZE agreed 100% in terms of percent composition for albumin, and α1- and α2-globulins. Concordance for γ-globulin was 92-100% in terms of both percent composition and absolute concentration. Three discordant cases according to the results for β-globulin by CAE and CZE were found in patients with monoclonal gammopathy, who also showed discordant results for γ-globulin. An M-protein spike observed in either the β-globulin or γ-globulin fraction was 100% detected by both CAE and CZE analyses. In another six discordant cases for the β-globulin fraction observed in patients with cirrhosis and polyclonal gammopathy, part of the tail of the β-globulin fraction tended to be measured as γ-globulin by CAE. In three of the four bisalbuminemia cases, the double band of albumin observed by CZE was not detected by CAE. The results show that the electrophoretic patterns and clinical information derived from the five principal fractions of the Beckman Paragon CZE 2000 system are comparable to the results obtained by classical CAE.
{"title":"Concordance between results of serum protein electrophoresis of pathological serum by cellulose acetate and capillary zone electrophoresis","authors":"H. Ihara, Takayuki Matsumoto, Takashi Kakinoki, Y. Aoki, N. Hashizume, Yoshinobu Inada, S. Nanba, G. Yoshino, Mitsutaka Yoshida","doi":"10.2198/JELECTROPH.49.45","DOIUrl":"https://doi.org/10.2198/JELECTROPH.49.45","url":null,"abstract":"We investigated whether the electrophoretic patterns and clinical information derived from the five principal fractions of the Beckman Paragon capillary zone electrophoresis (CZE) 2000 system are comparable to the results obtained by the cellulose acetate electrophoresis (CAE: Olympus AES 320) system. Concordance studies between the results obtained by CAE and CZE were performed on sera from pregnant women and from patients with nephrotic syndrome, acute and chronic inflammatory diseases, cirrhosis, monoclonal and polyclonal gammopathy, and bisalbuminemia. In all subjects except for the pregnant women, the results of CAE and CZE agreed 100% in terms of percent composition for albumin, and α1- and α2-globulins. Concordance for γ-globulin was 92-100% in terms of both percent composition and absolute concentration. Three discordant cases according to the results for β-globulin by CAE and CZE were found in patients with monoclonal gammopathy, who also showed discordant results for γ-globulin. An M-protein spike observed in either the β-globulin or γ-globulin fraction was 100% detected by both CAE and CZE analyses. In another six discordant cases for the β-globulin fraction observed in patients with cirrhosis and polyclonal gammopathy, part of the tail of the β-globulin fraction tended to be measured as γ-globulin by CAE. In three of the four bisalbuminemia cases, the double band of albumin observed by CZE was not detected by CAE. The results show that the electrophoretic patterns and clinical information derived from the five principal fractions of the Beckman Paragon CZE 2000 system are comparable to the results obtained by classical CAE.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"29 1","pages":"45-51"},"PeriodicalIF":0.0,"publicationDate":"2005-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"81311770","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-06-01DOI: 10.2198/JELECTROPH.49.39
Yumiko Hashimoto
We analyzed the differential expression pattern of intracellular proteins in tumor necrosis factor-α (TNF-α)-sensitive KDH-8/YK and TNF-α-resistant cKDH-8/11 rat hepatoma cell lines by means of two-dimensional electrophoresis (2-DE). The 2-DE protein patterns of KDH-8/YK cells demonstrated an increase in the protein spot of relative molecular mass (Mr) 36,000 and isoelectric point (pI) 4.9, which was determined by mass spectrometry (MS). Peptide mass fingerprinting (PMF) suggested that aldose reductase (AR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and tropomyosin were the candidate components of the spot. By immunoblot using specific antibodies, the spot was identified to be AR with a pI that was lower than the theoretical value.
{"title":"Differential expression of aldose reductase in TNF-α responsive rat hepatoma cell lines","authors":"Yumiko Hashimoto","doi":"10.2198/JELECTROPH.49.39","DOIUrl":"https://doi.org/10.2198/JELECTROPH.49.39","url":null,"abstract":"We analyzed the differential expression pattern of intracellular proteins in tumor necrosis factor-α (TNF-α)-sensitive KDH-8/YK and TNF-α-resistant cKDH-8/11 rat hepatoma cell lines by means of two-dimensional electrophoresis (2-DE). The 2-DE protein patterns of KDH-8/YK cells demonstrated an increase in the protein spot of relative molecular mass (Mr) 36,000 and isoelectric point (pI) 4.9, which was determined by mass spectrometry (MS). Peptide mass fingerprinting (PMF) suggested that aldose reductase (AR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and tropomyosin were the candidate components of the spot. By immunoblot using specific antibodies, the spot was identified to be AR with a pI that was lower than the theoretical value.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"78 1","pages":"39-43"},"PeriodicalIF":0.0,"publicationDate":"2005-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73665180","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-06-01DOI: 10.2198/JELECTROPH.49.53
K. Shimura, Takuma Waki, Masaki Okada, T. Toda, Izumi Kimoto, K. Kasai
The utility of an automated 96-capillary electrophoresis instrument equipped with a laser-induced fluorescence detector was evaluated in the analysis of affinophoresis using concanavalin A and trypsin as models. Affinophores for concanavalin A and trypsin were prepared by coupling the affinity ligands, p-aminophenylmannoside and p-aminobenzamidine to soluble succinylpolylysine, respectively at a density of about 1/5 of the carboxyl groups. A dual buffer system, 60 mM borate-Na (pH 9.35) as an electrophoresis buffer and 60 mM 3-morpholinopropanesulfonic acid-Na (pH 7.35) containing 0.1% Tween 20 as a sample buffer, was employed to allow the interactions to occur at a physiological pH and also to maintain a high level of electroosmosis and reproducibility in the bare silica capillaries. The electrophoresis of the fluorophore-labeled proteins was carried out in the presence of affinophores, and changes in the mobility of the labeled proteins were analyzed in terms of the mobility moment, which represents the average mobility of the proteins, thus permitting the dissociation constants for the protein-affinophore interactions to be determined. The affinophoresis system was then used to evaluate the extent of binding of low molecular mass compounds based on the inhibition of the affinophoresis. In combination with the mobility moment analysis and the dual buffer system, the multi-capillary electrophoresis instruments proved to be useful for the analysis of biomolecular interactions by electrophoresis.
以豆豆蛋白a和胰蛋白酶为模型，对配备激光诱导荧光检测器的全自动96毛细管电泳仪在亲和电泳分析中的实用性进行了评价。将亲和配体对氨基苯基甘露苷和对氨基苯并脒分别与可溶性琥珀基聚赖氨酸偶联，以1/5的羧基密度制备了刀豆蛋白A和胰蛋白酶的亲和载体。采用双缓冲系统，60 mM硼酸钠(pH 9.35)作为电泳缓冲液，60 mM 3- morpholinopropanesonic - na (pH 7.35)含0.1% Tween 20作为样品缓冲液，使相互作用在生理pH下发生，并保持高水平的电渗透和裸二氧化硅毛细血管的再现性。在亲和团存在的情况下对荧光团标记的蛋白质进行电泳，并根据代表蛋白质平均迁移率的迁移矩分析标记蛋白质迁移率的变化，从而确定蛋白质-亲和团相互作用的解离常数。然后使用亲和性电泳系统来评估基于亲和性电泳抑制的低分子质量化合物的结合程度。与迁移矩分析和双缓冲系统相结合，证明了多毛细管电泳仪在电泳分析生物分子相互作用方面的作用。
{"title":"Mobility-moment analysis of protein-ligand interactions using a multi-capillary electrophoresis instrument : Competitive affinophoresis of concanavalin A and trypsin","authors":"K. Shimura, Takuma Waki, Masaki Okada, T. Toda, Izumi Kimoto, K. Kasai","doi":"10.2198/JELECTROPH.49.53","DOIUrl":"https://doi.org/10.2198/JELECTROPH.49.53","url":null,"abstract":"The utility of an automated 96-capillary electrophoresis instrument equipped with a laser-induced fluorescence detector was evaluated in the analysis of affinophoresis using concanavalin A and trypsin as models. Affinophores for concanavalin A and trypsin were prepared by coupling the affinity ligands, p-aminophenylmannoside and p-aminobenzamidine to soluble succinylpolylysine, respectively at a density of about 1/5 of the carboxyl groups. A dual buffer system, 60 mM borate-Na (pH 9.35) as an electrophoresis buffer and 60 mM 3-morpholinopropanesulfonic acid-Na (pH 7.35) containing 0.1% Tween 20 as a sample buffer, was employed to allow the interactions to occur at a physiological pH and also to maintain a high level of electroosmosis and reproducibility in the bare silica capillaries. The electrophoresis of the fluorophore-labeled proteins was carried out in the presence of affinophores, and changes in the mobility of the labeled proteins were analyzed in terms of the mobility moment, which represents the average mobility of the proteins, thus permitting the dissociation constants for the protein-affinophore interactions to be determined. The affinophoresis system was then used to evaluate the extent of binding of low molecular mass compounds based on the inhibition of the affinophoresis. In combination with the mobility moment analysis and the dual buffer system, the multi-capillary electrophoresis instruments proved to be useful for the analysis of biomolecular interactions by electrophoresis.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"13 1","pages":"53-60"},"PeriodicalIF":0.0,"publicationDate":"2005-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88176914","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ryo Yamashita, Y. Fujiwara, Xunmei Yuan, K. Yasuda, Y. Kaburagi
Two-dimensional liquid chromatography coupled with tandem mass spectrometry (2-D LC-MS/MS) technique has high capability of resolving peptides, and is used for analysis of tryptic peptides derived from highly complex protein mixtures such as plasma. However, the detection of low-abundant proteins and low-molecular-weight proteins is often very difficult in this system, because major peptides from high-abundant proteins such as albumin mostly disturb the separation of minor peptides from low-abundant proteins. To resolve these problems, different methods of sample preparation for in-solution trypsin digestion were tested, and the optimized method was applied to the 2-D LC-MS/MS analysis of proteins secreted from human hepatoma cell line, HepG2. We could identify 247 and 142 proteins from the chemically and thermally denatured samples, respectively. Among them, 71 proteins were identified in common to both methods, while most of the proteins were identified using either of the two procedures. In addition to these denaturation methods, the molecular-mass cutoff via ultrafiltration improved the efficiency in identifying low-molecular-weight proteins. Finally, we could identify 478 secreted proteins in total using the combination of these processes. These data indicate that in sample preparation the combination of various denaturation methods, as well as molecular-mass cutoff, are very critical for the identification of a wider range of low-abundant proteins via 2-D LC-MS/MS analysis.
{"title":"2-D LC-MS/MS Analysis of secreted proteins from HepG2 cells: Combination with various sample preparation methods before in-solution trypsin digestion","authors":"Ryo Yamashita, Y. Fujiwara, Xunmei Yuan, K. Yasuda, Y. Kaburagi","doi":"10.2198/JELECTROPH.49.1","DOIUrl":"https://doi.org/10.2198/JELECTROPH.49.1","url":null,"abstract":"Two-dimensional liquid chromatography coupled with tandem mass spectrometry (2-D LC-MS/MS) technique has high capability of resolving peptides, and is used for analysis of tryptic peptides derived from highly complex protein mixtures such as plasma. However, the detection of low-abundant proteins and low-molecular-weight proteins is often very difficult in this system, because major peptides from high-abundant proteins such as albumin mostly disturb the separation of minor peptides from low-abundant proteins. To resolve these problems, different methods of sample preparation for in-solution trypsin digestion were tested, and the optimized method was applied to the 2-D LC-MS/MS analysis of proteins secreted from human hepatoma cell line, HepG2. We could identify 247 and 142 proteins from the chemically and thermally denatured samples, respectively. Among them, 71 proteins were identified in common to both methods, while most of the proteins were identified using either of the two procedures. In addition to these denaturation methods, the molecular-mass cutoff via ultrafiltration improved the efficiency in identifying low-molecular-weight proteins. Finally, we could identify 478 secreted proteins in total using the combination of these processes. These data indicate that in sample preparation the combination of various denaturation methods, as well as molecular-mass cutoff, are very critical for the identification of a wider range of low-abundant proteins via 2-D LC-MS/MS analysis.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"8 1","pages":"1-4"},"PeriodicalIF":0.0,"publicationDate":"2005-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90112631","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-03-01DOI: 10.2198/JELECTROPH.49.23
Yukio Yamamoto, Y. Akita, Shigeyuki Tai, S. Fukasaku, Teruhide Yamaguchi, T. Oshizawa, K. Yamaoka, M. Shimamura, T. Hazato
Comparing protein expression in the cerebrospinal fluid (CSF) of rheumatoid arthritis (RA) patients with that of controls, makes possible the uncovering of proteins that affect disease progression and regulate responsiveness to drugs. Two-dimensional gel electrophoresis (2-DE) and silver staining were used for identifying disease-associated CSF proteins in RA patients. First, to enhance the detection of CSF proteins and to improve the separation of their isoforms by 2-DE, CSF samples were pre-treated with an albumin and IgG removal kit, then by acetone precipitation. The 2-DE analysis revealed more than 1600 spots by the removal of albumin and immunoglobulin from CSF. The expression of the protein spots was not greatly changed in either group, but some notable changes in protein spots were observed in two RA samples. In particular, the expression of an approximately 50 kD protein increased markedly, whereas that of two sequential protein spots of 10-15 kD and with neutral pI decreased in the RA samples. These preliminary results suggest that the proteomic method is conducive to clarifying the mechanism of RA crises, and that some of the expression-changed proteins may be new candidates for disease-associated proteins of RA.
{"title":"Two-dimensional electrophoretic analysis of disease-associated proteins in human cerebrospinal fluid from patients with rheumatoid arthritis","authors":"Yukio Yamamoto, Y. Akita, Shigeyuki Tai, S. Fukasaku, Teruhide Yamaguchi, T. Oshizawa, K. Yamaoka, M. Shimamura, T. Hazato","doi":"10.2198/JELECTROPH.49.23","DOIUrl":"https://doi.org/10.2198/JELECTROPH.49.23","url":null,"abstract":"Comparing protein expression in the cerebrospinal fluid (CSF) of rheumatoid arthritis (RA) patients with that of controls, makes possible the uncovering of proteins that affect disease progression and regulate responsiveness to drugs. Two-dimensional gel electrophoresis (2-DE) and silver staining were used for identifying disease-associated CSF proteins in RA patients. First, to enhance the detection of CSF proteins and to improve the separation of their isoforms by 2-DE, CSF samples were pre-treated with an albumin and IgG removal kit, then by acetone precipitation. The 2-DE analysis revealed more than 1600 spots by the removal of albumin and immunoglobulin from CSF. The expression of the protein spots was not greatly changed in either group, but some notable changes in protein spots were observed in two RA samples. In particular, the expression of an approximately 50 kD protein increased markedly, whereas that of two sequential protein spots of 10-15 kD and with neutral pI decreased in the RA samples. These preliminary results suggest that the proteomic method is conducive to clarifying the mechanism of RA crises, and that some of the expression-changed proteins may be new candidates for disease-associated proteins of RA.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"1 1","pages":"23-27"},"PeriodicalIF":0.0,"publicationDate":"2005-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83028335","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-03-01DOI: 10.2198/JELECTROPH.49.35
H. Morisawa, H. Hisatomi, M. Hirota, T. Toda
SUMMARY We developed a collaborative proteomics framework with XML-based 2DPAGE database and XML viewer optimized for the proteome database. Prior to the development of the framework, we optimized the XML format of our TMIG-XML-2DPAGE database for efficient data sharing in a collaborative research community. The data format is comprised with 2 layers of 2-D gel map and spot protein information. The function of clickable imagemap for representing spot protein information is available both in the proteome data management system with PHP and in the XML viewer with Object Pascal. Individual spots on an experimental 2-D gel image are easily matched to corresponding spots on a standard 2-D gel map in the TMIG-XML-2DPAGE database using the function of the XML viewer.
{"title":"Collaborative proteomics framework with XML databases and an integrated XML viewer for 2DPAGE","authors":"H. Morisawa, H. Hisatomi, M. Hirota, T. Toda","doi":"10.2198/JELECTROPH.49.35","DOIUrl":"https://doi.org/10.2198/JELECTROPH.49.35","url":null,"abstract":"SUMMARY We developed a collaborative proteomics framework with XML-based 2DPAGE database and XML viewer optimized for the proteome database. Prior to the development of the framework, we optimized the XML format of our TMIG-XML-2DPAGE database for efficient data sharing in a collaborative research community. The data format is comprised with 2 layers of 2-D gel map and spot protein information. The function of clickable imagemap for representing spot protein information is available both in the proteome data management system with PHP and in the XML viewer with Object Pascal. Individual spots on an experimental 2-D gel image are easily matched to corresponding spots on a standard 2-D gel map in the TMIG-XML-2DPAGE database using the function of the XML viewer.","PeriodicalId":15059,"journal":{"name":"Journal of capillary electrophoresis","volume":"34 1","pages":"35-38"},"PeriodicalIF":0.0,"publicationDate":"2005-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82618116","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2005-03-01DOI: 10.2198/JELECTROPH.49.29
Shenge Ahmed, J. Thiemann, Sandra Kurzawski, Jana Rykl, J. Jankowski, B. Wittmann-Liebold, T. Pohl, H. Schlüter
Peptides play a central role as reaction specific probes for screening protein libraries for target enzymes with protein-modifying activities with the automated platform technology named mass spectrometry assisted enzyme screening (MES). The protein libraries are generated by fractionating protein extracts and by immobilizing the proteins covalently to activated affinity beads. The innovative idea of the MES-system is to incubate the fractions of the protein library with special designed peptides that serve as substrates for protein modifying enzymes and to detect the resulting reaction products by mass spectrometry, if the target enzyme is present in a fraction of the protein library. The peptide sequences, selected for the reaction specific probes, are usually parts of the endogenous protein substrates. The main advantage of the MES approach is that even complex protein mixtures can be screened for enzymatic activities. The MES technique is suited for the search for unknown target enzymes with defined catalytic reaction profiles, for investigating the metabolism of defined substrates in cells or tissues and for comparing defined enzyme activities in organisms in different states. Therefore the application of the MES system includes a wide area, from the identification of new drug targets to the identification of enzymes relevant for biotechnological processes. Here the MES system is demonstrated for the screening for urotensin-II (U-II) metabolising enzymes in renal tissue. With MES des-Val-U-II was determined as major metabolite of U-II of renal tissue proteins.
在质谱辅助酶筛选(MES)的自动化平台技术中，多肽作为反应特异性探针，在筛选具有蛋白质修饰活性的靶酶的蛋白质文库中发挥着核心作用。蛋白质文库是通过分离蛋白质提取物和将蛋白质共价固定在活化的亲和珠上产生的。mes系统的创新思想是用特殊设计的肽孵育蛋白质文库的部分，作为蛋白质修饰酶的底物，如果目标酶存在于蛋白质文库的一部分中，则通过质谱法检测得到的反应产物。为反应特异性探针选择的肽序列通常是内源性蛋白质底物的一部分。MES方法的主要优点是，即使是复杂的蛋白质混合物也可以筛选酶活性。MES技术适用于寻找具有特定催化反应特征的未知靶酶，研究细胞或组织中特定底物的代谢，以及比较不同状态下生物体中特定酶的活性。因此，MES系统的应用范围很广，从新药物靶点的鉴定到与生物技术过程相关的酶的鉴定。在这里，MES系统被证明用于筛选肾组织中的尿紧张素- ii (U-II)代谢酶。与MES des-Val-U-II被确定为肾组织蛋白U-II的主要代谢物。
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