Comprehensive Profiling by Non-targeted Stable Isotope Tracing Capillary Electrophoresis-Mass Spectrometry
ABSTRACT: We developed a stable-isotope tracing capillary electrophoresis (CE)-MS metabolomics approach to cover polar metabolites as well as isotopologues in a non-targeted way. An in-house developed software enables high throughput processing of complex multi-dimensional data. The practicability is demonstrated analysing 13C-U-glucose exposed prostate cancer and non-cancer cells.
Project description:The aim of this study was to investigate the presence of intact O- and N-glycopeptides in profiles of normal human urine analyzed by capillary electrophoresis coupled to mass spectrometry (CE-MS).CE and liquid chromatography coupled to tandem mass spectrometry (CE- and LC-MS/MS) were both used to identify glycopeptide sequences. Furthermore, we estimated the abundance levels of these glycopeptides in urine from five different cancer types i.e. bladder cancer (BCa), prostate cancer (PCa), pancreatic cancer, cholangiocarcinoma (CC) and renal cell carcinoma (RCC).
Project description:A non-optimal fetal environment is known to cause low birth weight, which has been epidemiologically associated with a greater risk of adult diseases. Maternal undernutrition in animal models has also revealed the increased risks for adult diseases in the offspring. In this study, pregnant mice underwent overnight food deprivation at gestation day (GD)17 or 50% food restriction (FR) from GD10 to GD17, and the fetal brains were examined for global changes in gene expression by DNA microarray analysis utilizing the dye-swap approach. We present here a list of candidate genes from the fetal brain that might be responsible for developmental origins of health and disease. For food deprivation (FD), the pregnant mice were deprived of the food for overnight before lights were turned off on GD17. For food restriction (FR), pregnant mice were exposed to 50% food restriction (FR) from GD 10 to 17 and caesarean section was performed between 10:00-12:00 AM on GD18. Amount of CE-2 chow supplied to FR group was calculated as 50% of CE-2 consumed by control group each gestation day. Control group was supplied with chow ad libitum. Pregnant mice were sacrificed by cervical dislocation, and the fetuses were taken out and anesthetized on ice cold phosphate-buffered saline. The fetuses were dissected under a dissection microscope, and fetal tissues are carefully removed avoiding any other tissues contamination. The brain was collected from control (n=5) and FD (n=5) or FR (n=5) fetuses, and immediately frozen by immersion in liquid nitrogen. For DNA microarray analyses, the total RNA was extracted, quality and quantity determined, and total RNA from each sample (control and treatment) in each group was pooled, followed by established protocols for genome wide expression changes for both FD and FR samples using a 60-mer probes (4 x 44K (41,090 gene probes), mouse whole genome, Agilent) DNA chip by the dye-swap approach.
Project description:A large number of rodent studies have supported the developmental origins of health and disease (DOHaD) hypothesis that interauterine undernutrition (IU) is a risk factor for non-communicable diseases. The effect of IU is considered to be induced thorough epigenetic programming in the fetal tissues. We have recently carried out global transcriptome expression and promoter DNA methylation analyses on the fetal mouse liver following maternal 50% food restriction (FR) during late gestation, and reported a list of fetal liver genes that were transcriptionally or epigenetically regulated by IU (Ogawa et al., 2014, Congenital Anomalies). We have already reported the genes that were regulated oppositely between maternal and fetal livers (Ogawa et al., 2014, Congenital Anomalies). Here, we considered that the fetal liver is a hematopoietic organ, and exposed nine-week-old male mice (C57BL/6J from Japan SLC, Hamamatsu, Japan; n=8 / group) to 50% FR (chow; CE-2, CLEA, Tokyo Japan) for two weeks and carried out global gene expression analysis on the whole blood by a method reported previously (Ogawa et al., 2014, Congenital Anomalies). Male C57BL/6J mice at 9 weeks (Japan SLC, Hamamatsu, Japan ; n=8 / group) were exposed to 50% FR (chow; CE-2, CLEA, Tokyo Japan) for 2 weeks. After 2 weeks, the mice were anesthetized with somunopentyl®(Kyoritsu eiyaku, Tokyo) at a dose of 0.5 mL/kg, and blood was taken from the right ventricle of the heart. Blood samples were immediately immersed in liquid nitrogen and stored at -80°C. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Showa University, and caried out at animal facilities in Showa University. DNA Microarray analysis was carried out on 3 pools, respectively. Pool 1 included samples from animal No. 1-8 in each group (control and 50% FR). Pool 2 included samples from animal No. 1-4 in each group. Pool 3 included samples from animal No. 5-8 in each group.
Project description:This study reports long-range and high-resolution repeat sizing of the C9orf72 repeat region for 2095 samples from the National Institute of Neurological Disorders and Stroke (NINDS) repository's ALS collection. Genomic DNA samples were analyzed with a PCR assay (AmplideX Registered Trademark PCR/CE C9orf72 Kit) and resolved on capillary electrophoresis. Expanded samples were also analyzed using a gene-specific assay and resolved by capillary electrophoresis (up to 145 GGGGCC hexanucleotide repeats) and AGE agarose gel electrophoresis (up to 950 hexanucleotide repeats, respectively).
Project description:Non-optimal fetal environments resulting in low birth weight have epidemiologically been associated with adult diseases. In animal models, maternal undernutrition has successfully demonstrated increased risks for adult diseases. In the present study, we treated pregnant mice with 50% food restriction (FR), and performed global gene expression and promotor DNA methylation profiling on the fetal livers. Considering that effects of food restriction is opposite between before and after birth, we further searched genes which are regulated oppositely against adult calorie restriction and commonly against aging. Searched genes were included in groups related to the immune system, obesity and heart disease. Among these genes, trib1 has already been demonstrated to contribute to an increased risk of cardiovascular disease. The present result suggests that trib1 is a target of DOHaD. In addition, lepr was also dow-regulaed by maternal FR, suggested a potential role of this gene in induction of obesity. Promotor DNA methylation profiling as well as gene expression profiling revealed glucocorticoid target genes were regulated by maternal FR, supported previous reports suggesting important role of glucocorticoid in DOHaD. C57BL/6J mice were purchased from Japan SLC Co. Ltd. (Hamamatsu, Japan). The animals were housed at the Animal Institution in Showa University. They were maintained in cages in a ventilated animal room with controlled temperature and relative humidity with a 12 h light: 12 h dark schedule (lights turned on at 8:00 AM) and had access to chow (CE-2, CLEA Japan) and tap water ad libitum. As per the supplier’s (CLEA) information, CE-2 includes 51.4% nitrogen free extracts, 24.9% crude protein, 8.6% moisture, 6.7% crude ash, 4.6% crude fat, and 3.7% crude fiber. The physiological fuel value is 346.7 kcal per 100 g chow. All animal experiments were started after a period of acclimation of at least one week. Pregnant animals were obtained by housing females with males (1-2 females/male). The day the vaginal plug was observed was designated embryonic day 0 (ED0) and gestation day 0 (GD0). Pregnant mice were exposed to 50% food restriction (FR) from GD 10 to 17 and caesarean section was performed between 10:00-12:00 AM on GD18. Amount of CE-2 chow supplied FR group was calculated as 50% of CE-2 consumed by control group each gestation day. Control group was supplied with chow ad libitum. Pregnant mice were sacrificed by cervical dislocation, and the fetuses were taken out and anesthetized on ice cold phosphate buffered saline. The fetuses were dissected under a dissection microscope, and fetal tissues were carefully removed avoiding any other tissues contamination. The liver was collected from six mothers in each group. Fetal liver was collected from two male fetuses from each mother. The liver was also collected from mothers to compare with fetal one in gene expression analysis. The harvested livers were immediately immersed in liquid nitrogen (N2) and stored at -80ºC. In all combinations, total RNA (1000 ng) was labeled with either Cy3 or Cy5 dye using an Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies Inc., CA, USA). Fluorescently labeled targets of control as well as treated samples were hybridized to the same microarray slide with 60-mer probes (4 x 44K (41,090 gene probes), mouse whole genome, Agilent). A flip labelling (dye-swap or reverse labelling with Cy3 and Cy5 dyes) procedure was followed to nullify the dye bias associated with unequal incorporation of the two Cy dyes into cDNA. Hybridization and wash processes were performed according to the manufacturer’s instructions, and hybridized microarrays were scanned using an Agilent Microarray scanner. For the detection of significantly differentially expressed genes between control and exercise samples each slide image was processed by Agilent Feature Extraction software (version 126.96.36.199).
Project description:Although thousands of intact proteins can be feasibly identified in recent years, global quantification of intact proteins is still challenging. Herein, we develop a high-throughput strategy for global intact protein quantification based on chemical isotope labeling. The isotope incorporation efficiency is as high as 99.2% for complex intact protein samples extracted from HeLa cells. Further, the pTop 2.0 software is developed for automated quantification of intact proteoforms in a high-throughput manner. The high quantification accuracy and reproducibility of this strategy have been demonstrated for both standard and complex cellular protein samples. A total of 2,283 intact proteoforms originated from 660 protein accessions are successfully quantified under anaerobic and aerobic conditions and the differentially expressed proteins are observed to be involved into the important biological processes such as stress response.
Project description:We investigated a contaminant-degrading microbial community by sequencing total RNA (without rRNA depletion) from microcosms containing sediment from a hypoxic contaminated aquifer fed with isotopically labeled toluene. Overall design: Microcosms were made using sediment from a hypoxic, hydrocarbon-contaminated aquifer in Siklos, Hungary. They were incubated under hypoxic/microoxic conditions and provided with isotopically-labeled toluene as a substrate. RNA was extracted and subjected to isopycnic centrifugation to separated heavy (isotopically labeled) and light (unlabeled) RNA, representing microbes which did or did not metabolize toluene, respectively. Total RNA was sequenced to provide both taxonimic (16S) and functional (mRNA) data on this microbiota, the first instance of total-RNA-stable isotope probing used to investigate a contaminated environment.
Project description:The goal of this study was to enable the analysis of anionic and cationic metabolites from the same identified single cell in Xenopus laevis embryos. A 10 nL portion of identified animal-ventral (V1) cells was aspirated from 8-cell embryos, and metabolites were extracted from the aspirate, before characterization of cationic and anionic compounds using a custom-built capillary electrophoresis (CE) electrospray ionization (ESI) mass spectrometry platform. A total of ~250 cationic molecular features and ~150 anionic molecular features were detected, including 76 metabolites that were identified in this study. Pathway analysis of the identified metabolites highlighted arginine-proline metabolism of significance.
Project description:To identify novel Peroxisome Proliferator-Activated Receptor gamma (PPARg) responsive secretory and/or transmembrane genes that is related to obesity, we integrated the expression data from the adipose tissue derived from obese mice with the other two data sets: expression profiling of adipocyte differentiation using ST2 cells and siRNA-mediated knockdown of Pparg during ST2 cell adipogenesis. We used microarrays to detect the up-regulated genes in adipose tissue derived from mice fed a high fat diet compared to a control. Total RNA from adipose tissue was obtained and from mice fed a high fat diet HFD32 (MOUSE_HFD) from 6 week-old to 18 week-old, or a normal diet CE-2 (MOUSE_ND) as a control. Pooled RNAs of each three animals were analyzed by the Affymetrix GeneChip microarray system.