NanoFreeProteomics (NFP) Compared to Conventional nanoLC based proteomics
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): Cell Suspension Culture, Permanent Cell Line Cell
DISEASE(S): Colon Cancer
SUBMITTER: 
Ben Orsburn
LAB HEAD: Benjamin Orsburn
PROVIDER: PXD016704 | Pride | 2020-02-25
REPOSITORIES: Pride
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2014-09-01 | GSE59892 | GEO
Project description:Adult female mice (C57BL/6) were super-ovulated by intraperitoneal hormone injections with the interval of 48 hours between injections. The hormones administered were PMSG followed by hCG (5IU/0.1ml, Sigma-Aldrich). Following the second hormonal treatment, the females were mated with males (1:1, C57BL/6). Zygotes, 2-cell and 4-cell and blastocysts were collected 26, 48 and 52 hours post hCG. Prior to cell collection, the zona pelucida was removed by Tyrode s acid solution. The 2- and 4-cell embryos were then immersed in Trypsin solution (Invitrogen) with RNase inhibitor (1 IU/ul, Clontech) and BSA (50 ug/ul) for the separation of the blastomeres. The separated blastomeres were snap frozen, and maintained at -80 C until processed. Twelve zygotes, 20 2-cell and 9 4-cell embryos were collected for single cell qPCR. In addition, we prepared with a pool of 10 zygotes and a pool of 50 zygotes as positive controls. The cells and pools were subjected to reverse transcription with SuperScript VILO cDNA Synthesis kit (Life Technologies) as recommended by the manufacturer. DELTAgene Assays were custom designed for 94 genes associated with embryonic development or regulation of gene expression, as well as 2 housekeeping genes. The cDNA from cells and pools were subjected to target specific amplification for 20 cycles. Pre-amplified template was diluted and subjected to primer specific amplification with SooFast EvaGreen Supermix (Biorad) on 96.96 array chip and BiomarkHD System (Fluidigm). Relative quantification of gene expression (log2 space) was obtained by subtracting the Ct values from the baseline value of 22. 12 zygotes, 10 2-cell, and 9 4-cell mouse (C57BL/6) embryos were collected and multi-cell embryos were separated into blastomeres
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Project description:Limiting contamination of LC-MS systems and reducing downtime associated with maintenance and cleaning is essential for productivity. We developed a simple device that creates a gas curtain barrier to prevent ions entering the MS inlet. The gas can be quickly and easily applied when certain contaminant ions are known to elute. We show the device can prevent the build up of contaminants on the heated transfer capillary following >100 injections of a crude tissue lysate and improves peptide identifications. The device may provide a promising approach towards improving instrument robustness.
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Project description:Data analyzed within Procedure 2 in Kantnerova et al. 2024 in Nature Protocols (https://doi.org/10.1038/s41596-024-00981-5). Procedure 2 describes the stable isotope analysis of the model analyte nitrate (NO3-) by applying the flow injection using an HPLC system with an autosampler. The nitrate data were collected using a Thermo Scientific Orbitrap Exploris 240 Isotope Solutions. Sample = USGS32, Reference = USGS35 (nitrate isotopic reference materials from the United States Geological Survey), 50 uM nitrate in methanol. Isotopocule data collected "with M0" base peak. RAW files were processed using the IsoX software (version 2022) using the isotopologs.tsv files as input. There are three datasets with the following prefixes: "01_" - USGS32 vs USGS35, optimal ion source conditions, 13 alternating injections of USGS35 and USGS32 + 2 blank methanol measurements "02_" - USGS32 vs USGS35, non-optimal ion-source conditions, 13 alternating injections of USGS35 and USGS32 + 2 blank methanol measurements "03_" - USGS32 vs USGS35, alternating injections of USGS35 and USGS32 extended to a total of 315 injections. Before rerunning the IsoX data analysis, remove the prefixes "01_", "02_" and "03_" from the respective .tsv files.
2023-11-01 | MSV000093223 | MassIVE
Project description:Data analyzed within Procedure 2 in Kantnerova et al. 202X in Nature Protocols. Procedure 2 describes the stable isotope analysis of the model analyte nitrate (NO3-) by applying the flow injection using an HPLC system with an autosampler.
The nitrate data were collected using a Thermo Scientific Orbitrap Exploris 240 Isotope Solutions.
Sample = USGS32, Reference = USGS35 (nitrate isotopic reference materials from the United States Geological Survey), 50 uM nitrate in methanol.
Isotopocule data collected "with M0" base peak.
RAW files were processed using the IsoX software (version 2022) using the isotopologs.tsv files as input.
There are three datasets with the following prefixes (to be removed before re-analysis using the IsoX software and the R scripts):
"01_" - USGS32 vs USGS35, optimal ion source conditions, 13 alternating injections of USGS35 and USGS32 + 2 blank methanol measurements
"02_" - USGS32 vs USGS35, non-optimal ion-source conditions, 13 alternating injections of USGS35 and USGS32 + 2 blank methanol measurements
"03_" - USGS32 vs USGS35, alternating injections of USGS35 and USGS32 extended to a total of 315 injections
2023-08-17 | MSV000092683 | MassIVE
Project description:Uterine leiomyomas (UL) are the most common benign tumors in women of reproductive age. Despite the high prevalence, its pathology remains unclear, which hampers the development of safe and effective treatments. Several works have pointed the involvement of epigenetic mechanisms in UL development. Specifically, DNA methylation plays an important role in gene expression regulation. We aim to determine the relationship between DNA methylation and gene expression in UL compared to adjacent myometrium (MM) to describe molecular mechanisms involved in UL formation that are under epigenetic control. Our results showed a different DNA methylation profile between UL and MM, leading to hypermethylation of UL, as well as a different global transcriptome profile. Integration of DNA methylation and transcriptome data resulted in 93 genes regulated by methylation, 22 hypomethylated/upregulated and 71 hypermethylated/downregulated. Functional enrichment analysis showed dysregulated biological processes involved in metabolism and cell physiology, response to extracellular signals, invasion, and proliferation in UL. Cellular components as cell membranes, vesicles, extracellular matrix, and cell junctions were dysregulated in UL. In addition, we found hypomethylation/upregulation of oncogenes (PRL, ATP8B4, CEMIP, ZPMS2-AS1) and hypermethylation/downregulation of tumor suppressor genes (EFEMP1, FBLN2, ARHGAP10, HTATIP2) in UL, which were related to proliferation, invasion, altered metabolism, deposit of extracellular matrix and Wnt/β-catenin pathway dysregulation. This confirms that key processes of UL development are under DNA methylation control. Finally, 5-aza-CdR treatment increased expression of hypermethylated/downregulated genes in UL cells. In conclusion, DNA methylation gene regulation is implicated in UL pathogenesis, and its reversion could be a potential therapeutic option.
2022-05-31 | GSE192353 | GEO
Project description:Uterine leiomyomas (UL) are the most common benign tumors in women of reproductive age. Despite the high prevalence, its pathology remains unclear, which hampers the development of safe and effective treatments. Several works have pointed the involvement of epigenetic mechanisms in UL development. Specifically, DNA methylation plays an important role in gene expression regulation. We aim to determine the relationship between DNA methylation and gene expression in UL compared to adjacent myometrium (MM) to describe molecular mechanisms involved in UL formation that are under epigenetic control. Our results showed a different DNA methylation profile between UL and MM, leading to hypermethylation of UL, as well as a different global transcriptome profile. Integration of DNA methylation and transcriptome data resulted in 93 genes regulated by methylation, 22 hypomethylated/upregulated and 71 hypermethylated/downregulated. Functional enrichment analysis showed dysregulated biological processes involved in metabolism and cell physiology, response to extracellular signals, invasion, and proliferation in UL. Cellular components as cell membranes, vesicles, extracellular matrix, and cell junctions were dysregulated in UL. In addition, we found hypomethylation/upregulation of oncogenes (PRL, ATP8B4, CEMIP, ZPMS2-AS1) and hypermethylation/downregulation of tumor suppressor genes (EFEMP1, FBLN2, ARHGAP10, HTATIP2) in UL, which were related to proliferation, invasion, altered metabolism, deposit of extracellular matrix and Wnt/β-catenin pathway dysregulation. This confirms that key processes of UL development are under DNA methylation control. Finally, 5-aza-CdR treatment increased expression of hypermethylated/downregulated genes in UL cells. In conclusion, DNA methylation gene regulation is implicated in UL pathogenesis, and its reversion could be a potential therapeutic option.
2022-05-31 | GSE192352 | GEO