Project description:Capillary zone electrophoresis-mass spectrometry (CZE-MS) has been recognized as a valuable technique for proteomics of mass-limited biological samples (i.e., single cells) due to its high efficiency and high sensitivity for peptide separation and detection. However, its broad adoption for single cell proteomics (SCP) of human cells has been impeded by the low sample loading capacity of CZE, only allowing to use less than 5% of the available peptide material for measurements. Here we present a reversed-phase based solid phase microextraction (RP-SPME)-CZE-MS platform to solve the low sample loading capacity issue of CZE, paving the way for SCP of human cells using CZE-MS. The RP-SPME-CZE system was constructed in one fused silica capillary with zero dead volume for connection via in-situ synthesis of a frit, followed by packing C8 beads into the capillary to form a roughly 2-mm-long SPME section. Peptides were captured by SPME, and then eluted with a buffer containing 30%(v/v) acetonitrile and 50 mM ammonium acetate (pH 6.5), followed by dynamic pH junction-based CZE-MS. The SPME-CZE-MS enabled the injection of nearly 40% of the available peptide sample for measurements. The system identified 257 ± 24 proteins and 523 ± 69 peptides (N=2) when only 0.25 ng of a commercial HeLa cell digest was available in the sample vial and 0.1 ng of the sample was injected. The available peptide amount is equivalent to the protein mass of two HeLa cells. The data indicates that SPME-CZE-MS offers sufficient sensitivity for SCP of human cells.
Project description:The demo datasets available for MSCohort analysis. You can download to inspect their formats and practice using the software tool. This dataset contains raw files of 7 urine QC samples, spectronaut analysis results and MSCohort report results
Project description:Obtaining multiple sample types from the same exhaled breath condensate (EBC) sample can reduce the number of samples needed for diagnostics purposes, allowing for sampling to be completed quicker and making it even easier to collect breath from patients. In this study, we performed analysis for volatile organic compounds (VOCs) and proteins from the same EBC sample. Pooled EBC samples were split into two groups: three samples that utilized immersion thin film-solid phase microextraction (TF-SPME) sampling for VOCs analysis and three samples that did not undergo TF-SPME sampling (non-TF-SPME). All six EBC samples were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS) for proteomics analysis. VOCs were analyzed via two-dimensional gas chromatography-mass spectrometry (GC x GC-MS). One hundred and eighty-four VOCs were found to be more abundant in EBC samples compared to blank or controls. There was no significant difference in the number of proteins detected in the TF-SPME samples compared to the non-TF-SPME samples and 144 of the 206 total unique proteins detected were found in both sample groups. These results indicate that TF-SPME sampling does not negatively affect the number of proteins that can be detected in EBC. This work is a step towards linking VOC and protein data together to obtain multi-omics breath data from a single breath sample.
Project description:In this manuscript, we have used RNA-Sequencing experiment to obtain and integrate a variety of genomic features in order to identify signaling pathways that are associated to mutant KRAS lung tumors. 8 lung adenocarcinomas with mutant KRAS in lung tumors and 8 lung adenocarcarcinomas without KRAS mutation of which one sample is ran twice for QC purposes.
Project description:These files represent single cell RNA-Seq data generated on a 10x Chromium genomics platform from three biological replicates from the embryonic day (E)18.5 developing mouse kidney and three biological replicates of iPSC-derived human kidney organoids differentiated according to our published protocol (Takasato et al., Nature Protocols 2016). When aggregated, the mouse data represents >6000 cells that passed our QC, containing most major cell types known to exist in the developing mouse kidney. The aggregated human organoid data contains of >7000 cells that passed our QC and contains populations representing endothelial cells, podocytes, stroma, nephron, and off-target populations with similarity to neurons.
Project description:This dataset contains single nucleus Assay for Transposase-Accessible Chromatin (ATAC) sequencing results from rat nucleus accumbens tissue. Rats received repeated cocaine injections (20mg/kg, intraperitoneal injection), or saline injections as a control. Single-nucleus ATAC-seq was carried out with FACS-sorted nuclei using the 10X Genomics Chromium single cell sequencing platform using the Chromium Next GEM Single Cell ATAC Kit v2.
Project description:Identification of repeat-associated non-AUG (RAN) translation in trinucleotide (CAG) repeat diseases leads to an emerging concept that CAG repeat diseases are caused by non-polyglutamine products. Nonetheless, the exact contribution of RAN translation to the pathogenesis of CAG repeat diseases remains elusive. Via CRISPR/Cas9-mediated genome editing, we established new knock-in mouse models that harbor expanded CAG repeats in the mouse huntingtin gene, which express RAN translated products or polyglutamine products respectively. Here we report that RAN translation is not detected in the knock-in mouse models, and that only the expanded polyglutamine products can cause neuropathology and behavioral phenotypes. Therefore, polyglutamine products, rather than RAN translated products, play a major role in the pathogenesis of CAG repeat diseases.
Project description:The small GTPase RAN plays a role in the biogenesis of mature miR-126, which is supplied by the bone marrow arterioles to leukemic stem cells (LSCs). MiR-126 supports the homeostasis of LSCs that initiate and maintain acute myeloid leukemia (AML). While therapeutic targeting of RAN has been difficult due to its structural features, through molecular dynamics simulations and docking studies, we have identified MAR-3.6.2 as a novel allosteric inhibitor that binds in a cryptic pocket in the C-terminal domain of RAN. We showed that MAR-3.6.2 disrupted RAN interaction with its guanine nucleotide exchange factor RCC1 and prevented the nuclear switch of RAN-GDP to RAN-GTP. This in turn led to RAN nuclear retention and reduced the RAN/XPO5-mediated export of pre-miR-126, thereby limiting mature miR-126 biogenesis in endothelial cells and their exogenous supply of mature miR-126 to LSCs. In a MllPTD/WT/Flt3ITD/ITD AML murine model, MAR-3.6.2 reduced leukemia burden, prolonged survival, and decreased LSC frequency in secondary transplants. These findings highlight MAR-3.6.2 and future, potential derivates as a promising small molecule-based approach to eradicate AML LSCs via inhibition of RAN/XPO5 trafficking and block of miR-126 biogenesis.
Project description:This dataset contains single-nucleus RNA sequencing results from adult rat brain (nucleus accumbens) and serves as the basis for characterization of transcriptional response to cocaine (20mg/kg, intraperitoneal injection). The goal of this experiment was to define the transcriptional response to cocaine across distinct cell types in the nucleus accumbens. Single-cell sequencing was carried out on FACS-sorted nuclei isolated from these experiments using the 10X Genomics Chromium single cell sequencing platform.
Project description:Nematostella vectensis is a venomous organism with a complex life cycle. This dataset is to complement the dataset PXD008218 where proteoms of several Nematostella life stages (unfertilised eggs, planulae, primary polyp, adult male and female) were analyzed to compare toxins abbunances. Here we re-ran unfertilized eggs LC-MSMS (4 replicates) separately from other life stages to eliminate cross-contamination with abbundant toxins expressed at adult stage. To control for comparability to the older PXD008218 dataset we also re-ran adult female LC-MSMS (1 replicate). Same samples as inPXD008218 were used.