Project description:Metabolomics analysis of mice stool samples. Data was acquired using a Thermo q-Exactive and C18 RP-UHPLC. Positive polarity acquisition of LC-MS/MS.
Project description:40 µL plasma from a pool of 20 neurologically healthy donors was separated into a bound and a depleted fraction using a MARS Hu-14 column. This experiment represents the bound fraction. Sample was in-solution trypsin digested and peptides were fractionated into 10 fractions by mixed-mode reversed phase anion exchange (MM(RP-AX) using a Promix MP column. Each fraction was analyzed separately by LC-MS/MS on an Orbitrap Velos Pro mass spectrometer. Resulting data was searched using SeachGui, summarized in PeptideShaker and exported to the CSF-PR.
Project description:40 µL plasma from a pool of 20 neurologically healthy donors was separated into a bound and a depleted fraction using a MARS Hu-14 column. This experiment represents the depleted fraction. Sample was in-solution trypsin digested and peptides were fractionated into 67 fractions by mixed-mode reversed phase anion exchange (MM(RP-AX) using a Promix MP column. Each fraction was analyzed separately by LC-MS/MS on an Orbitrap Velos Pro mass spectrometer. Resulting data was searched using SeachGui, summarized in PeptideShaker and exported to the CSF-PR.
Project description:The anterior silk gland in the silkworm plays an important role in the process of liquid fibroin to solid silk fiber .In view of this,the proteomics analysis was applied to to study the relationship between the function of proteins in the anterior silk gland and the mechanism of spinning. The anterior silk glands on the 3rd day of fifth instar were dissected.Aftter 1D SDS-PAGE ,one gel lane was cut into 10 bands and each band further sliced into small pieces was subjected to in-gel tryptic digestion for 20 hours.The digested peptides were separated by RP nanoscale capillary liquid chromatography and analyzed using a surveyor LC system (Thermo Figgigan, San Jose, CA).The eluate from the RP column was analyzed by Finnigan LTQ(Thermo Electron Corporation)linear ion trap Mass equipped with a nanospray souce in the positive ion mode. The MS analysis was performed with one full MS scan followed by three MS/MS scans on the most intense ions from the MS spectrum with the dynamic exclusion settings: repeat count 2, repeat duration 30s, exclusion duration 90s. Data were acquired in data-dependent mode using Xcalibur software.Ten raw datasets from LC-MS/MS were searched against the predicted silkworm database by Xia.et al which consists of 21312 silkworm proteins.The searching was carried out with the Turbo SEQUEST(Bioworks version 3.2, Thermo Electron).
Project description:1.6 mL CSF from a pool of 21 neurologically healthy donors was separated into a bound and a depleted fraction using a MARS Hu-14 column. This experiment represents the depleted fraction. Sample was in-solution trypsin digested and resulting peptides were fractionated into 66 fractions by mixed-mode reversed phase anion exchange (MM(RP-AX) using a Promix MP column. Each fraction was analyzed separately by LC-MS/MS on an Orbitrap Velos Pro mass spectrometer. Resulting data was searched using SeachGui, summarized in PeptideShaker and exported to the CSF-PR.
Project description:1.6 mL CSF from a pool of 21 neurologically healthy donors was separated into a bound and a depleted fraction using a MARS Hu-14 column. This experiment represents the bound fraction. Sample was in-solution trypsin digested and peptides were fractionated into 10 fractions by mixed-mode reversed phase anion exchange (MM(RP-AX) using a Promix MP column. Each fraction was analyzed separately by LC-MS/MS on an Orbitrap Velos Pro mass spectrometer. Resulting data was searched using SeachGui, summarized in PeptideShaker and exported to the CSF-PR.
Project description:The Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) is an essential epigenetic modifier able to methylate lysine 27 on histone H3 (H3K27) to induce chromatin compaction, protein complex recruitment and ultimately transcriptional repression. Hematologic malignancies, including Diffuse Large B cell lymphoma (DLBCL) and Acute myeloid leukemia (AML) have shown a high EZH2-mutation frequency (>20%) associated with poor clinical outcomes. Particularly, two distinct oncogenic mutations, so-called gain-of-function (Y641F and A677G) and loss-of-function (H689A and F667I) are found in the catalytic domain of EZH2. In this study, a comprehensive multi-omics approach was employed to characterize downstream effects of H3K27me3 deposition driven by EZH2 mutations. Human embryonic kidney cells (HEK293T) were transfected to generate three stable EZH2 mutants: EZH2(Y641F), EZH2(A677G), and EZH2(H689A/F667I), which were validated via immunoblotting and DIA-MS-based histone profiling assay. Subsequently, constructs were analyzed under a comprehensive multi-omics approach including: 1) Cleavage Under Targets and Tagmentation (CUT&Tag); 2) chromatin accessibility characterization using the assay for transposase-accessible chromatin with sequencing (ATAC-Seq); 3) transcriptomics (RNA-Seq); 4) label-free whole-cell proteomics, acquired with a Bruker timsTOF Pro HPLC-MS/MS with Ion Mobility, and 5) MS-based untargeted metabolomics, in positive and negative ionization MS/MS mode, acquired with an Agilent 6545 QTOF with a 1290 UHPLC system and HILIC column. Total coverage comprised over 21,000 chromatin regions, 18,000 transcripts, 6,000 proteins and 400 metabolic features. Pair-wise comparison using univariate and supervised multivariate statistical methods revealed significant differences between constructs in each omic level. Furthermore, effector pathway analysis combining omics data revealed distinctive reprogramming effects for each EZH2 mutant.