ABSTRACT: DSS crosslinking of mLST8-CCT purified from HEK293T cells using a tandem affinity approach. First cell extract was run over HisTrap column then a streptactin column.
Project description:DSS crosslinking of mLST8-CCT purified from HEK293T cells using a tandem affinity approach. First cell extract was run over HisTrap column then a streptactin column.
Project description:To understand the functional roles of YTHDF1 in cellular senescence and aging, we lead the RNA sequence in WT or Ythdf1 deficiency colon epithelial cells. The results showed that the cholesterol biosynthesis related genes were upregulated in Ythdf1 KO mice. We also purified FLAG-YTHDF1 protein in HEK293T cells and did protein mass spectrometry, and found that the mTORC1 and TSC complex constituents mTOR, RPTOR, and TSC1/2 were identified in the YTHDF1-complex. Methylated RNA immunoprecipitation(MeRIP) with specific m6A antibody and used for library construction and the next generation sequencing, to identify m6A modified transcripts in WT or Ythdf1 deficiency colon epithelial cells after DSS treatment
Project description:The Nucleosome Remodeling and Deacetylase (NuRD) complex is essential for development in complex animals but has been refractory to detailed analysis because of its low abundance and resistance to recombinant production. In combination with other techniques, crosslinking mass spectrometry was used to elucidate the structure of the Nucleosome Remodelling and Deacetylase (NuRD) complex. Natively-purified NuRD, and four recombinantly-expressed NuRD subcomplexes, and a PWWP2A-MTA-HDAC-RBBP alternative ‘NuRD-like’ complex were subjected to crosslinking with DSS, ADH, BS3 and DMTMM.
Project description:Chemical crosslinking (DSS) of proteins in conjunction with mass spectrometry was performed to identify protein-protein interactions between the light-harvesting proteins of PSI and PSII.
Project description:KKT23 is a kinetoplastid kinetochore protein that has structural similarity to the GCN5 acetyltransferase domain. We performed crosslinking mass spectrometry of the recombinant KKT3-KKT22-KKT23 complex purified from Sf9 insect cells.
Project description:Co-Immunoprecipitation with Crosslinking Using DSS for Exo84 Subunit Sample Preparation: Yeast strains expressing Exo84-3xGFP and FRB-tagged Sec18 or Sec9 were cultured in YPD at 30°C until OD600 0.8-1. Cells were harvested, washed with resuspension buffer (50 mM HEPES, 150 mM NaCl, protease inhibitors), and frozen in liquid nitrogen. Lysis was performed using a Freezer/Mill, followed by clarification via centrifugation. Crosslinking with DSS: Lysates were crosslinked with DSS (0.5 mM, 1h, RT) and quenched with glycine (100 mM, 15 min). Supernatants were clarified by centrifugation. Immunoprecipitation: GFP-Trap beads were used to capture Exo84 complexes. Lysates were pre-cleared with blocking beads (1h, 4°C) before incubation with GFP-Trap beads (overnight, 4°C). Beads were washed with a stringent buffer (2M NaCl, 8M urea, 1% Triton X-100) and PBS. Mass Spectrometry Sample Preparation: Immunoprecipitated complexes were reduced (DTT), alkylated (IAA), digested with trypsin (37°C, 8h), and desalted before LC-MS/MS. LC-MS/MS Analysis: Samples were analyzed on an Orbitrap Fusion Lumos coupled to an EASY-nLC 1200 with a 50 cm C18 column. Peptides were separated with a gradient from 5% to 40% acetonitrile over 60 min. MS1 scans were acquired at 120,000 resolution, and the Top Speed method was used for data-dependent MS2 acquisition with HCD fragmentation.
Project description:In this project we are describing a novel mono- and intralink filter (mi-filter) that is applicable to any kind of crosslinking data and workflow. It stipulates that only proteins for which at least one monolink or intra-protein crosslink has been identified within a given dataset are considered for an inter-protein cross-link and therefore participate in a PPI. We show that this simple and intuitive filter has a dramatic effect on all types of crosslinking-data ranging from single protein complexes, over medium-complexity affinity enrichments to proteome-wide settings and significantly improves false-discovery rates for inter-protein links in all types of XL-MS data.
Project description:Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone whose urinary (u-hCG) and recombinant (r-hCG) forms differ in glycosylation and stability. To test whether this reflects a structural reorganization of the α/β subunit interface, the two forms were compared by chemical crosslinking mass spectrometry (XL-MS). Crosslinking was performed on the intact, natively glycosylated heterodimers using two complementary chemistries: the lysine-reactive reagent DSS and the zero-length coupling reagent DMTMM (targeting Lys-Asp/Glu pairs, each in biological triplicate for both forms. Identified inter-subunit crosslinks were mapped onto the hCG structure to evaluate whether the assembled core interface is conserved between u-hCG and r-hCG. The dataset comprises the raw LC-MS/MS files and the corresponding crosslink identifications for all conditions and replicates, and accompanies a broader native-MS, ion-mobility and collision-induced-unfolding study of the two hCG forms.