ABSTRACT: Peptide pulldowns with C-man antibodies to demostrate the utelity of reagents for proteomic analysis. Using Murine brain samples Novel C-man sites are readily identifiable.
Project description:Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine (Ser) and threonine (Thr) residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, e.g Saccharomyces cerevisiae. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation, and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway (HBP) to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-Mannose (O-Man) glycoproteome, and used this to identify a pleothora of O-linked mannose glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and doing so we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic and mitochondrial proteins in Saccharomyces cerevisiae. The type of nucleocytoplasmic proteins and the localization of identified O-Man residues mirrors that of the O-GlcNAc glycoproteome in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzyme machinery that is predicted to regulate the discovered nucleocytoplasmic O-Man glycosylation. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing.
Project description:Repo-Man chromatin binding sites were obtained by expression of GST:Repo-Man and incubation with nucleosomes extracted from HeLa cells (GST alone signal was used as a negative control and subtracted from the GST:Repo-Man chromatin bound fraction)
Project description:Protein O-linked mannose (O-Man) glycosylation is an evolutionary conserved post-translational modification, whose biosynthesis is initiated by three non-redundant enzyme families, POMT1/POMT2, TMTC1-4 and TMEM260. In this study, we applied a targeted workflow for membrane glycoproteomics to five human cell lines and to a panel of genetically engineered cells with individual and combinatorial knock-out of O-Man glycosyltransferase genes to extensively map substrates, specificities, and crosstalk of O-Man enzymes. Our quantitative glycoproteomics results demonstrate new protein targets for the POMT1/POMT2 pathway and show that TMTC1-4 and TMEM260 widely target distinct Ig-like protein domains of plasma membrane proteins. This new evidence adds further knowledge on the emerging concept of domain-specific O-Man glycosylation and establishes a platform for functional studies of O-Man glycosylated adhesion molecules and receptors.
Project description:GST:Repo-Man(C-terminus) or GST:alone were expressed as in Vagnarelli et al. 2011. Before elution, beads were first incubated with nucleosomes extracted from HeLa. HeLa cells were lysed and nucleosomes were digested with MNase (20min, 37C). Nucleosomes were firstly incubated with GST alone for 2hrs. This cleared lysate was then incubated with GST:Repo-Man and GST alone. Beads were then eluted and mix with 3x Laemli Buffer. Histone bands were sent to Mass Spec.
Project description:With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.
Project description:mzXML files of MS/MS data from skin swab samples of 1 man volunteer. Samples were collected from 17 spots on a man face, everyday during 10 days. MS/MS data collected from beauty products used by this subject are also included.
Project description:Bacterial infectious diseases have posed a serious challenge to public health, often resulting in treatment failure and infection recurrence due to the emergence of drug-resistant bacteria. Owing to inaccessible binding sites, pathogens can evade attack from host immune cells and traditional antibiotics, leading to local immunosuppressive status. Our study reports a novel bacteriophage-based immune scavenger labeling nanoplatform (Mn2+@Man-phage) to combat immune-evasive bacteria and reverse immunosuppressive status. Our nanosystem utilizes the inherent bacterium-targeting ability of bacteriophages to aggregate at infection sites and mediates mannose-dependent recognition, phagocytosis, and killing of bacteria by macrophages, while the released Mn2+ amplifies the antibacterial immune efficacy. Consequently, macrophages polarize towards M1 and secrete various pro-inflammatory factors, effectively clearing bacteria. Moreover, reprogramming macrophages directly activate T cells at infection sites, eliciting potent adaptive antibacterial immune responses and ultimately achieving bacterial eradication. Overall, we demonstrate a universal strategy for pathogen targeting and immunomodulation of macrophages against bacterial infection.