Project description:Protein glycosylation and phosphorylation are two of the most common post-translational modifications (PTMs), which plays an important role in many biological processes. However, low abundance and poor ionization efficiency of phosphopeptides and glycopeptides make direct MS analysis challenging. Previously, we explored the electrostatic and hydrophilic properties of commercial centrifuge-assisted-extraction Titanium (IV) IMAC (CAE-Ti-IMAC) material and its application in simultaneously enriching and separating common glycopeptides, phosphopeptides, and M6P glycopeptides in dual-mode. In this study, we developed a hydrophilicity enhanced dual-functional Ti-IMAC material with adenosine triphosphate as grafted group (denoted as: epoxy-ATP-Ti4+) to achieve better enrichment performance in dual-mode separation. The epoxy-ATP-Ti4+ IMAC material was prepared from commercially available epoxy functionalized silica particles in a facile way, which only required two steps of reaction. The ATP molecule not only provided superiorly strong and active metal phosphate sites to bind phosphopeptides, but also contributed significantly to the hydrophilicity to enrich glycopeptides. The epoxy-ATP-Ti4+ IMAC material showed great selectivity and sensitivity for phosphopeptide enrichment in conventional IMAC mode. With optimized buffer and fractionation, the material successfully separated glycopeptides and phosphopeptides with high specificity. Besides standard protein samples, the material was further applied to HeLa cells and mouse lung tissue samples. Our method allows simple and effective enrichment and separation of glycopeptides and phosphopeptides, which paves the way for studying the potential crosstalk between these two PTMs.
Project description:Cortical pathology contributes to chronic cognitive impairment of patients suffering from the neuroinflammatory disease multiple sclerosis (MS). How such gray matter inflammation affects neuronal structure and function is not well understood. Here we use functional and structural in vivo imaging in a mouse model of cortical MS to demonstrate that bouts of cortical inflammation disrupt cortical circuit activity coincident with a widespread, but transient loss of dendritic spines. Spines destined for removal show local calcium accumulations and are subsequently removed by invading macrophages or activated microglia. Targeting phagocyte activation with a new antagonist of the colony-stimulating factor 1 receptor prevents cortical synapse loss. Overall, our study identifies synapse loss as a key pathological feature of inflammatory gray matter lesions that is amenable to immunomodulatory therapy.
Project description:Cell membrane proteins are densely decorated with surface glycosylation and intracellular phosphorylation whose interplay determines the cell-cell communication and signaling cascades. However, their concomitant characterization remains extreme challenges due to complexity of glycan structures, low abundance of glycopeptides (particularly sialylation), labile, dynamic nature and low detectability of both phosphopeptides and glycopeptides in mass spectrometry. In addition, the interplay and dynamic change between glycosylation and phosphorylation in tyrosine kinase inhibitor (TKI)-resistant non-small cell lung cancer (NSCLC) cells is still unclear. In this study, we introduce a streamlined metal ion-decorated ZIC-cHILIC strategy, featuring a simple pH control, to allow simultaneous enrichment and stepwise separation of intact (sialo-)glycopeptides and phosphopeptides.