ADAM10 hyperactivation acts on piccolo to deplete synaptic vesicle stores in Huntington’s disease

Synaptic dysfunction and cognitive decline in Huntington’s disease (HD) involve hyperactive A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10). To identify the molecular mechanisms through which ADAM10 is associated with synaptic dysfunction in HD, we performed an immunoaffinity purification-mass spectrometry (IP-MS) study of endogenous ADAM10 in the brains of wild-type and HD mice. In the normal brain, proteins implicated in synapse organization, synaptic plasticity, and vesicle and organelles trafficking interact with ADAM10, suggesting that it may act as a hub protein at the excitatory synapse. Importantly, the ADAM10 interactome is enriched in presynaptic proteins and ADAM10 coimmunoprecipitates with piccolo (PCLO), a key player in the recycling and maintenance of synaptic vesicles (SVs). In contrast, reduced ADAM10/PCLO immunoprecipitation occurs in the HD brain, with decreased density of SVs in the reserve and docked pool at the HD presynaptic terminal. Conditional heterozygous deletion of ADAM10 in the forebrain of HD mice reduces active ADAM10 to wild-type level, and normalizes ADAM10/PCLO complex formation and SVs density and distribution. The results indicate that presynaptic ADAM10 and PCLO are a relevant component of HD pathogenesis.

Improving the throughput of immunoaffinity purification and enzymatic digestion of therapeutic proteins using membrane-immobilized reagent technology

Rapid spin membrane technology decreases the time for IP and digestion of therapeutic proteins.

A non-acidic method using hydroxyapatite and phosphohistidine monoclonal antibodies allows enrichment of phosphopeptides containing non-conventional phosphorylations for mass spectrometry analysis

Four types of phosphate-protein linkage generate nine different phosphoresidues in living organisms. Histidine phosphorylation is a long-time established but largely unexplored post-translational modification, mainly because of the acid-lability of the phosphoramidate bonds. This lability means that standard phosphoproteomic methods used for conventional phosphate esters (phospho-Ser/Thr/Tyr) must be modified to analyze proteins containing the phosphoramidate-amino acids - phospho-His/Arg/Lys. We show that a non-acidic method allows enrichment of non-conventional phosphoresidue-containing peptides from tryptic digests of human cell lines, using hydroxyapatite binding and/or immobilized 1-pHis and 3-pHis monoclonal antibodies for enrichment. 425 unique non-conventional phosphorylation sites (i.e. pHis, pLys and pArg) were detected with a high probability of localization by LC-MS/MS analysis and identified using a customized MaxQuant configuration, contributing to a new era of study in post-translational modification and cell signaling in humans. This is the first fully non-acidic method for phosphopeptide enrichment which uses immunoaffinity purification and remains compatible with mass spectrometry analysis for a wider coverage of potential protein phosphorylation events.