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:Settlement-inducing protein complex (SIPC) is a protein that acts as a phoromone cue to attract conspecific of the barnacle Amphibalanus amphitrite to settle on a substrate. Recombinant settlement-inducing protein complex was expressed in insect Sf9 cells through a baculovirus overexpression system, purified and analysed by mass spectrometry to confirm its identity.
Project description:Analysis of recombinant mouse Ric-8B protein phosphorylation. Ric-8B was purified from insect cells and E.coli and both treated with protein kinase CK2 and re-purified prior to LC-MS/MS. Another Ric-8B sample from insect cells was not treated with CK2 and also subjected to LC-MS/MS.
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:We performed chemical crosslinking mass spectrometry of native protein complexes purified from Trypanosoma brucei procyclic cells to obtain protein-protein interaction information for the chromosomal passenger complex (CPC) and kinetochore complex. Crosslinks among CPC subunits were detected using YFP-AUK1, while those among KKT8 complex subunits were detected in the YFP-KKIP1 sample. We also performed immunoprecipitation and mass spectrometry of CPC subunits (full-length proteins and fragments) to identify co-purifying proteins.
Project description:We conducted a screening for nuclear proteins which interact with α-synuclein by mass-spectrometry. As a result, we found that α-synuclein interact with BAF complex. Furthermore, overexpressed α-synuclein increased global H4R3me2s by promoting interaction between BAF complex and PRMT5 in cultered cells. In this study, we explored the influence of overexpressed α-synuclein on H4R3me2s by ChIP-seq using anti-H4R3me2s antibody.
Project description:Aberrant forms of the SWI/SNF chromatin remodeling complex are associated with human disease. Loss of the Snf5 subunit of SWI/SNF is a driver mutation in pediatric rhabdoid cancers and forms aberrant sub-complexes that are not well characterized. We determined the effects of loss of Snf5 on the composition, nucleosome binding, recruitment and remodeling activities of yeast SWI/SNF. The Snf5 subunit interacts with the ATPase domain of Snf2 and forms a submodule consisting of Snf5, Swp82 and Taf14 as shown by mapping SWI/SNF subunit interactions by crosslinking-mass spectrometry and subunit deletion followed by immunoaffinity chromatography. Snf5 promoted binding of the Snf2 ATPase domain to nucleosomal DNA, enhanced its catalytic activity and facilitated nucleosome remodeling. Snf5 was required for acidic transcription factors to recruit SWI/SNF to chromatin. RNA-seq analysis suggested that both the recruitment and catalytic functions mediated by Snf5 are required for SWI/SNF regulation of gene expression.