Project description:During DNA replication initiation, the Origin Recognition Complex (ORC) and Cdc6 co-associate on DNA to load replicative helicases onto origins of replication. We apply DSSO crosslinking mass spectrometry of reconstituted ORC-DNA-Cdc6 to identify interactions between different subunits and domains in the complex.
Project description:Cells organize their actions partly through tightly controlled protein-protein interactions – collectively termed the interactome. Here we use crosslinking mass spectrometry (XL-MS) to chart the interactome of intact human nuclei. We overall identified ~8700 crosslinks, of which 2/3 represent links connecting distinct proteins. From this data we constructed an overview of the nuclear interactome. We observed that the histone proteins on the nucleosomes expose well-defined crosslinking hot-spots. For several nucleosome-interacting proteins, such as USF3 and Ran GTPase, the data allowed us to build models of their binding mode to the nucleosome. For HMGN2 the data guided the construction of a refined model of the interaction with the nucleosome, based on complementary NMR, XL-MS and modeling. Excitingly, several isoform-specific interactors seem to exist for distinct histone H1 variants and the analysis of crosslinks carrying post-translational modifications allowed us to extract how specific modifications influence the nucleosome interactome. Overall, our data depository will support future structural and functional analysis of cell nuclei, including the nucleoprotein assemblies they harbor.
Project description:We have created a synthetic crosslinked peptide library to benchmark crosslinking mass spectrometry search engines. The unique benefit of the library is knowing which identified crosslinks are true and which are false. The data collected from mass spectrometry measurements of the peptide library were used to assess the most frequently used search algorithms. The datasets included will provide an important resource for the crosslinking community to evaluate and optimise search engines, results from which have far-reaching implications.
Project description:Chemical-Crosslinking Mass Spectrometry (XLMS) using MS-cleavable crosslinkers is fast becoming an established technique in the study of protein-protein interactions for both small and large scale samples. With the increased uptake of XLMS as a technique, different combinations of crosslinker types, fragmentation strategies and analysis programs are being applied to a diverse array of biological samples. This study is focused on understanding how the three variables of an XLMS experiment – crosslinker type, fragmentation and program – could generate differences in identifications, leading to increased biological coverage of a sample. Here we probe for the first time, the known enzyme:substrate interaction between yeast arginine methyltransferase Hmt1p and its substrate, heterologous nucleolar protein Npl3p. We use this known interaction as a platform to compare two analysis programs, MeroX and XlinkX2.0, using two crosslinker types, DSSO and DSBU, and two mass-spectrometry fragmentation strategies, CID/ETD and SteppedHCD. Through these we also compare different algorithm strategies, Precursor and Reporter-Ion, as well as assess the impact of restricting data searches to lysine only crosslinks versus the inclusion of serine, threonine and tyrosine as reactive residues. From this analysis we show direct evidence of Hmt1p in contact with its known methylation sites on Npl3p, the intrinsically disordered “SRGG” region. We also show through our multi-crosslinker, multi-fragmentation and multi-software approach that two approaches leads to greater understanding and depth of a crosslinked sample, and this is due to some combinations of experimental variables creating greater coverage of crosslinks than others.
Project description:As part of an in-depth characterization of nanobodies directed against the human Leucine-rich repeat kinase 2 (LRRK2), epitopes of the nanobodies bound to LRRK2 were mapped by chemical crosslinking combined with mass spectrometry using the CID-cleavable crosslinker disuccinimidyl sulfoxide (DSSO).
Project description:As part of an in-depth characterization of nanobodies directed against the Chlorobium tepidum protein CtRoco, a bacterial LRRK2 homologue, chemical crosslinking combined with mass spectrometry (CX-MS) was performed. Two allosteric nanobodies binding to two different epitopes in the LRR and the Roc domain of CtRoco, respectively were analysed using the CID-cleavable crosslinker disuccinimidyl sulfoxide (DSSO).
Project description:The associated files are mass spec data from size exclusion chromatography fractions that were subsequently crosslinked with DSSO and digested with one of two enzymes, Trypsin or Chymotrypsin. The starting material was a native extract prepared from Chlamydomonas reinhardtii. The mass spectrometry used a data-dependent MS2-MS3 method to identify crosslinks.
Project description:Studies using crosslinking coupled to mass spectrometry on the proteome-wide level have spurred great interest as they facilitate structural probing of protein interactions in living cells or even organisms. Here we show, by using both an in-vitro mimic of a crowded cellular environment and eukaryotic cell lysates, that current proteome-wide crosslinking protocols have a bias for high abundant proteins. We demonstrate that this bias can be explained by kinetics that govern the formation of a crosslink between two polypeptides. We further show that optimized parameter settings, in particularly an excess of crosslinker, leadto a significant overall increase in the detection of lower abundant proteins within cellular lysates on a proteome-wide scale. Our study therefore explains the cause of a major limitation in current proteome-wide crosslinking studies and demonstrates a way forward how to address a larger part of the proteome by crosslinking