Project description:Chemical cross-linking coupled to mass spectrometry was used to study binary and ternary complexes involving cyclin-dependent kinase 8 (CDK8), cyclin-C, and subunit 12 of the Mediator complex (MED12). Cross-linking was performed using different cross-linking chemistries: (1) disuccinimidyl suberate (DSS); (2) a combination of pimelic acid dihydrazide (PDH) and the coupling reagent, DMTMM.
Project description:Chemical cross-linking coupled to mass spectrometry was used to study two tRNA ligase complexes, one consisting of subunits RTCB, DDX1, CGI-99, FAM98B and Ashwin (“full tRLC”) and one lacking Ashwin (“core tRLC”). Cross-linking was performed using the homobifunctional, noncleavable reagent, disuccinimidyl suberate (DSS).
Project description:Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) is widely used in protein structural analysis. In this study we develop a new class of non-hydrolyzable amine-selective di-ortho-phthalaldehyde (DOPA) cross-linkers, one of which is DOPA2. Cross-linking of proteins with DOPA2 is 60-120 times faster than that with the N-hydroxysuccinimide ester cross-linker DSS. Compared with DSS cross-links, DOPA2 cross-links have a higher degree of agreement with the crystal structures of tested proteins. More importantly, DOPA2 has unique advantages of working at low pH, low temperature, or in the presence of denaturants such as 8 M urea or 6 M guanidine hydrochloride. Using staphylococcal nuclease, bovine serum albumin, and bovine pancreatic ribonuclease A, we demonstrate that DOPA2 cross-linking provides abundant spatial information about the conformations of proteins denatured to varying degrees. Lastly, we show that CXMS with only 10 seconds of DOPA cross-linking uncovers conformational changes ass
Project description:Chemical cross-linking coupled to mass spectrometry was used to study the interaction between fibronectin (Fn; full-length or gelatin-binding domain fragment = Fn 45) with tissue transglutaminase 2 in oxidized and non-oxidized form. The complexes were cross-linked with disuccinimidyl suberate (DSS) or a combination of pimelic dihydrazide (PDH) in combination with the zero-length cross-linking reagent, DMTMM.
Project description:Chemical cross-linking coupled to mass spectrometry was used to study the folding of the client protein, beta-tubulin, by the chaperonin TRiC/CCT. Different complexes containing TRiC/CCT and/or the chaperone prefoldin were cross-linked in absence or presence of nucleotides with the homobifunctional, noncleavable reagent, disuccinimidyl suberate (DSS).
Project description:Chemical cross-linking coupled to mass spectrometry was used to study the complex between the Integrator complex subunits INTS13 (Asunder) and INTS14. Cross-linking was performed using disuccinimidyl suberate (DSS).
Project description:In cross-linking mass spectrometry (XL-MS), the depth and sensitivity of cross-link detection is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of cross-links, here we present a gas-phase separation strategy using high field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in gas phase using the FAIMS device, we increase the number of cross-link identification by 22% for a medium complex sample and 59% for strong cation exchange-fractionated HEK293T cell lysate in XL-MS experiments using disuccinimidyl sulfoxide (DSSO) cross-linker. When disuccinimidyl suberate (DSS) cross-linker is in use, we are able to boost cross-link identification by 89% for the medium and 100% for the highly complex sample comparing to the analyses without FAIMS . Furthermore, we show that for medium complex samples, FAIMS enables the collection of single-shot XL-MS data with comparable depth to the corresponding sample fractionated by chromatography-based approaches. Altogether, we demonstrate FAIMS is highly beneficial for XL-MS studies by expanding the proteome coverage of cross-links while improving the efficiency and confidence of cross-link identification.
Project description:Chemical cross-linking coupled to mass spectrometry was used to study the organization of the Radial Spoke protein complex from Chlamydomonas. Experiments were performed by on-bead cross-linking of the immobilized complex with the amine-reactive cross-linking reagent disuccinimidyl suberate (DSS).
Project description:Chemical cross-linking coupled to mass spectrometry was used to study the complex between the nucleolar factor, Puf6, and the yeast 60S ribosome. Disuccinimidyl suberate (DSS) was used as the cross-linking reagent.
Project description:In this study, we used bottom-up proteomics and cross-linking MS (XL-MS) to study the composition and structure of soluble membrane attack complex (sMAC). For bottom-up proteomics sMAC was digested using trypsin and analyzed on Orbitrap Fusion Lumos. For the cross-linking analysis sMAC was cross-linked using DMTMM or DSS. The cross-linked proteins were digested using trypsin and the data was acquired using an Ultimate 3000 system coupled on-line to an Orbitrap Fusion. Proteomics raw data was searched using MaxQuant and cross-linking raw data was searched using pLink.