Project description:Here we describe a cross-linking-aided MS workflow for isolation and identification of signal-dependent membrane protein interactomes using EGFR as an example. Employing formaldehyde as a cross-linking reagent, we performed immuno-precipitation of endogenous EGFR upon EGF treatment, facilitating the capture of weak and transient interactions in the proximity of the receptor, and analyzed the associated proteins by quantitative mass spectrometry.
Project description:Formaldehyde is a widely used fixative in biology and medicine. The current chemical model for formaldehyde cross-linking of proteins is the formation of a methylene bridge that incorporates one carbon atom into the link. Here, we present mass spectrometry data that largely refute this model. Instead, our data show that cross-linking of structured proteins mainly involves a reaction that incorporates two carbon atoms into the link. Under MS/MS fragmentation, the link cleaves symmetrically to yield unusual fragments with a modification of one carbon atom. We apply this new understanding of the underlying cross-linking chemistry to the structural approach of cross-linking coupled to mass spectrometry. First, we cross-linked a mixture of purified proteins with formaldehyde. Our new analysis readily identified tens of cross-links from these proteins, which fit well with their atomic structures. We then perform in-situ cross-linking of human cells in culture. We identified 469 intra-protein and 90 inter-protein cross-links, which also fit well with available atomic structures. Interestingly, many of these cross-links could not be mapped onto a known structure and thus provide new structural insights. We highlight an example in which formaldehyde cross-links localize the binding site of βNAC on the ribosome. We also find several interactions of actin with auxiliary proteins. Our findings not only expand our understanding of formaldehyde reactivity and toxicity, but also clearly demonstrate how to use this potent reagent for structural studies.
Project description:In-gel digest of a cross-linked product band was used to identify an internal isopeptide cross-link formed in A2M TR K704 upon bait region cleavage by trypsin. PRM was used to quantify the thiol ester in native PAGE bands of A2M nEXT and EXT mutants.
Project description:Laminin, a ~800 kD heterotrimeric protein, is a major functional component of the extracellular matrix, contributing to tissue development and maintenance. We used cross-linking and mass spectrometry to determine the order of subunits in the ~750 Å trimeric coiled-coil oligomerization domain of laminin and to achieve an estimate of the register of the three subunits along the length of the coiled coil.
Project description:Immuno-LC-PRM assay was developed to simultaneously quantify the expression levels of six immune markers (CD8A, CD4, LAG3, PD1, PD-L1 and PD-L2) using as little as 1-2 mg of fresh frozen tissue.