CPTR - Mass Spectrometry Unit

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Overall, the expertise of the Mass Spectrometry Unit is being used to further the research of multiple groups within the NIH. In FY23, we collaborated in 65 different projects from 40 different investigators, with over 2700 samples run. Among these are projects to characterize the post-translational modifications of target proteins, including sites of phosphorylation, acetylation, and methylation, to better understand signal transduction, protein regulation, and the effects of small molecule inhibitors. The resource is also being used to identify protein interactors of both proteins and nucleic acids. Mass spectrometry is additionally being used extensively for large-scale quantitative proteomics projects, using both isotopic labeling and label-free approaches. Structural mass spectrometry applications, such as crosslinking and limited proteolysis methods, are used to investigate protein conformation. Finally, the resource is using inductively-coupled plasma mass spectrometry (ICP-MS) to quantify the level of metals in biological samples. In the past year, seven collaborative studies have been published; several other projects are nearing completion or manuscripts are under review. In collaboration with Ettore Appella, Laboratory of Cell Biology, we investigated the effects and mechanism of action of NSC59984, a small molecule inhibitor of mutant p53. Mass spectrometry analysis revealed that p53 is covalently modified by NSC59984 on specific cysteine residues, which likely affects the conformation of p53 R248W and returns it to a more wild-type like state. These findings are reported in one manuscript in Frontiers in Oncology and in a second in preparation. With Maria Morasso, NIAMS and adjunct in the Laboratory of Cancer Biology and Genetics, mass spectrometry was used to investigate the role of keratin-75 in the wound healing response. Among the interactors was SUN2, an inner nuclear membrane protein and component of the linker of nucleoskeleton and cytoskeleton complex (LINC). LINC transduces mechanical signals from the plasma membrane to the nucleus and influencing gene expression, suggesting that this interaction may regulate the wound healing response by regulating gene expression. A proximity ligation assay confirmed the keratin-75-SUN2 interaction and showed that it occurred primarily at the cytoplasmic face of the nuclear periphery. This research was published in the Journal of Investigative Dermatology. Another publication resulted from collaboration with Yves Pommier, Developmental Therapeutics Branch. Mass spectrometry was used to characterize the protein interactors of topoisomerase-3B (TOP3B) to help characterize its role in the resolution of R-loops, non-B-DNA structures consisting of RNA-DNA hybrids with displaced single-stranded DNA segments. In this work, published in Cell Reports, mass spectrometry analyses identified the R-loop helicase DDX5 as an interactor of TOP3B and showed that this interaction occurs independently of TDRD3, an auxiliary factor of TOP3B. Further experiments demonstrated that TOP3B and DDX5 function together to resolve R-loops. Thermal proteome profiling mass spectrometry methods were used to examine the targets of the small molecule inhibitor NAV-2729. This project, a collaboration with Paul Randazzo, Laboratory of Cellular and Molecular Biology, focused on the selectivity of NAV-2729, which was originally identified as a specific inhibitor of Arf6. Cell-based experiments showed that Arf6 expression did not correlate with NAV-2729 sensitivity, and mass spectrometry approaches identified 48 other possible targets. These results were published in the Journal of Biological Chemistry. Crosslinking mass spectrometry was used to explore the complexes of J-domain proteins with Hsp90 and Hsp70 in collaboration with the group of Sue Wickner, Laboratory of Molecular Biology. This study, published in the Journal of Molecular Biology, explored the role of J-domain proteins in protein reactivation by chaperones. It was found that a J-domain protein, CbpA, was able to form a ternary complex with E. coli Hsp70 (DnaK) ad E. coli Hsp90. Analysis of crosslinking experiments by mass spectrometry confirmed the ternary complex and identified sites of binding for binary interactions. Collaborative research with the lab of Deborah Hinton, NIDDK, examined the conformation of the Bordetella pertussis BvgAS system that regulates virulence gene expression. Crosslinking mass spectrometry was used to identify changes in the BvgA conformation when unmodified or phosphorylated. The findings suggest that phosphorylation of BvgA causes a structural rearrangement that relocates its N-terminal domain to allow better interaction of the C-terminal domain with DNA. The findings were published in the Computational and Structural Biotechnology Journal. In addition to the above projects, mass spectrometry has been used to investigate the mechanism of action of SAMT-247, a thioester molecule developed to prevent and treat HIV infection. Working with Drs. Genoveffa Franchini, Vaccine Branch, and Daniel Appella, NIDDK, mass spectrometry experiments have demonstrated that SAMT-247 covalently modifies cysteine and lysine residues in HIV Gag, resulting in protein aggregation and loss of function. Additionally, new work has identified a host protein that can also be targeted by SAMT-247, resulting in decreased HIV infection of cells.