CPTR - Mass Spectrometry Unit

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Overall, the expertise of the Mass Spectrometry Resource is being used to further the research of multiple groups within the NIH. In FY25, we collaborated in more than 90 separate projects from 60 different investigators, with over 7000 sample runs. Among these are projects to characterize the post-translational modifications of target proteins, including sites of phosphorylation, acetylation, methylation, and ubiquitination 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, is used to investigate protein conformation. Finally, the resource is using inductively-coupled plasma mass spectrometry (ICP-MS) and time-of-flight (TOF) mass spectrometry for targeted quantification of metals and small molecules in biological samples. In the past year, more than ten collaborative studies have been published or accepted for publication; several other projects are nearing completion or manuscripts are under review. Highlights of three of these are described below. In work that continued a long-standing collaboration with Dr. Yves Pommier, Developmental Therapeutics Branch, mass spectrometry was used to better understand DNA-protein crosslinks (DPCs) produced by formaldehyde (FA) and how they are repaired. First, we profiled the proteins that were localized to DPCs, identifying several DNA processing enzymes {TOP1, TOP2alpha, TOP2beta, DNMT1, and poly[adenosine diphosphate (ADP)-ribose] polymerase1 (PARP1)} among the most abundant. Further experiments found that flap endonuclease 1 (FEN1) was critical for resolution of the FA-induced DPCs. As poly(ADP)ribosylation (PARylation) is critical for sensing sensing DNA damage and we identified PARP1 at the DPCs, we tested whether PARylation could be important for DPC repair. We found that FEN1 was PARylated upon FA exposure on E285, a modification that is critical for its recruitment to DPCs. The results of this study were published in Science Advances. Mass spectrometry was the driving method of a project aiming to understand the mechanism of reaction of NSC59984, a small molecule reactivator of mutant p53. TP53 is commonly mutated in cancer, giving rise to loss of wild-type tumor suppressor function and increases in gain-of-function oncogenic roles. Thus, inhibition of mutant p53 and reactivation of wild-type function represents a potential means to target diverse tumor types. Working with Dr. Ettore Appella, Laboratory of Cell Biology, we mapped the reaction of NSC59984 with p53, identifying p53 Cys124 and Cys229 to be modified by NSC59984 following vitro reaction and upon treatment of cells. Two mass spectrometry approaches were next used to probe for other targets of NSC59984. First, a biotinylated form of the inhibitor was used for pulldown experiments and second thermal proteome profiling, a global approach, was used. Several metabolic proteins were identified, including TIGAR, a protein known to be a transcriptional target of p53. Mass spectrometry was finally used to map reaction of NSC59984 with TIGAR, showing that it too could be modified. These findings were published in ACS Pharmacology and Translational Science. Protein crosslinking coupled with mass spectrometry (XL-MS) is a powerful approach to map sites of protein complex formation. This method can be particularly useful for transient complexes or those that involve a functional activity. We applied XL-MS to investigate the binding of chaperones with cochaperone partners. Focusing on bacterial proteins as model systems, our collaborators found that ClpB bound DnaJ in a concentration-dependent manner. We found that the majority of the crosslinking occurred within the NBD I and MD domains of of ClpB and the J-domain and CTD I domain of DnaJ. These results are consistent with several sites of interaction on both ClpB and DnaJ. The project was performed in collaboration with Dr. Sue Wickner, Laboratory of Molecular Biology, and is published in Proceedings of the National Academy of Sciences USA.