CPTR - Mass Spectrometry Unit

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

The expertise of the Mass Spectrometry Unit is being used to further the research of multiple groups within the NIH. In FY21, we collaborated in 61 different projects from 41 different investigators, with more than 3000 samples processed and analyzed. 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, including copper, iron, lead, tungsten, and platinum. In the past year, ten collaborative studies have been published; several other projects are nearing completion or manuscripts are under review. Working with Dr. Ashish Lal, Genetics Branch, we collaborated on three studies to investigate the functions of long-noncoding (lnc) RNAs. In 2020, three papers originating from work with Dr. Lal were published. The first, published in RNA, studied the roles of lncRNAs during cellular quiescence and cell cycle reentry. Mass spectrometry was used to characterize the interactors of MIR222HG, a microRNA-host-gene lncRNA that was found to have serum-stimulated RNA processing that promoted cell cycle reentry. Among the identified interactors, binding of the ILF3/ILF2 complex was shown to facilitate the complex of MIR222HG with DNM3OS, another lncRNA that promotes cell cycle reentry; this complex stabilizes DNM3OS and thereby enhances DNM30S functions. The second paper investigated the roles of lncRNAs in cell cycle progression. SUNO1 is a lncRNA found to be upregulated in S phase that facilitated cell cycle progression by promoting YAP1-mediated gene expression. Mass spectrometry analysis of the protein interactors of SUNO1 identified DDX5, which is known to be a transcriptional co-activator of cell cycle genes; this complex promoted DDX5-mediated stabilization of RNA polymerase II on chromatin. These studies were published in eLife. Finally, the resource helped study the novel small protein FORCP, which was transcribed by the putative gastrointestinal-tract-specific lncRNA LINC00675. Mass spectrometry used to demonstrate the existence of this new protein and investigate its function by identification of its interactors. These results were published in eLife. Working with the lab of Dr. Yves Pommier, Developmental Therapeutics Branch, mass spectrometry analyses were used in three published studies. First, mass spectrometry was used to investigate the role of sumoylation in the repair of trapped topoisomerase DNA-protein crosslinks (TOP-DPCs). Mass spectrometry analyses further identified PIAS4 as a shared SUMO ligase for TOP1-, TOP2alpha-, and TOP2beta-DPCs. A paper reporting on this collaborative work was published in Science Advances. Second, mass spectrometry was used to help investigate the mechanism of chemoresistance following SLFN11 inactivation; analysis of SLFN11 interactors identified that it is a cofactor of CUL4(CDT2) ubiquitin ligase and promotes degradation of CDT1, blocking replication reactivation. These findings were published in the Proceedings of the National Academy of Sciences. The third study continued investigations into SLFN11 inactivation. Following a screen for therapeutic inhibitors in SLFN11-knockout leukemia cells, TAK-243 was identified as an inhibitor of UBA1 with preferential activity in the SLFN11-knockout cells. Proteomic analysis demonstrated physical interactions of SLFN11 with translation initiation complexes and protein folding machinery, a previously unknown function for SLFN11. This study was published in Cancer Research. In work published in Biosensors, mass spectrometry was used to demonstrate the utility of a new high-sensitivity label for protein gels. This research, performed in collaboration with the group of Dr. Jennifer Jones, Laboratory of Pathology, showed that the Bio-Rad stain-free technology for visualizing proteins with UV light has similar sensitivity to silver stain and was fully compatible with downstream mass spectrometry analysis without extensive destaining, as is required for silver stain. Working with the research group of Dr. Stavroula Mili, Laboratory of Cellular and Molecular Biology, mass spectrometry was used to investigate the protein interactors of wildtype and mutant RAB13 to better understand the mechanism by which RAB13 activity and function are regulated by the subcellular location of RAB13 mRNA translation. Whereas RAB13 RNA is enriched at peripheral protrusions, the expressed protein is concentrated perinuclearly. Mass spectrometry analysis was performed to identify interactors of wildype RAB13 and a nucleotide-free mutant that binds to GEFs. Further research demonstrated that RAB13 translation leads to a co-translational association of nascent RAB13 with RABIF and the RAB13-RABIF complex at the cell periphery. These findings were published in EMBO Journal. Rigosertib is a benzyl-styryl-sulfone compound originally developed as a non-ATP competitive, multi-kinase inhibitor which induces apoptosis and cell-cycle arrest in many human cancer cell lines. The precise mechanism of action of rigosertib has remained elusive despite rigorous investigation. To help delineate its mechanism in pediatric cancers, mass spectrometry was used to identify the proteins that specifically interacted with the active form of rigosertib, as compared with an inactive isomer. Among the proteins identified was beta-tubulin; further experiments demonstrated that rigosertib treatment resulted in decreased levels of acetylated tubulin and induced mitotic spindle defects. This research, performed in collaboration with Dr. Marielle Yohe, Pediatric Oncology Branch, was published in Molecular Cancer Therapeutics.