CPTR - Mass Spectrometry Unit

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Overall, the expertise of the Mass Spectrometry Unit is being used to further the research of multiple groups within the NIH. In FY22, we collaborated in 64 different projects from 36 different investigators, with over 2500 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. In the past year, ten collaborative studies have been published; several other projects are nearing completion or manuscripts are under review. One publication resulted from collaboration with Dr. Yves Pommier, Developmental Therapeutics Branch. Both used mass spectrometry to characterize interactors of proteins as a means to better understand their function. In the first, the role of poly(ADP)-ribosylation (PARylation) in the regulation of the repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) was investigated. Mass spectrometry analysis of TOP1 interactors revealed that PARP1 is pulled down with TOP1 even in the absence of CPT, consistent with the fact that PARP1 and TOP1 are associated under unperturbed conditions and that PARP1 promptly PARylates TOP1-DPCs without the requirement for replication or transcription. Other work showed that PARylation recruits the deubiquitylating enzyme USP7 to reverse the ubiquitylation of PARylated TOP1-DPCs. Poly(ADP-ribose) glycohydrolase (PARG) was shown to be a repair factor for TOP1-DPCs by enabling the proteasomal digestion of TOP1-DPCs, which suggests the potential regulatory role of PARylation for the repair of a broad range of DPCs. This research was published in Nature Communications. Global proteomics methods were used in a collaborative project with Dr. Roberto Weigert, Laboratory of Cellular and Molecular Biology, to understand the effects of Butyrophilin 1A1 (BTN1A1) knockout in mammary epithelium. BTN1A1 has been implicated in the secretion of lipid droplets from mammary epithelial cells as a membrane receptor in a secretion complex with the redox enzyme, xanthine oxidoreductase (XDH). Combination of RNAseq and global proteomics analysis of wildtype and Btn1a1-/- knock-out mice demonstrated effects on proteins involved in inflammation and IL-6 signaling, autophagy, the cell cycle, apoptosis, membrane-bounded organelles, lipid metabolism, and transport functions. Evidence was found for the involvement of several pathways responsible for cell death in the knock-out mice, including caspase signaling, autophagy, and pStat3-mediated lysosomal lysis. This research was published in FABSEB BioAdvances. In collaboration with Dr. David Roberts, Laboratory of Pathology, mass spectrometry was used to investigate potential mechanisms by which CD47 regulates the trafficking of specific RNAs to extracellular vesicles. Interaction of CD47 and its cytoplasmic adapter ubiquilin-1 with components of the exportin-1/Ran nuclear export complex were identified and shown to have functional consequences for nuclear to cytoplasmic trafficking of 5'-7-methylguanosine-modified microRNAs and further transport into extracellular vesicles. This research was published in the Journal of Cell Communication and Signaling. SIRT1 chromatin interactome analyses were performed in collaboration with Dr. Mirit Aladjem, Developmental Therapeutics Branch, to better understand the differential molecular cues that distinguish dormant origins from baseline origins. In examining the interactome of SIRT1, the MCM protein were identified as binding partners for SIRT1 on chromatin; these proteins are known to be key components of pre-replication complexes. Further analysis of the interactome of MCM2 revealed the reciprocal interaction; phosphorylated SIRT1 was also able to interact with several MCM proteins. Further experiments demonstrated that dormant origins can be distinguished from baseline origins by their preferential association with two phosphoforms of MCM2. This research was published in Nucleic Acids Research. In collaboration with Dr. Alexander Kelly, Laboratory of Molecular Biology, mass spectrometry was used to investigate the mechanism of condensing engagement with and action on chromatin. Mass spectrometry analysis of immunoprecipitations of the XPB subunit of TFIIH revealed that it was responsible for TFIIH recruitment to condensed chromatin, helping to demonstrate a new role for the TFIIH complex in chromosome condensation. Further experiments demonstrated that the TFIIH complex is continuously required to establish and maintain a compacted chromosome structure in transcriptionally silent Xenopus egg extracts. This research was published in eLife. The transcription factor Thpok is essential for development of CD4+ T cells, but the mechanisms by which it do so are not well defined. Working with of Dr. Remy Bosselut, Laboratory of Immune Cell Biology, mass spectrometry was used to analyze the proteins that interact with Thpok in primary CD4+ T cells. These experiments identified components of the nucleosome remodeling and deacetylase complex (NuRD). Further investigation revealed three residues of Thpok that are required for interaction with NuRD and subsequent repression of CD8+ lineage genes including Runx3. These results were published in Science Immunology. With the lab of Dr. Michael Bustin, Basic Research Laboratory, mass spectrometry was used to investigate whether HMGN proteins regulate higher order chromatin status. ChIP-MS experiments were performed to identify HMGN1 and HMGN2 interactors on chromatin. Among proteins consistently observed and enriched above background in the analysis were proteins related to epigenetic regulation and histone modification, transcriptional regulation, chromatin remodeling, and DNA damage repair. Hi-C, Promoter Capture Hi-C, and ChIP-seq experiments revealed that HMGN proteins occupy genomic regions involved in cell-type-specific long-range promoter-enhancer interactions but do not cause structural changes in higher order chromatin. These findings were published in Epigenetics & Chromatin. 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.