In the last year, the mass spectrometry facility added a new mass spectrometer for use - a nanospray Orbitrap Fusion (Thermo) tribrid mass spectrometer. This instrument combines a quadropole mass filter with ion trap and Orbitrap mass analyzers, which allows the two detectors to operate in parallel, improving both sensitivity and selectivity. In addition to CID and HCD methods for peptide fragmentation, the Orbitrap Fusion is configured with an ETD source that facilitates analysis of larger peptides and post-translational modifications, particularly phosphorylation and glycosylation. Overall, the expertise of the mass spectrometry facility is being widely used to further the research of multiple groups within the CCR. In FY2014, the mass spectrometry facility within CPTR collaborated in 35 different projects, with more than 1100 samples processed and analyzed. These projects are being performed in collaboration with 23 different CCR investigators, including 4 tenure-track investigators. These projects include several to identify sites of post-translational modification, including phosphorylation, ubiquitination, acetylation, and methylation, to better understand signal transduction and protein regulation. The facility is also being used to identify protein interactors, including identification of those that change following post-translational modification. These studies will further research into protein function and regulation. Finally, mass spectrometry is being used extensively for large-scale quantitative proteomics projects. In these, labeled or label-free methods are used to comprehensively identify the proteome of a vesicle, fluid, or protein interaction network. These discovery-oriented studies, which are sample- and time-intensive, provide broad information for defining new hypotheses and provide new insight into global protein activities and cellular responses. In the past year, two studies incorporating results obtained in the mass spectrometry facility have been published. In addition, several other projects are nearing completion and will be prepared as manuscripts for publication. The first study, published in the Journal of Biological Chemistry, is a collaboration with Dr. Michael Gottesmann in the Laboratory of Cell Biology. Here, mass spectrometry was used as one technique to investigate the mechanism by which tiopronin elicits collateral sensitivity in multidrug-resistant (MDR) cells. Collateral sensitivity is the selective targeting of MDR cells, while the sensitive parental cells are unaffected. Previously, the group had observed that some MDR cell lines showed collateral sensitivity towards tiopronin, a thiol-substituted N-propanoyl form of glycine and an Food and Drug Administration-registered orphan drug used for over 30 years to treat a diverse range of pathophysiological conditions. Although tiopronin showed MDR-selective activity against some P-glycoprotein (P-gp) and multidrug resistance protein 1 (MRP1)-expressing cells, it did not kill all P-gp-expressing cell lines tested, and inhibition of P-gp did not abrogate selective killing. Rather, using mass spectrometry, we showed that tiopronin inhibits glutathione peroxidase (Gpx) by reacting with the selenocysteine residue in the active site, which is followed by intramolecular transfer to a proximal lysine residue, resulting in its covalent modification. This inhibition of Gpx resulted in increased production of reactive oxygen species (ROS), leading to cell death. The second study is the result of a collaboration with Dr. Curtis Harris, Laboratory of Human Carcinogenesis, to study the biological regulation of the delta133 isoform of p53. This isoform lacks the N-terminal transactivation domain and acts as a dominant negative inhibitor of full-length p53. Previously, we had observed that endogenous delta133p53 protein was down-regulated during replicative senescence, but not upon oncogene-induced senescence, in human fibroblasts;this down-regulation was not due to changes in mRNA level. Since delta133p53 was not degraded by the proteasome, we investigated whether it could be degraded by autophagic mechanisms. Using cell-based methods, we demonstrated that indeed delta133p53 was degraded by selective autophagy, and mass spectrometry experiments identified two ubiquitinated lysine residues in the C-terminal regulatory domain that were responsible for this effect. Further, mass spectrometric identification of interacting proteins indicated that delta133p53 forms a complex with the Hsp70 chaperone complex and the STUB1 E3 ubiquitin ligase. Contrary to initial hypotheses, the interaction with the Hsp70 complex and STUB1 actually protects delta133p53 from degradation, rather than promoting degradation. When STUB1 was knocked down, the delta133p53 level was decreased and p53-dependent senescence increased. Further studies to identify the ubiquitin ligase and mechanism for delta133p53 autophagic degradation are ongoing. This work has been accepted for publication in Nature Communications.

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Kriebel, Paul W; Majumdar, Ritankar; Jenkins, Lisa M et al. (2018) Extracellular vesicles direct migration by synthesizing and releasing chemotactic signals. J Cell Biol 217:2891-2910
Murai, Junko; Tang, Sai-Wen; Leo, Elisabetta et al. (2018) SLFN11 Blocks Stressed Replication Forks Independently of ATR. Mol Cell 69:371-384.e6
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