Session TOB. There are 3 abstracts in this session.

Session: Protein Interactions and Signaling, time: 09:50 - 10:15 am

Spatial proteomics and transcriptomics with promiscuous labeling enzymes

Alice Ting
Stanford University, Stanford, CA


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Session: Protein Interactions and Signaling, time: 10:40 - 10:55 am

Biochemical Reduction of the Topology of the Diverse WDR76 Interactome

Dayebgadoh Gerald1; Mihaela E. Sardiu1; Laurence Florens1; Michael P. Washburn1, 2
1Stowers Institute for Med Res, Kansas City, ; 2The University of Kansas Medical Center, Kansas City, Kansas

WD40 repeat proteins form a diverse protein family constituting about 1% of the protein coding genome with functions ranging from DNA damage repair, cell cycle progression, apoptosis, and autophagy. Thus, characterizing novel WD40 repeat proteins is of great interest. Here we rigorously carry out a series of Affinity‚Äźpurification coupled to mass spectrometry (AP-MS) to reduce the topology of the WDR76 interactome using different biochemical conditions. Our high confidence results uncovered macromolecular diversity and the stability of WDR76 interactions. Our data links human WDR76 to multiple biological processes: protein folding, gene silencing, DNA damage, mitotic cell cycle, and metabolism. For example, we connect WDR76 to GAN, HELLS, and SIRT1 and show that these interactions are outside of the WD40 repeat domain of WDR76. Taken together, GAN, HELLS, and SIRT1 are possible hubs that confer WDR76 its biological specificity.

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Session: Protein Interactions and Signaling, time: 10:55 - 11:10 am

An antibody-free method for identification of protein complexes using RNA aptamers

Angela Kruse1; Judhajeet Ray1; Abdullah Ozer1; Richard Johnson2; Michael MacCoss2; John Lis1; Michelle Heck1, 3
1Cornell University, Ithaca, NY; 2University of Washington, Seattle, WA; 3USDA ARS, Ithaca, NY

Affinity purification mass spectrometry (AP-MS) has revolutionized the study of protein interactions in vivo. A major challenge in AP-MS experiments is the high abundance of peptides derived from the antibodies used for protein complex isolation. We present a method to use RNA aptamers in place of antibodies to pull down target proteins and protein complexes from living cells. RNA aptamers are short oligonucleotides developed using an in vitro process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX), and have affinity for proteins, small molecules, and even carbohydrates that is comparable to antibodies. Aptamers are small and easily penetrate animal tissues. To test the hypothesis that RNA aptamers could be used in protein complex isolation, we performed five replicate isolations of two different protein complex isolations from human cells: heat shock factor-1 (HSF1) fused to GFP and negative elongation factor E (NELF E) using GFP and NELF E aptamers, respectively. Protein complex isolations were analyzed using 1-D gels and high resolution mass spectrometry. Gel analysis revealed clean and reproducible banding patterns enriched for the target proteins. Mascot and Scaffold were used for protein identification and spectral counting. Significance Analysis of Interactomes (SAINT) was used to identify members of these protein complexes. The use of multiple aptamers allowed us to make a database of common contaminant RNA binding proteins useful for aptamer pulldown experiments. We confidently identified many known HSF1-interacting proteins and all of the known NELF E-interacting proteins using this method. New proteins assigned to these protein complexes may represent novel discoveries in the molecular regulation of these proteins. This method will improve the development and testing of RNA aptamers for medical and industrial applications, and offers an alternative to antibody-based pulldown methods that improves the identification of low-abundance binding partners in protein complexes without the need for antibody development.

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