Project description:The in vivo labeling technique Split-Biotin identification (Split-BioID, De Munter et al., 2017, Schopp et al., 2017, Cho et al., 2020) was used to capture the common microenvironment of Bre5 and the ribosomal protein Rps2 (uS5). Biotinylated proteins were enriched from cell lysates using affinity purification. After SDS-PAGE, proteins were digested in-gel with trypsin and the resulting peptides were analyzed by liquid chromatography-mass spectrometry (LC-MS) for identification and relative quantification against controls. An aliquot of the cell lysate that was used for the affinity purification was taken as an input control and analyzed as well by LC-MS to account for possible differences in the input abundance of enriched proteins. Stable isotope labeling with amino acids in cell culture (SILAC) was used for relative quantification of proteins according to the 2nSILAC approach (Dannenmaier et al., 2018). To evaluate the labeling efficiency, an aliquot of each culture was harvested separately prior to the pooling of the cells for the Split-BioID experiment. Cell lysates from these samples were used for SDS-PAGE, and a part of each gel lane was used for an in-gel digest of proteins. These peptides were also analyzed with LC-MS and the percentages of SILAC-labeled peptides (based on MSMS counts) were calculated. The captured proteins belong to a common cellular microenvironment of Bre5 and Rps2. References: Cho, K.F., Branon, T.C., Rajeev, S., Svinkina, T., Udeshi, N.D., Thoudam, T., Kwak, C., Rhee, H.W., Lee, I.K., Carr, S.A., Ting, A.Y. (2020). Split-TurboID enables contact-dependent proximity labeling in cells. Proc Natl Acad Sci U S A. 117, 12143-12154. Dannenmaier, S., Stiller, S.B., Morgenstern, M., Lübbert, P., Oeljeklaus, S., Wiedemann, N., Warscheid, B. (2018). Complete Native Stable Isotope Labeling by Amino Acids of Saccharomyces cerevisiae for Global Proteomic Analysis. Anal Chem 90, 10501-10509. De Munter, S., Görnemann, J., Derua, R., Lesage, B., Qian, J., Heroes, E., Waelkens, E., Van Eynde, A., Beullens, M., Bollen, M. (2017). Split-BioID: a proximity biotinylation assay for dimerization-dependent protein interactions. FEBS Lett 591, 415-424. Schopp, I.M., Amaya Ramirez, C.C., Debeljak, J., Kreibich, E., Skribbe, M., Wild, K., Béthune, J. (2017). Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes. Nat Commun 8,15690.
2021-11-10 | PXD028879 | Pride
