April 2021 Issue
Mapping Isoform Abundance and Interactome of the Endogenous TMPRSS2-ERG Fusion Protein by Orthogonal Immunoprecipitation–Mass Spectrometry AssaysIn Brief Orthogonal immunoprecipitation-mass spectrometry assays quantified TMPRSS2-ERG fusion protein (∼27,000 copies/cell) and its four distinct isoforms, and revealed that T1E4-ERG isoform accounted for 52 ± 3% of the total ERG in VCaP cells and 50 ± 11% in FFPE prostate cancer tissues. Methionine-truncated and N-acetylated peptide TASSSSDYGQTSK unique for T1/E4 TMPRSS2-ERG fusion was identified. Unlike the N-terminal antibodies, C-terminal antibodies identified 29 ERG-interacting proteins, including mutually exclusive BRG1- and BRM-associated canonical SWI/SNF chromatin remodeling complexes. Clinical perspectives of assays were discussed.
Stable Isotope Labeling of Amino Acids in Flies (SILAF) Reveals Differential Phosphorylation of Mitochondrial Proteins Upon Loss of OXPHOS SubunitsIn Brief We advanced a fully composed food source for Drosophila melanogaster into a highly efficient, versatile, and cheap labeling method, termed SILAF. The larval proteome incorporates more than 99% heavy lysine-6 label within 6 days, while the adult fly metabolism allows protein turnover studies. We obtained a phosphoproteome with site-specific occupancy in a fly model of mitochondrial metabolic disease, highlighting the regulation of two novel conserved phosphosites on subunits of the electron transport chain.