ABSTRACT: MLF2-APEX2 fusion-based proximity labeling of nuclear envelope bleb composition. APEX reactions were conducted in WT and TorsinKO HeLa cells.
Project description:Datasets generated using WT or TorsinKO HeLa cells expressing APEX2-MLF2-HA or d133 ORF10 from the KSHV. Data were used to generate Table S1.
Project description:Proximity labeling approach to identify protein inside nuclear envelope blebs arising in Torsin-deficient HeLa cells. WT and TorsinKO cells were engineered to express MLF2-APEX2 fusion. APEX reaction was carried out via 1 mM H2O2 for 1 minute in the presence of biotin phenol. Biotinylated protein were captured via streptavidin beads. Each APEX reaction was accompanied by an untreated (no H2O2) control.
Project description:The truncated d133-ORF10-FLAG from KSHV was expressed and immunoprecipitated from WT and TorsinKO HeLa cells. The datasets were compared to an untransfected control. As d133-ORF10 localizes to blebs in TorsinKO cells, this approach allows probing of bleb protein contents.
Project description:HSPA1A was immunoprecipitated from WT or TorsinKO HeLa cells. A band around 27 kDa was uniquely found to co-immunoprecipitate with HSPA1A in TorsinKO cells. To identify this band, it was extracted and subjected to mass spectrometry.
Project description:HSPA1A was immunoprecipitated from WT or TorsinKO HeLa cells. A band around 27 kDa was uniquely found to co-immunoprecipitate with HSPA1A in TorsinKO cells. To identify this band, it was extracted and subjected to mass spectrometry.
Project description:MLF2 was purified from mammalian Expi293 cells as a fusion with MBP. As MLF2 associates tightly with HSP70/HSPA1A, this resulted in a stoichiometric co-purification with HSP70 members. To identify which ones, we subjected the whole purification or a gel-extracted band around 70 kDa to mass spectrometry.
Project description:Using APEX2, an engineered peroxidase, as an imaging tag, we successfully located iPSC-derived cardiomyocytes (iPSC-CMs) engrafted in post-myocardial infarction mouse hearts for more than 6 months. APEX2 caused clear contrast in X-ray microscopy and electron microscopy, and relatively well organized sarcomere structures were formed in iPSC-CMs. Electron microscopic tomography further found the development of T-tubules and dyads in APEX2-labelled cells, which are the signature characteristics of the maturation of cardiomyocytes.
Project description:MLF2 was purified from mammalian Expi293 cells as a fusion with MBP. As MLF2 associates tightly with HSP70/HSPA1A, this resulted in a stoichiometric co-purification with HSP70 members. To identify which ones, we subjected the whole purification or a gel-extracted band around 70 kDa to mass spectrometry.
Project description:Proximity labeling has emerged as a prominent, reliable tool for obtaining local proteomes from a wide range of cell-types. Two major classes of labeling reagents, peroxidase based (APEX family), or biotin-ligase based (BioID family) have been developed in parallel. These two approaches are often used interchangeably, or chosen based on availability of reagents, however each may produce a biased proteome which should be considered during experimental design. We compared proximity labeling with TurboID or APEX2 in HEK293 cells across cytosol, nucleus, and membrane compartments. Both enzymes enriched compartment-specific proteomes, validated by GO terms, but showed distinct protein profiles. TurboID identified more membrane proteins, favoring identification of proteins associated with RNA processing and protein localization, while APEX2 enriched for proteins involved in metabolic pathways. Trypsin digestion highlighted biases from TurboID’s lysine biotinylation, which we show can be mitigated by an endoproteinase GluC digestion during sample prep, yet these differences persist to some degree. We find that TurboID suits broader proteomic studies whereas APEX2 targets specific signaling pathways. We therefore show that strategic enzyme and protease selection is critical for optimizing proximity labeling-based proteomic studies, advancing cellular proteome mapping.