Human cell lines pulldown interactions CL-MSMS

Using a combination of GFP-trap affinity purification, native gel band identification and APEX2-mediated BioID pulse-chase analysis, we investigate an import pathway and folding mechanism for histones H3 and H4. We investigated proteins associating with H3 and H4 bearing mutations along alpha-helix 2 using affinity-capture followed by on-beads trypsin digestion and label-free mass spectrometry. In addition, we investigated the composition subcomplexes of affinity-purified histone chaperone NASP separated by native-PAGE, followed by in-gel trypsin digestion and label-free mass spectrometry. Finally, we investigated the proximity in vivo to histone H3 construct under a pulse-chase system (RAPID-release) using two different time points for APEX2-based biotin-phenol biotinylation, 0 minutes and 120 minutes. Samples were quenched and streptavidin-purified, followed by on-beads digestion and label-free mass spectrometry.