Article Title: Serine ADP-ribosylation marks nucleosomes for ALC1-dependent chromatin remodeling
Figure Lengend Snippet: The recombinant PARP1:HPF1 complex installs linear poly-ADP-ribose chains at biologically relevant target sites. ( A ) Summary of LC-MS/MS analysis of enzymatic digestion products from PAR chains installed on a peptide using our technology. The spectra were produced on a Sciex QTRAP 6500+ mass spectrometer. ( B ) RP-HPLC and MS analysis of substrate peptides (H2B wild-type or S6A mutant, amino acids 1–16) and corresponding PARP1:HPF1 reaction products. RP-HPLC gradients are from 0 to 35% Solvent B (2–22 min). ( C ) RP-HPLC traces from PARG- or ARH3-treated H2B peptide ADPr reactions that were optimized for ADP-ribose chain elongation. The number of ADP-ribose units was verified by MS analysis. ( D ) Representative RP-HPLC analyses of ADPr reactions from Figure 1E containing PARP1 (1 μM), unmodified H3 substrate peptide (amino acids 1–20; 180 μM), NAD + (2 mM), and various HPF1 concentrations (indicated on corresponding trace). RP-HPLC gradients are from 0 to 35% Solvent B (2–22 min). For peak area integration values, see Supplementary Dataset.
Article Snippet: The lyophilized powder containing a mixture of the digestion products was then resuspended in water to a final concentration of 20 μg/mL and 12 μL was injected onto Phenomenex Synergi Polar-RP column (150 × 2 mm, 4 μm packing) and analyzed by LC-MS/MS using a Sciex QTRAP 6500+ mass spectrometer coupled to a Shimadzu Nexera X2 UPLC.
Techniques: Recombinant, Liquid Chromatography with Mass Spectroscopy, Produced, Mass Spectrometry, High Performance Liquid Chromatography, Mutagenesis