Journal: Science Advances

Article Title: Sorbate induces lysine sorbylation through noncanonical activities of class I HDACs to regulate the expression of inflammation genes

doi: 10.1126/sciadv.adv1071

Figure Lengend Snippet: ( A ) Illustrations for the chemical structures of sorbic acid and the formation of protein Ksor. ( B ) Extracted ion high-performance liquid chromatography (HPLC) chromatograms and MS spectra for the validation of Ksor with HPLC co-elution analysis. Histone H4 K12 sorbylation peptide “GLGK(So)GGAK(Ac)R” was used as an example (So, Ksor; Ac, lysine acetylation). “-So” in MS/MS spectra indicates the neutral loss signal of Ksor. Histones were extracted for HCT116 cells treated with 10 mM of sorbate for 24 hours and subject to SDS–polyacrylamide gel electrophoresis (PAGE) and in-gel digestion by trypsin. Diluted synthetic peptide, endogenous histone peptides from in-gel digestion, and their equal mixture were analyzed with the same HPLC gradient to compare chromatography retention time (left column), MS2 fragmentation (middle column), and peptide MS1 mass/charge ratio ( m / z ) = 490.2804 to 490.2952 (right column). ( C ) Validation of Ksor with isotopic labeling in HCT116 cells. HCT116 cells were treated with 2 mM 13 C 2 -sorbate for 24 hours, and histones were extracted for in-gel tryptic digestion. Peptides were analyzed by LC-MS and compared with Ksor peptides from sorbate-treated HCT116 cells. ( D ) Illustration of representative Ksor sites identified on core histones from the LC-MS analysis of histones extracted from sorbate-treated 293T cells (blue), Raw264.7 cells (orange), and mouse liver tissue (green).

Article Snippet: Briefly, digested peptides dissolved in HPLC buffer A [0.1% formic acid in water (v/v)] were separated on an in-house packed capillary HPLC column (length of 20 cm and inner diameter of 75 μm) from CoAnn Technologies (Richland, WA) with Luna C18 beads (particle size of 5 μm and pores of 100 Å) from Phenomenex (Torrance, CA).

Techniques: High Performance Liquid Chromatography, Biomarker Discovery, Co-Elution Assay, Tandem Mass Spectroscopy, Polyacrylamide Gel Electrophoresis, Chromatography, Isotopic Labeling, Liquid Chromatography with Mass Spectroscopy