Journal: Nature chemical biology

Article Title: Functional Elucidation of TfuA in Peptide Backbone Thioamidation

doi: 10.1038/s41589-021-00771-0

Figure Lengend Snippet: ( a ) Top , MS spectrum of Mt ThiS ( m/z 8,001 Da). Bottom , MS spectrum of Mt ThiS-COSH (thiocarboxylated C-terminus, m/z 8,017 Da). ( b ) The same sample as above was digested with endoproteinase GluC. Shown is the spectral window surrounding the C-terminal peptide: VIRVIYGG.

Article Snippet: For the hydrolysis reaction, 5 μM of Mt TfuA (1 μL) was incubated with 45 μM Mt ThiS-COSH in [ 18 O]-H 2 O (9 μL) at room temperature for 1 h. The reaction was stopped by the addition of endoproteinase GluC (NEB) at the final concentration of 5 ng/μL and incubation at 37 °C for 16 h. The resulting fragment of Mt ThiS-COSH was analyzed by MALDI-TOF-MS (desalted using a ZipTip and eluted into 70% aq.

Techniques:

Journal: PLoS ONE

Article Title: Sequestering HMGB1 via DNA-Conjugated Beads Ameliorates Murine Colitis

doi: 10.1371/journal.pone.0103992

Figure Lengend Snippet: A) Schematic representation of fully reduced, disulfide and sulfonyl forms of HMGB1 and their biological functions. B) DNA beads bind to disulfide HMGB1 in feces from colitis mice. The redox state of HMGB1 captured by DNA beads B1 or B2 from feces of colitis mice was assessed by trypsin digestion followed by LC/MS analysis. Cell lysate from colorectal epithelial (Caco 2) cells was used as control for intracellular HMGB1. Diagnostic peptides showed with the presence of molecular weights 1496 and 550 (Da) indicates reduced cysteines 23 and 45 respectively, whereas the appearance of molecular weights of 1564 and 618 (Da) indicates forming of an intramolecular disulfide linkage between cysteines 23 and 45 (arrows). Cys 106 (MW = 2002) is in reduced form in all samples presented here. Data are representative from two separate experiments. C) DNA beads bind to hyper-acetylated form of HMGB1 in feces from colitis mice. Representative spetra of the LC-MS traces of the above samples digested with endopeptidase GluC. The presence of peptides with molecular weights 1624 and 1132 Da (arrows) indicates the hypo-acetylation of lysine residues in NLS1 and NLS2 regions respectively; whereas the presence of peptides of 1749 and 1342 Da (arrows) indicates hyper-acetylation of lysines within NLS1 and NLS2 regions. Data are representative from two separate experiments. D) B2 beads bind to redox modified HMGB1 proteins. Increasing amounts of various HMGB1 proteins (100 or 250 ng) were added to B2 beads (20 µl) and the mixture (50 µl total volume) was incubated at room temperature for two hours. The mixture was then centrifuged and HMGB1 bound to beads were revealed by western blotting with anti-HMGB1 antibodies. N = 3 experiments. E) B2 beads do not bind to TNF. To examine the specificity of DNA beads and HMGB1 binding, human TNF (200 ng) was incubated with increasing amounts of B2 beads (as indicated) at room temperature for two hours. After incubation, TNF remaining in the supernatants and bound to the beads were analyzed by western blot probed with anti-human TNF antibodies. Data are representative from three separate experiments. F) B2 beads do not bind to human histone H3. B2, B1 or control beads (5 µl) in each tube were incubated with increasing amounts of human histone H3 (0, 0.01, 0.1, 0.2, 0.5, 1, 2, 3 µg) for two hours at room temperature. The amount of histone H3 bound to DNA beads was analyzed by western blot using anti-histone H3 antibodies. Histone H3 protein was used as a positive control in western blot. Data are representative of 3 repeats.

Article Snippet: Samples were subjected to digestion by trypsin (Promega, Madison, WI) or endopeptidase GluC (New England Bio Labs., Ipswich, MA) according to the manufacturer's instructions.

Techniques: Liquid Chromatography with Mass Spectroscopy, Diagnostic Assay, Modification, Incubation, Western Blot, Binding Assay, Positive Control