Journal: bioRxiv

Article Title: An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS

doi: 10.1101/2024.06.11.596744

Figure Lengend Snippet: A) Sequence alignment of the C-terminal regions, beginning with the most C-terminal arginine residue, of selected variants of human histone H2A (top) and H2B (bottom). Canonical ubiquitination sites H2AK119ub and H2BK120ub are indicated. B) Schematic of spike-in experiment. Synthetic, isotope-labeled peptides were spiked into histone extracts before or after trypsin digestion and then analyzed by LC-MS/MS. *, heavy amino acid; [gg], diglycine remnant from ubiquitin; /, observed tryptic cleavage site. C) Extracted ion chromatograms of labeled synthetic peptides (blue lines) bearing H2AK118ub (bottom) or H2AK119ub (top) and their unlabeled endogenous forms (red lines) with spike-in occurring before or after digestion. The synthetic H2AK118ub and H2AK119ub peptides are distinguished by the presence of one (*) or two (**) isotopically labeled amino acids while the endogenous light forms of these sequences are isobaric (#). D) Extracted ion chromatograms of two synthetic peptides (blue lines) bearing H2BK120ub and their endogenous forms (red lines) with spike-in occurring before or after digestion.

Article Snippet: The antibodies used are as follows: mouse anti-V5 (CST, 80076), rabbit anti-GAPDH (CST, 5174), rabbit anti-H2A (CST, 12349), rabbit anti-H2AK119ub (CST, 8240), rabbit anti-H2BK120ub (CST, 5546), anti-mouse IgG-Alexa488 Plus (Thermo, A32723), anti-rabbit IgG-Alexa647 Plus (Thermo, A32733).

Techniques: Sequencing, Residue, Labeling, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS

doi: 10.1101/2024.06.11.596744

Figure Lengend Snippet: A, B) Mixtures containing the H2AK119ub (A) and H2BK120ub (B) synthetic peptides were subjected to trypsin digestion with or without subsequent propionylation and then analyzed by LC-MS/MS. Extracted ion chromatograms are presented below each expected product sequence for the digestion with or without propionylation. *, heavy amino acid; [gg], diglycine remnant from ubiquitin; pr, propionyl group.

Article Snippet: The antibodies used are as follows: mouse anti-V5 (CST, 80076), rabbit anti-GAPDH (CST, 5174), rabbit anti-H2A (CST, 12349), rabbit anti-H2AK119ub (CST, 8240), rabbit anti-H2BK120ub (CST, 5546), anti-mouse IgG-Alexa488 Plus (Thermo, A32723), anti-rabbit IgG-Alexa647 Plus (Thermo, A32733).

Techniques: Liquid Chromatography with Mass Spectroscopy, Sequencing

Journal: bioRxiv

Article Title: An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS

doi: 10.1101/2024.06.11.596744

Figure Lengend Snippet: A) Histone extracts were digested with or without subsequent propionylation. Extracted ion chromatograms are shown for the derivatized H2AK119ub peptide from the derivatized and underivatized conditions. The HCD IT MS/MS fragmentation spectrum is shown at right using the IPSA tool . [gg], diglycine remnant from ubiquitin; pr, propionyl group. B) As in A except for the derivatized and underivatized H2BK120ub peptide.

Article Snippet: The antibodies used are as follows: mouse anti-V5 (CST, 80076), rabbit anti-GAPDH (CST, 5174), rabbit anti-H2A (CST, 12349), rabbit anti-H2AK119ub (CST, 8240), rabbit anti-H2BK120ub (CST, 5546), anti-mouse IgG-Alexa488 Plus (Thermo, A32723), anti-rabbit IgG-Alexa647 Plus (Thermo, A32733).

Techniques: Tandem Mass Spectroscopy

Journal: bioRxiv

Article Title: An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS

doi: 10.1101/2024.06.11.596744

Figure Lengend Snippet: A) Schematic of workflow for relative quantification of H2AK119ub and H2BK120ub by encoding samples and reference pools with either heavy (Pr-D5) or light (Pr-D0) propionic anhydride. Hypothetical mass spectra representing the precursor ion pairs of the H2AK119ub and H2BK120ub peptides (lower left) and the unmodified peptides from H2A and H2B (lower right) used for normalization are also shown. In this example, the sample appears in the light channel while the reference pool appears in the heavy channel. Based on the sample/pool ratio of the unmodified peptide, the normalized abundance of the ub-modified peptide is 0.5. B, C) Histone digests derivatized with either heavy or light propionic anhydride were combined in a 1:1 mixture and analyzed by PRM-based LC-MS/MS. Extracted ion chromatograms (left) and intact mass spectra (center) for the precursor ion pairs (light in red, heavy in blue) representing H2AK119ub (B) and H2BK120ub (C) are shown. Extracted ion chromatograms for selected fragments from the heavy and light precursors are also presented (right).

Article Snippet: The antibodies used are as follows: mouse anti-V5 (CST, 80076), rabbit anti-GAPDH (CST, 5174), rabbit anti-H2A (CST, 12349), rabbit anti-H2AK119ub (CST, 8240), rabbit anti-H2BK120ub (CST, 5546), anti-mouse IgG-Alexa488 Plus (Thermo, A32723), anti-rabbit IgG-Alexa647 Plus (Thermo, A32733).

Techniques: Modification, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS

doi: 10.1101/2024.06.11.596744

Figure Lengend Snippet: A) Schematic of experiment in which the H2BK120ub synthetic peptide was mixed with the total H2B synthetic peptide at two-fold serially increasing molar ratios from 0.01 to 0.32 as indicated. Oppositely labeled samples and reference pools were combined for each of the two reciprocal labeling schemes and analyzed by PRM-based LC-MS/MS. B) The theoretical log2 change in the relative level of H2BK120ub, normalized to the 0.01 molar ratio, is plotted against the log2 of the observed sample/reference pool ratio for both the H2BK120ub peptide and the total H2B peptide. The color indicates whether the sample was labeled with heavy (H, red) or light (L, blue) propionic anhydride with the reference pool receiving the opposite isotopic label. C) The theoretical log2 change in the relative level of H2BK120ub is plotted against the observed log2 of the relative change after normalization to the total H2B peptide. As in B, the color indicates the isotope used for sample labeling. The line of best fit (solid, y = 1.134x + 0.089, R 2 = 0.994) after linear regression analysis is drawn against the expected trend (dashed, y = x).

Article Snippet: The antibodies used are as follows: mouse anti-V5 (CST, 80076), rabbit anti-GAPDH (CST, 5174), rabbit anti-H2A (CST, 12349), rabbit anti-H2AK119ub (CST, 8240), rabbit anti-H2BK120ub (CST, 5546), anti-mouse IgG-Alexa488 Plus (Thermo, A32723), anti-rabbit IgG-Alexa647 Plus (Thermo, A32733).

Techniques: Labeling, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS

doi: 10.1101/2024.06.11.596744

Figure Lengend Snippet: A) Histone extracts from parental (two left panels) and sgRING1A/B 10T1/2 cells (two right panels) were digested and derivatized for relative quantification of H2AK119ub and H2BK120ub. Extracted ion chromatograms, in which the light channel (L, red) encodes the reference pool and the heavy channel (H, blue) encodes individual samples, are shown for the paired precursor ions for the total H2A peptide and the H2AK119ub peptide. B) Relative quantification of H2AK119ub and H2BK120ub in RING1A/B-deficient cells and in response to inhibitors, including actinomycin D, panobinostat, Ei1, mitomycin C, and etoposide. 293T cells were treated with the indicated doses of inhibitors for 24 hrs.

Article Snippet: The antibodies used are as follows: mouse anti-V5 (CST, 80076), rabbit anti-GAPDH (CST, 5174), rabbit anti-H2A (CST, 12349), rabbit anti-H2AK119ub (CST, 8240), rabbit anti-H2BK120ub (CST, 5546), anti-mouse IgG-Alexa488 Plus (Thermo, A32723), anti-rabbit IgG-Alexa647 Plus (Thermo, A32733).

Techniques:

Journal: bioRxiv

Article Title: An optimized and robust workflow for quantifying the canonical histone ubiquitination marks H2AK119ub and H2BK120ub by LC-MS/MS

doi: 10.1101/2024.06.11.596744

Figure Lengend Snippet:

Article Snippet: The antibodies used are as follows: mouse anti-V5 (CST, 80076), rabbit anti-GAPDH (CST, 5174), rabbit anti-H2A (CST, 12349), rabbit anti-H2AK119ub (CST, 8240), rabbit anti-H2BK120ub (CST, 5546), anti-mouse IgG-Alexa488 Plus (Thermo, A32723), anti-rabbit IgG-Alexa647 Plus (Thermo, A32733).

Techniques:

Journal: Journal of Fungi

Article Title: SAGA Complex Subunit Hfi1 Is Important in the Stress Response and Pathogenesis of Cryptococcus neoformans

doi: 10.3390/jof9121198

Figure Lengend Snippet: Hfi1 influences histone deubiquitination and acetylation in C. neoformans . ( A ) Ubiquitination of histone 2B was detected using anti-H2BK120ub antibody. ( B ) Acetylation of histone H3 was detected using anti-H3K9ac, -H3K14ac, and -H3K18ac antibodies. ( C ) Acetylation of histone H4 and methylation of H3 was detected using anti-H4K8ac, -H4K12ac, -H4K16ac, and -H3K4trimethyl antibodies. Arrows indicate the 17 kDa (black arrow) and 26 kDa (white arrow) bands in the Broad Range Color Prestained Protein Standard (NEB). ( D ) Summary of Western blot results.

Article Snippet: For detecting histone modification, a 1:5000 dilution of anti-H2B (#12364), anti-H2BK120ub (#5546), anti-H3 (#4499), anti-H3K9ac (#9649), anti-H3K14ac (#7627), anti-H3K18ac (#13998), anti-H4K8 (#2594), anti-H4K12 (#13944), anti-H4K16 (#13534), or anti-H3K4trimethl (#9751) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) was used.

Techniques: Methylation, Western Blot

Journal: Journal of Virology

Article Title: KSHV RTA utilizes the host E3 ubiquitin ligase complex RNF20/40 to drive lytic reactivation

doi: 10.1128/jvi.01389-23

Figure Lengend Snippet: RNF20 regulates RNA polymerase II occupancy on RTA promoter during lytic reactivation of KSHV. (A–D) iBCBL1-3xFLAG-RTA cells were transduced with lenti-shControl or lenti-shRNF20 then reactivated for 12 h, and ChIP-qPCR analysis was performed at the indicated sites of the RTA promoter and gene body. (A) H2BK120ub ChIP. (B) H3K4me3 ChIP. (C) RNA polymerase II (RNAP II) ChIP. (D) ChIP for RNAP II Serine 2 phosphorylation. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 (n = 3).

Article Snippet: To induce KSHV lytic reactivation in 293TBAC16 cells, we transfected the cells with a plasmid expressing 3xFLAG-RTA. . Antibodies The following primary antibodies were used in the study: anti-RTA (gift from Yoshihiro Izumiya, UC Davis), anti-ORF6 (gift from Gary S. Hayward, Johns Hopkins University), anti-ORF45 (Santa Cruz sc-53883), anti-K8.1 (Santa Cruz sc-65446), anti-RNF20 (Cell Signaling 11974S), anti-RNF40 (Cell Signaling 12187S), anti-FLAG (Sigma F1804), anti-HA (BioLegend 901501), anti-V5 (Abcam ab27671), anti-tubulin (Sigma T5326), anti-H2B (Active Motif 39210), anti-H2BK120ub (Cell Signaling 5546S), anti-H3 (Abcam ab1791), anti-H3K4me3 (Active Motif 39159), anti-RNA polymerase II CTD (Cell Signaling 2629), and anti-RNA polymerase II CTD phospho Ser2 (Abcam ab5095). . Plasmids, DNA transfection, and luciferase assays We used pCDH-CMV-MCS-EF1-puro vector to express the different epitope-tagged full-length and mutant versions of RTA, RNF20, and RNF40 in cells.

Techniques: Transduction

Journal: Journal of Virology

Article Title: KSHV RTA utilizes the host E3 ubiquitin ligase complex RNF20/40 to drive lytic reactivation

doi: 10.1128/jvi.01389-23

Figure Lengend Snippet: RNF20 regulates RNA polymerase II occupancy on RTA promoter during lytic reactivation of KSHV. (A–D) iBCBL1-3xFLAG-RTA cells were transduced with lenti-shControl or lenti-shRNF20 then reactivated for 12 h, and ChIP-qPCR analysis was performed at the indicated sites of the RTA promoter and gene body. (A) H2BK120ub ChIP. (B) H3K4me3 ChIP. (C) RNA polymerase II (RNAP II) ChIP. (D) ChIP for RNAP II Serine 2 phosphorylation. The degree of statistical significance calculated by t-tests is indicated as *P ≤ 0.05 (n = 3).

Article Snippet: To induce KSHV lytic reactivation in 293TBAC16 cells, we transfected the cells with a plasmid expressing 3xFLAG-RTA. . Antibodies The following primary antibodies were used in the study: anti-RTA (gift from Yoshihiro Izumiya, UC Davis), anti-ORF6 (gift from Gary S. Hayward, Johns Hopkins University), anti-ORF45 (Santa Cruz sc-53883), anti-K8.1 (Santa Cruz sc-65446), anti-RNF20 (Cell Signaling 11974S), anti-RNF40 (Cell Signaling 12187S), anti-FLAG (Sigma F1804), anti-HA (BioLegend 901501), anti-V5 (Abcam ab27671), anti-tubulin (Sigma T5326), anti-H2B (Active Motif 39210), anti-H2BK120ub (Cell Signaling 5546S), anti-H3 (Abcam ab1791), anti-H3K4me3 (Active Motif 39159), anti-RNA polymerase II CTD (Cell Signaling 2629), and anti-RNA polymerase II CTD phospho Ser2 (Abcam ab5095). . Plasmids, DNA transfection, and luciferase assays We used pCDH-CMV-MCS-EF1-puro vector to express the different epitope-tagged full-length and mutant versions of RTA, RNF20, and RNF40 in cells.

Techniques: Transduction