nm parp14  (BPS Bioscience)

Journal: Journal of Proteome Research

Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

doi: 10.1021/acs.jproteome.8b00895

Figure Lengend Snippet: Gas phase segmentation (GPS) improves ADPr peptide detection. (A) A screenshot of the ADP-ribose product ion triggered EThcD and HCD data acquisition method on the Orbitrap Fusion Lumos. (B) Area under the curve (AUC) of the four ADPr fragment ions (136.06, 250.09, 348.07, and 428.04 m / z ) dissociated from an ARTD8/PARP14-MARylated STAT1 peptide using HCD. (C) Pilot Af1521 enrichment study to determine the optimal collision energy for ADP-ribose product ion screening and ADPr peptide identification. Only high confidence (HCD and EThcD combined peptides) were used in this plot (more details in Figure ). (D) A schematic showing the principle of GPS using multiple injections. (E) The extracted ion chromatograms of the ADPr fragment peak (348.07 m / z ) in each full scan and GPS injection. (F,G) Precursor ion m / z and retention time of triggered EThcD spectra in full scan and combined GPS scans.

Article Snippet: ARTD8/PARP14 recombinant human protein (amino acids 1470–1801, catalytic domain only; 4.0 μg, BPS Bioscience, San Diego, CA, Cat# 80514) alone, and 1.0 μg of ARTD8/PARP14 with signal transducer and activator of transcription 1 (STAT1) recombinant human protein (4.0 μg, Thermo Fisher Scientific, Waltham, MA, Cat# PHF0011) were incubated in a reaction buffer [50 μM β-nicotinamide adenine dinucleotide (β-NAD, Sigma-Aldrich, Cat# N0632), 50 mM Tris-HCl pH 7.4 (Boston Bio Products, Ashland, MA, Cat# BM-314)] for 1 h at room temperature.

Techniques: Injection

Journal: Journal of Proteome Research

Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

doi: 10.1021/acs.jproteome.8b00895

Figure Lengend Snippet: GPS leads to the identification of ADP-ribosylated ARTDs/PARPs other than ARTD1/PARP1. (A) A comparison using Venn diagrams for ADPr peptides found in two replicates for full scan (400–1500 m / z ) and combined 4× GPS scans (GPS-1, 400–605; GPS-2, 595–805; GPS-3, 795–1005; GPS-4, 995–1200 m / z ). (B) A comparison of ADPr peptides found in control and IFN-γ-treated THP-1 cells for the full scan and combined 4× GPS scans. (C) Sequence motif analysis for ADPr acceptor amino acids (N, number of ADPr peptides used for the analysis). (D) A plot of the number of ADP-ribosylation sites per protein. (E) Comparison of ADPr peptide abundances between control and IFN-γ in each replicate; regression lines, 95% confidence interval, and standard error of estimate (SEE) are provided (red dots are outliers). (F) MS/MS spectra of an ARTD8/PARP14 ADPr peptide using PRM acquisitions. Black peaks were manually annotated. *, ADPr site. (G) A comparison of the number of proteins identified in the Af1521 elution (ADPr proteins) and input samples (backbone proteins) per replicate. (H) A comparison of the relative changes to ADPr peptides versus their backbone proteins in response to IFN-γ (IFN-γ/control).

Article Snippet: ARTD8/PARP14 recombinant human protein (amino acids 1470–1801, catalytic domain only; 4.0 μg, BPS Bioscience, San Diego, CA, Cat# 80514) alone, and 1.0 μg of ARTD8/PARP14 with signal transducer and activator of transcription 1 (STAT1) recombinant human protein (4.0 μg, Thermo Fisher Scientific, Waltham, MA, Cat# PHF0011) were incubated in a reaction buffer [50 μM β-nicotinamide adenine dinucleotide (β-NAD, Sigma-Aldrich, Cat# N0632), 50 mM Tris-HCl pH 7.4 (Boston Bio Products, Ashland, MA, Cat# BM-314)] for 1 h at room temperature.

Techniques: Sequencing, Tandem Mass Spectroscopy

Journal: Journal of Proteome Research

Article Title: A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation

doi: 10.1021/acs.jproteome.8b00895

Figure Lengend Snippet: IFN-γ increased ARTD8/PARP14 and ARTD9/PARP9 ADP-ribosylation. (A) A pan ADP-ribose Western blot analysis of control and IFN-γ-treated THP-1 cells after 10H IP or incubation with IgG. (B) A comparison of enriched proteins using a Venn diagram between 10H and IgG. The plot showing the log 2 (abundance ratio (10H/IgG)) and −log( p -value) of 551 shared proteins (Venn diagram). 114 proteins passed the threshold of abundance ratio (10H/IgG) > 10-fold and p -value < 0.05. (C) Abundance ratio (IFN-γ/control) of 10H-enriched proteins, ARTD9/PARP9, DNAJC13, and ITGB5, were unique to IFN-γ and filled with maximum. (D) Western blot analysis of Af1521- and 10H antibody-enriched ARTD/PARP enzymes from control, IFN-γ-treated, and PJ34 plus IFN-γ-treated THP-1 cells. (E) Comparing ADP-ribosylome from Af1521 (ADPr proteins) and 10H (backbone proteins) data set and showing the list of 39 common proteins between the two data sets.

Article Snippet: ARTD8/PARP14 recombinant human protein (amino acids 1470–1801, catalytic domain only; 4.0 μg, BPS Bioscience, San Diego, CA, Cat# 80514) alone, and 1.0 μg of ARTD8/PARP14 with signal transducer and activator of transcription 1 (STAT1) recombinant human protein (4.0 μg, Thermo Fisher Scientific, Waltham, MA, Cat# PHF0011) were incubated in a reaction buffer [50 μM β-nicotinamide adenine dinucleotide (β-NAD, Sigma-Aldrich, Cat# N0632), 50 mM Tris-HCl pH 7.4 (Boston Bio Products, Ashland, MA, Cat# BM-314)] for 1 h at room temperature.

Techniques: Western Blot, Incubation