4-nitrophenyl-n-acetyl- beta-d-galactosaminide; pnp-beta-d-galnac  (Gold Biotechnology Inc)

Journal: Journal of the American Chemical Society

Article Title: Development of a GalNAc-Tyrosine-Specific Monoclonal Antibody and Detection of Tyrosine O ‑GalNAcylation in Numerous Human Tissues and Cell Lines

doi: 10.1021/jacs.2c04477

Figure Lengend Snippet: (A) Structures of GalNAc-Tyr and select structurally similar glycans. (B) Synthesis of the GalNAcα-Tyr hapten―CRM197 conjugate: (a) methyl 3-(4-hydroxyphenyl)propanoate, DTBMP, AgOTf, 4 Å MS, DCM/Tol. (1.5, 1 v/v) −78 °C to rt, overnight, 79%, (1:1 α/β); (b) AcSH, pyridine, (1:1 v/v). 0 °C―rt, overnight, 84%, (1:1 α/β); (c) 2 M NH3 in MeOH, rt, 2 h, Dowex H+ resin; (d) LiOH, H2O/MeOH (4:1, v/v), rt, overnight, 98% (1:1 α/β), (e) N-hydroxy-succinimide, 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride, rt, 90 min, then protein, borate buffer pH 8, rt, 90 min.

Article Snippet: A 4.91 mM solution of GalNAc α -PNP (Cayman Chemical) ligand was prepared in PBS (pH 7.4, 2 mL) and molecular-biology-grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich, 200 μ L).

Techniques:

Journal: Journal of the American Chemical Society

Article Title: Development of a GalNAc-Tyrosine-Specific Monoclonal Antibody and Detection of Tyrosine O ‑GalNAcylation in Numerous Human Tissues and Cell Lines

doi: 10.1021/jacs.2c04477

Figure Lengend Snippet: Characterization data and glycan microarray results for G10C. Evaluation of G10C purity by (A) SDS-PAGE visualized using Ponceau total protein stain and (B) SEC HPLC. (C) Bar graph illustrating selectivity of G10C on our 815-component glycan microarray. Signals are in relative fluorescence units. Data shown are at a concentration of 1 nM, which is about 10-fold higher than the apparent KD value for GalNAcα-Tyr glycopeptides. Positive and negative controls have been excluded.

Article Snippet: A 4.91 mM solution of GalNAc α -PNP (Cayman Chemical) ligand was prepared in PBS (pH 7.4, 2 mL) and molecular-biology-grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich, 200 μ L).

Techniques: Glycoproteomics, Microarray, SDS Page, Staining, Fluorescence, Concentration Assay

Journal: Journal of the American Chemical Society

Article Title: Development of a GalNAc-Tyrosine-Specific Monoclonal Antibody and Detection of Tyrosine O ‑GalNAcylation in Numerous Human Tissues and Cell Lines

doi: 10.1021/jacs.2c04477

Figure Lengend Snippet: Structural basis for the recognition of GalNAcα-Tyr by G10C. (A) Surface rendering of the pocket looking from the top down. The G10C heavy chain is in teal, and the light chain is in salmon. The N-acetyl group of GalNAc is positioned at the bottom of the pocket, while the C6-OH is positioned closer to the top. C6-OH and C3 of GalNAc have been labeled for orientation. (B) Side angle of the binding pocket of G10C (green) in complex with GalNAcα-PNP (yellow) illustrating key molecular interactions. An Fo–Fc omit map calculated without inclusion of the glycan in the phasing model was contoured at 3σ (blue mesh).

Article Snippet: A 4.91 mM solution of GalNAc α -PNP (Cayman Chemical) ligand was prepared in PBS (pH 7.4, 2 mL) and molecular-biology-grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich, 200 μ L).

Techniques: Labeling, Binding Assay, Glycoproteomics

Journal: Journal of the American Chemical Society

Article Title: Development of a GalNAc-Tyrosine-Specific Monoclonal Antibody and Detection of Tyrosine O ‑GalNAcylation in Numerous Human Tissues and Cell Lines

doi: 10.1021/jacs.2c04477

Figure Lengend Snippet: Detection of GalNAc-Tyr modified proteins in lysates from human cell lines. 15 μg of cell lysate from each cell line was resolved on a 4–12% BT protein gel, transferred to a PVDF membrane, and probed with either G10C or isotype control.

Article Snippet: A 4.91 mM solution of GalNAc α -PNP (Cayman Chemical) ligand was prepared in PBS (pH 7.4, 2 mL) and molecular-biology-grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich, 200 μ L).

Techniques: Modification, Membrane, Control

Journal: Journal of the American Chemical Society

Article Title: Development of a GalNAc-Tyrosine-Specific Monoclonal Antibody and Detection of Tyrosine O ‑GalNAcylation in Numerous Human Tissues and Cell Lines

doi: 10.1021/jacs.2c04477

Figure Lengend Snippet: Prevalence of GalNAc-Tyr in various tissue lysates. Western blots of various soluble and insoluble fractions of normal and cancerous human tissues. 20 μg of tissue lysates from each cell line was resolved on a 4–12% or 8% BT protein gel, transferred to the PVDF membrane, and probed with either G10C or isotype control. Samples labeled T-1 or T-2 are tumor samples, whereas N-1 and N-2 are matched adjacent normal tissue. N-1 and T-1 are the soluble protein fraction. N-2 and T-2 are the insoluble protein fraction. mIgG is the mouse IgG isotype control.

Article Snippet: A 4.91 mM solution of GalNAc α -PNP (Cayman Chemical) ligand was prepared in PBS (pH 7.4, 2 mL) and molecular-biology-grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich, 200 μ L).

Techniques: Western Blot, Membrane, Control, Labeling

Journal: Journal of the American Chemical Society

Article Title: Development of a GalNAc-Tyrosine-Specific Monoclonal Antibody and Detection of Tyrosine O ‑GalNAcylation in Numerous Human Tissues and Cell Lines

doi: 10.1021/jacs.2c04477

Figure Lengend Snippet: IHC staining of various normal human tissues using G10C, isotype control, or G10C in the presence of a GalNAc-BSA inhibitor. Strong staining is observed in certain cell populations with G10C. Replacement of the primary (anti-G10C antibody) with nonspecific, isotype control antibody results in minimal to absent staining in all regions. Inhibition with a GalNAc-α-thioethylglutaramide-BSA-neoglycoprotein significantly reduces staining.

Article Snippet: A 4.91 mM solution of GalNAc α -PNP (Cayman Chemical) ligand was prepared in PBS (pH 7.4, 2 mL) and molecular-biology-grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich, 200 μ L).

Techniques: Immunohistochemistry, Control, Staining, Inhibition

Journal: Journal of the American Chemical Society

Article Title: Development of a GalNAc-Tyrosine-Specific Monoclonal Antibody and Detection of Tyrosine O ‑GalNAcylation in Numerous Human Tissues and Cell Lines

doi: 10.1021/jacs.2c04477

Figure Lengend Snippet: Summary of GalNAc-Tyr Frequencies on Different Cell Lines a

Article Snippet: A 4.91 mM solution of GalNAc α -PNP (Cayman Chemical) ligand was prepared in PBS (pH 7.4, 2 mL) and molecular-biology-grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich, 200 μ L).

Techniques:

Journal: Journal of the American Chemical Society

Article Title: Development of a GalNAc-Tyrosine-Specific Monoclonal Antibody and Detection of Tyrosine O ‑GalNAcylation in Numerous Human Tissues and Cell Lines

doi: 10.1021/jacs.2c04477

Figure Lengend Snippet: Confocal imaging of GalNAc-Tyr in LOX-IMVI cells. (A) Imaging of LOX-IMVI cells under permeabilizing conditions. Cells were first transfected with a vector encoding for a GFP-labeled Golgi resident enzyme (green), then fixed, and permeabilized prior to staining with G10C or isotype control conjugated to Alexa Fluor 647. Staining of nuclear DNA is shown in blue. (B) For surface staining, live cells were stained with labeled G10C or control, followed by fixation and imaging. The merged images include fluorescence and bright-field images.

Article Snippet: A 4.91 mM solution of GalNAc α -PNP (Cayman Chemical) ligand was prepared in PBS (pH 7.4, 2 mL) and molecular-biology-grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich, 200 μ L).

Techniques: Imaging, Transfection, Plasmid Preparation, Labeling, Staining, Control, Fluorescence

Journal: Journal of the American Chemical Society

Article Title: Development of a GalNAc-Tyrosine-Specific Monoclonal Antibody and Detection of Tyrosine O ‑GalNAcylation in Numerous Human Tissues and Cell Lines

doi: 10.1021/jacs.2c04477

Figure Lengend Snippet: Summary of GalNAc-Tyr Prevalence on Primary Cells

Article Snippet: A 4.91 mM solution of GalNAc α -PNP (Cayman Chemical) ligand was prepared in PBS (pH 7.4, 2 mL) and molecular-biology-grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich, 200 μ L).

Techniques:

Journal: Journal of the American Chemical Society

Article Title: Development of a GalNAc-Tyrosine-Specific Monoclonal Antibody and Detection of Tyrosine O ‑GalNAcylation in Numerous Human Tissues and Cell Lines

doi: 10.1021/jacs.2c04477

Figure Lengend Snippet: X-ray Crystallography Data

Article Snippet: A 4.91 mM solution of GalNAc α -PNP (Cayman Chemical) ligand was prepared in PBS (pH 7.4, 2 mL) and molecular-biology-grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich, 200 μ L).

Techniques: Structural Proteomics

Journal: Journal of the American Chemical Society

Article Title: Development of a GalNAc-Tyrosine-Specific Monoclonal Antibody and Detection of Tyrosine O ‑GalNAcylation in Numerous Human Tissues and Cell Lines

doi: 10.1021/jacs.2c04477

Figure Lengend Snippet: X-ray Crystallography Data

Article Snippet: A 4.91 mM solution of GalNAc α -PNP (Cayman Chemical) ligand was prepared in PBS (pH 7.4, 2 mL) and molecular-biology-grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich, 200 μ L).

Techniques: