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O-GlcNAc Containing Proteins in Normal and Idiopathic Pulmonary Fibrosis Human Fibroblasts

ABSTRACT: Isolated normal and IPF fibroblasts were homogenized in cold MilliQ water using a bullet blender. Samples were centrifuged and inhibitors were added: HALT (Thermo Fisher Scientific), Z-Pugnac (Tocris), Thiamet G (Cayman Chemicals), and benzonase (E1014, Millipore, Sigma). O-GlcNAc enzymatic labeling and protein capturing was performed as described using a Click-IT enrichment kit following the manufacturer's protocol (cat no: C33368, C33372, and C10416; Thermo Fisher Scientific). Following an alkyne agarose bead enrichment, proteins were reduced (5 mM DTT, 30 min at 37 C), alkylated (40 mM iodoacetamide, 1 h at 37C) and digested with trypsin (1:50 enzyme/protein ratio). 0.5 ug of samples were analyzed by LC-MS/MS on a Q-Exactive HF-X mass spectrometer. Data was searched using MSFragger in match between runs mode.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Jennifer E. Kyle

PROVIDER: MSV000093453 | MassIVE | Tue Nov 21 15:52:00 GMT 2023

REPOSITORIES: MassIVE

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