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Platelet carbamylated protein LC-MS/MS

ABSTRACT: Urea undergoes natural hydrolysis to form cyanate, a reactive species capable of inducing protein carbamylation. To determine whether elevated urea levels promote carbamylation of platelet proteins, whether this modification is mediated by the urea transporter B (UT-B) expressed on platelets, and which specific proteins are affected, we employed immunoprecipitation coupled with data-independent acquisition (DIA) mass spectrometry to profile carbamylated proteins. Platelets isolated from wild-type or UT-B knockout mice were incubated with urea or a control solvent. Subsequently, the platelets were lysed with RIPA, and carbamylated proteins were enriched via agarose beads conjugated with anti-carbamylated protein antibodies. Following on-bead enzymatic digestion, DIA mass spectrometric analysis was performed using an Orbitrap Astral mass spectrometer interfaced with the Vanquish Neo UHPLC system. These data represent the quantitative profiles of carbamoylated proteins detected by DIA mass spectrometry. The samples were categorized into four experimental groups: wild-type mouse platelets treated with solvent control (Ctrl), WT mouse platelets treated with urea (U), UT-B knockout mouse platelets treated with solvent control (PU), and UT-B knockout mouse platelets treated with urea stimulation (UPU), with three biological replicates per group.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Blood Platelet, Platelet

DISEASE(S): Chronic Kidney Disease

SUBMITTER: Zhiwei Qiu

LAB HEAD: Qian Xiang

PROVIDER: PXD066058 | Pride | 2026-05-28

REPOSITORIES: Pride

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Project description:Protein lysine residue carbamylation is an irreversible post-translational modification, resulting in the generation of protein-bound homocitrulline (N--carbamyllysine). Carbamylation alters protein structure and can thus also alter protein function because of the loss of the charged -amino lysine moiety. Two distinct pathways can promote protein carbamylation. The first pathway occurs as a result of urea decomposition forming an equilibrium mixture of cyanate (CNO-) and the reactive electrophile isocyanate; the second (enzymatic) pathway occurs from myeloperoxidase (MPO)–catalyzed oxidation of thiocyanate (SCN-), yielding CNO- and isocyanate. We and others have previously shown that apolipoprotein A-I (apoA-I), the major protein constituent of high density lipoprotein (HDL), is a target for MPO-catalyzed modification in vivo altering its function, converting the traditionally cardio-protective lipoprotein into a pro-atherogenic and pro-apoptotic one. We hypothesized that insights into the chemical environment within the human artery wall could be gained by monitoring site-specific carbamylation patterns of apoA-I recovered from human atherosclerotic aorta. Toward testing this hypothesis, we first mapped and quantified carbamyllysine obtained from in vitro carbamylation of apoA-I by: (i) the urea driven (cyanate) vs. the inflammatory driven (MPO) pathways; as well as (ii) in lipid-poor vs. lipidated apoA-I (reconstituted nascent HDL). Our in vitro studies, interpreted in the context of existing structural models, suggest lysine residues within regional proximity of the known MPO binding sites on HDL are preferentially targeted by the enzymatic (MPO) carbamylation pathway, whereas the non-enzymatic (cyanate) pathway leads to nearly uniform distribution of carbamylated lysine residues along the apoA-I polypeptide chain. Quantitative proteomic analyses of apoA-I recovered from human aortic atheroma identified 16 of the 21 total lysine residues as carbamylated and suggests that the majority of apoA-I carbamylation in vivo occurs via the non-enzymatic cyanate pathway, and on ‘lipid-poor’ apoA-I forms. Monitoring the patterns of post translational modification of apoA-I lysine residues in vivo can provide insights into the chemical environment within human atheroma.

			Action	DRS
	Ctrl1.raw	Raw
	Ctrl2.raw	Raw
	Ctrl3.raw	Raw
	PU1.raw	Raw
	PU2.raw	Raw

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Platelet carbamylated protein LC-MS/MS

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