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Human platelet carbamylated protein LC-MS/MS

ABSTRACT: The concentration of blood urea is markedly elevated in patients with advanced chronic kidney disease (CKD). 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 healthy volunteers were pre-incubated with PU-48 (an UT-B inhibitor) or vehicle. Subsequently, they were incubated with plasma from healthy volunteers or patients with advanced CKD. 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 three experimental groups: platelets pre-treated with vehicle and treat with plasma from healthy volunteers (HE), platelets pre-treated with vehicle and treat with plasma from patients with advanced CKD (EX), and platelets pre-treated with PU-48 and treat with plasma from patients with advanced CKD (EXPU), with three biological replicates per group.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Platelet, Blood Cell

DISEASE(S): Chronic Kidney Disease

SUBMITTER: Zhiwei Qiu

LAB HEAD: Qian Xiang

PROVIDER: PXD067646 | Pride | 2026-05-28

REPOSITORIES: Pride

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Project description:Coronary artery disease (CAD) is the leading cause of human morbidity and mortality worldwide, underscoring the need to improve diagnostic strategies. Platelets play a major role, not only in the process of acute thrombosis during plaque rupture, but also in the formation of atherosclerosis itself. MicroRNAs are endogenous small non-coding RNAs that control gene expression and are expressed in a tissue and disease-specific manner. Therefore they have been proposed to be useful biomarkers. The aim of this study was to investigate whether differences in miRNA expression levels in platelets can be found (i) between patients with premature CAD and healthy controls and (ii) within healthy controls after and before aspirin and statin administration. In this case-control study we measured expression levels of platelet miRNAs using microarrays from 40 male patients with premature CAD and 40 age- and sex-matched healthy controls. Premature CAD was defined as a cardiac event before the age of 51 years. The patients were selected from the outpatient clinic of the Academic Medical Center (AMC) of Amsterdam, which is specialised in premature CAD. The control cohort was composed of 40 healthy Caucasian male volunteers, who were recruited by advertisement and who were matched with the cases for age and smoking habits. Individuals of this control cohort did not have a history of CVD, nor did they have a positive family history of CVD and they were not allowed to use any medication. Patients and controls were excluded when they suffered from diabetes. Most CAD patients use aspirin and statins as secondary prevention. The influence of these drugs on miRNA profiles is unknown. To assess the possible influence of medication on miRNA expression and to control for medication as confounding factor, we asked 27 volunteers in our control cohort to also use these drugs. We administered simvastatin 40 mg, once daily, for 6 weeks and during the last two weeks we added 100mg of acetyl salicylic acid, once daily. Blood samples including isolated platelets were collected at baseline in the absence of aspirin and statins and after six weeks of medication use. We also assessed platelet function using the Multiplate® Analyzer (Roche) in the absence of aspirin and statin use. In short, 300 µl whole blood was diluted with 300 µl 0.9% saline and stirred for 3 minutes at 37 ºC. Adenosine diphosphate (ADP) was added in a final concentration of 2.5 ?mol/L to initiate platelet aggregation. Aggregation was measured for 6 minutes and was reported in arbitrary aggregation units plotted against time. Also, the area under the aggregation curve (AUC) was measured. All samples were measured in the absence and presence of 200 ?mol/L indomethacin (20 min incubation with blood) to mimic the effect of aspirin use. We calculated the percentage reduction in AUC after incubation with indomethacin as an in vitro measure of the effect of aspirin use on whole blood platelet aggregation.

			Action	DRS
	EX1.raw	Raw
	EX2.raw	Raw
	EX3.raw	Raw
	EXPU1.raw	Raw
	EXPU2.raw	Raw

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Human platelet carbamylated protein LC-MS/MS

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