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RESEARCH PRODUCT

Comparisons of detergents for purification of platelet membrane lipid rafts: a lipidomics and proteomics analysis focusing on P2XR localization

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Introduction: Platelet membrane contains cholesterol and sphingomyelin-rich microdomains also known as lipid rafts (LR). LRs have been considered as pharmacological target of anti-aggregation drugs as they suggested providing a platform for physiological activity of target proteins such as purinergic receptors (P2XR, P2YR) activated by nucleotides (ATP and ADP respectively). Localization of P2XR in these microdomains remains mostly unknown. The aim of this study was to compare the effectivity of different conventional detergents for lipid rafts isolation and investigation on the lipidomics and proteomics features of these microdomains focusing their potentiality of containing P2XR. Obviously, given the lack of well-established methods for platelet membrane studies, we also aimed at comparing the effectivity of different conventional detergents studies of plasma membrane studies. Material and methods: Platelets were obtained from healthy donors from Etablissement Franc_ais du Sang. Platelets were lysed with three different detergents (Brij 35, lubrol and TritonX100) in low and high doses (0.05% and 1%). After density separation of platelets lysate using ultracentrifugation with sucrose gradient (5, 35 and 45%), 12 equal sucrose fractions were collected and concentration of cholesterol (Ch), sphingomyelin (SM) and phosphatidyl-choline (PC) were quantified. Based on these lipidomics results, fractions containing raft and nonraft domains were subjected to proteomics analysis. Results: LRs mainly were observed in fractions 1–4 in all detergents except Triton 0.05% that was not being able to extract lipid rafts. Ch and SM enrichment analysis were performed by Ch/PC and SM/PC ratio in each fraction. It showed that Lubrol 1% and Triton 1% were more suitable detergents as they were able to isolate raft fractions enriched in Ch and SM about 1.5 times more than other detergents (Lubrol 1%: Ch/PC = 1.52 and SM/PC= 1.44; Triton 1%: Ch/PC = 1.77 and SM/PC= 1.48). By proteomics analysis; overall 822 proteins were identified in platelet membrane. Each detergent revealed a different profile of protein’s set. P2XR was found only in fractions contain lipid rafts by Brij 1% and Lubrol 0.05%. Discussion / Conclusion: Investigating on P2XR on the membrane of platelets, Brij 1% and Lubrol 0.05% would be suitable detergents. Our results suggest that depending on the focal objective of study, adopting a compatible detergent has a great importance.