Asymmetrical flow field-flow fractionation for improved characterization of human plasma lipoproteins
Asymmetrical flow field-flow fractionation for improved characterization of human plasma lipoproteins
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DOI：
10.1007/s00216-018-1499-3
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发表时间：
2019-01-01
影响因子：
4.3
通讯作者：
Williams, S. Kim Ratanathanawongs
中科院分区：
文献类型：
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作者：
Bria, Carmen R. M.;Afshinnia, Farsad;Williams, S. Kim Ratanathanawongs
高密度和低密度脂蛋白（HDL和LDL）是生物标志物发现的有吸引力的目标。然而，超离心（UC），目前选择用于分离HDL和LDL的方法，是繁琐的，需要大的样品体积，导致样品损失，并且不容易提供关于颗粒大小的信息。本工作采用半制备型非对称流场-流分级分离技术（SP-AF 4）和UC分离和收集人血浆HDL和LDL。比较SP-AF 4和UC分离条件、样品通量和液相色谱/质谱（LC/MS）脂质组学结果。在单次SP-AF 4运行中回收了超过600 g的总蛋白，并且Western印迹结果证实了apoA 1纯组分和apoB 100纯组分，分别与HDL和LDL一致。SP-AF 4分离每个样品需要60分钟，因此提供了比UC（可能持续数小时至数天）的显著改善。将SP-AF 4制备的HDL和LDL级分的脂质组分析与UC制备的HDL和LDL样品进行比较。通过两种样品制备技术鉴定了阳性MS模式下的270多种脂质和阴性MS模式下的140多种脂质，脂质组之间的重叠超过98%。此外，脂蛋白大小分布使用分析标度AF 4与多角度光散射（MALS）和动态光散射（DLS）检测器联用测定。这些进展将SP-AF 4定位为脂蛋白生物标志物表征和鉴定的首选样品制备方法。
High- and low-density lipoproteins (HDL and LDL) are attractive targets for biomarker discovery. However, ultracentrifugation (UC), the current methodology of choice for isolating HDL and LDL, is tedious, requires large sample volume, results in sample loss, and does not readily provide information on particle size. In this work, human plasma HDL and LDL are separated and collected using semi-preparative asymmetrical flow field-flow fractionation (SP-AF4) and UC. The SP-AF4 and UC separation conditions, sample throughput, and liquid chromatography/mass spectrometry (LC/MS) lipidomic results are compared. Over 600g of total proteins is recovered in a single SP-AF4 run, and Western blot results confirm apoA1 pure and apoB100 pure fractions, consistent with HDL and LDL, respectively. The SP-AF4 separation requires 60min per sample, thus providing a marked improvement over UC which can span hours to days. Lipidome analysis of SP-AF4-prepared HDL and LDL fractions is compared to UC-prepared HDL and LDL samples. Over 270 lipids in positive MS mode and over 140 lipids in negative MS mode are identified by both sample preparation techniques with over 98% overlap between the lipidome. Additionally, lipoprotein size distributions are determined using analytical scale AF4 coupled with multiangle light scattering (MALS) and dynamic light scattering (DLS) detectors. These developments position SP-AF4 as a sample preparation method of choice for lipoprotein biomarker characterization and identification.