Unravelling and Quantifying the "NMR-Invisible" Metabolites Interacting with Human Serum Albumin by Binding Competition and T2 Relaxation-Based Decomposition Analysis
Unravelling and Quantifying the "NMR-Invisible" Metabolites Interacting with Human Serum Albumin by Binding Competition and T2 Relaxation-Based Decomposition Analysis
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DOI：
10.1021/acs.jproteome.6b00814
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发表时间：
2017-05-01
影响因子：
4.4
通讯作者：
Correig, Xavier
中科院分区：
文献类型：
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作者：
Barrilero, Ruben;Ramirez, Noelia;Correig, Xavier
通过H-1 NMR对低分子量代谢物（LMWM）进行定量分析通常用于高通量血清代谢组学。首先，使用T2滤波器衰减蛋白质背景;然后，通过线形拟合解析LMWM信号。然而，蛋白质结合改变了LMWM的运动特性，并且它们的信号随着T2滤波器沿着蛋白质背景而部分衰减。因此，定量LMWM信号不反映血清中的总浓度，而是反映非结合部分。在这里，我们提出了一种新的策略，结合竞争的基础上，促进释放的“NMR看不见”的代谢物从血清蛋白质，并实现定量更接近总浓度。该研究重点关注具有不同结合特性的五种临床相关氨基酸（缬氨酸、异亮氨酸、亮氨酸、酪氨酸和苯丙氨酸）。我们分析了它们与人血清白蛋白（HSA）在血清模拟样品中的结合亲和力，并通过TSP滴定促进其结合部分的释放。此外，我们使用了伪二维CPMG和多变量曲线分辨率分析的新组合，允许分离LMWM和蛋白质信号，并提供针对横向弛豫效应校正的LMWM定量。我们发现TSP浓度大于3 mM时释放了大部分结合部分，并在真实的血清/血浆样本中验证了这些发现。
Quantitative profiling of low-molecular-weight metabolites (LMWMs) by H-1 NMR is routinely used in high throughput serum metabolomics. First, the protein background is attenuated using a T2 filter; then, the LMWM signals are resolved by line-shape fitting. However, protein binding modifies the motional properties of LMWM, and their signal partially attenuates with the T2 filter, along with the protein background. Consequently, the quantified LMWM signals do not reflect the total concentration in serum but the nonbinding part. Here we present a novel strategy based on binding competition to promote the release of the "NMR-invisible" metabolites from serum proteins and achieve quantifications closer to total concentrations. The study focuses on five clinically relevant amino acids with different binding properties (valine, isoleucine, leucine, tyrosine, and phenylalanine). We analyzed their binding affinity to human serum albumin (HSA) in serum mimic samples and promoted the release of their bound fraction by TSP titration. Furthermore, we used a novel combination of pseudo-2D CPMG and multivariate curve resolution analysis, allowing the separation of LMWM and protein signals and providing LMWM quantifications corrected for transverse relaxation effects. We found that TSP concentrations larger than 3 mM released most of the bound fraction and validated these findings in real serum/plasma samples.