iBiMED - Institute of Biomedicine, Department of Medical Sciences, University of Aveiro, Aveiro, Portugal.
iBiMED - Institute of Biomedicine, Department of Medical Sciences, University of Aveiro, Aveiro, Portugal; Unidade de Investigação Cardiovascular, Departamento de Cirurgia e Fisiologia, Faculdade de Medicina, Universidade do Porto, Portugal.
Talanta. 2017 Aug 1;170:81-88. doi: 10.1016/j.talanta.2017.03.087. Epub 2017 Apr 1.
Urine is a highly attractive source of biological information and disease biomarkers, whose proteome characterization is ongoing. To that end, depletion/enrichment strategies for protein analysis can be of great convenience. We have thus developed a method based on the use of EDTA-functionalized magnetic nanoparticles (NPs@EDTA), to fractionate urine samples before liquid chromatography-mass spectrometry analysis and compared the identified proteins with those obtained from ultrafiltrated/unfractionated (UF) urine samples. NPs@EDTA allowed larger urine volumes to be processed, resulting in a greater number of protein identifications (~2-fold) at a lower cost when compared to UF samples. Proteins of greater abundance (such as albumin and uromodulin) were, at least partially, depleted with NPs@EDTA while those of lower abundance were enriched. Bioinformatics analysis showed that approximately 27% of NPs@EDTA-enriched proteins were annotated as displaying enzymatic activity, most of these being hydrolytic enzymes (56%), particularly proteases/peptidases (48%). Also, post-translational modifications were prominently predicted across NPs@EDTA-enriched proteins (90%), particularly glycosylation (52%), phosphorylation (47%) and acetylation (30%). NPs@EDTA allowed the identification of 109 proteins in urine for the first time, showing high potential as a platform for urine's fractionation prior to proteomic analysis.
尿液是生物信息和疾病生物标志物的一个极具吸引力的来源，其蛋白质组学特征正在不断研究中。为此，蛋白质分析的耗竭/富集策略可能非常方便。因此，我们开发了一种基于使用 EDTA 功能化磁性纳米颗粒（NPs@EDTA）的方法，在进行液相色谱-质谱分析之前对尿液样本进行分级，并将鉴定的蛋白质与超滤/未分级（UF）尿液样本中获得的蛋白质进行比较。与 UF 样本相比，NPs@EDTA 允许处理更大体积的尿液，从而以更低的成本获得更多的蛋白质鉴定（约 2 倍）。NPs@EDTA 至少部分耗尽了白蛋白和尿调素等丰度较高的蛋白质，而丰度较低的蛋白质则得到了富集。生物信息学分析表明，约 27%的 NPs@EDTA 富集蛋白被注释为具有酶活性，其中大多数是水解酶（56%），特别是蛋白酶/肽酶（48%）。此外，NPs@EDTA 富集蛋白中预测到大量的翻译后修饰（90%），特别是糖基化（52%）、磷酸化（47%）和乙酰化（30%）。NPs@EDTA 首次在尿液中鉴定出 109 种蛋白质，显示出作为蛋白质组学分析前尿液分级平台的巨大潜力。
