Department of Chemistry, University of Waterloo, Ontario N2L 3G1, Canada.
Department of Chemistry, University of Waterloo, Ontario N2L 3G1, Canada.
Anal Chim Acta. 2014 Oct 3;845:45-52. doi: 10.1016/j.aca.2014.08.018. Epub 2014 Aug 13.
In this publication, a novel solid phase microextraction (SPME) coating functionalized with a DNA aptamer for selective enrichment of a low abundance protein from diluted human plasma is described. This approach is based on the covalent immobilization of an aptamer ligand on electrospun microfibers made with the hydrophilic polymer poly(acrylonitrile-co-maleic acid) (PANCMA) on stainless steel rods. A plasma protein, human α-thrombin, was employed as a model protein for selective extraction by the developed Apt-SPME probe, and the detection was carried out with liquid chromatography/tandem mass spectrometry (LC-MS/MS). The SPME probe exhibited highly selective capture, good binding capacity, high stability and good repeatability for the extraction of thrombin. The protein selective probe was employed for direct extraction of thrombin from 20-fold diluted human plasma samples without any other purification. The Apt-SPME method coupled with LC-MS/MS provided a good linear dynamic range of 0.5-50 nM in diluted human plasma with a good correlation coefficient (R(2)=0.9923), and the detection limit of the proposed method was found to be 0.30 nM. Finally, the Apt-SPME coupled with LC-MS/MS method was successfully utilized for the determination of thrombin in clinical human plasma samples. One shortcoming of the method is its reduced efficiency in undiluted human plasma compared to the standard solution. Nevertheless, this new aptamer affinity-based SPME probe opens up the possibility of selective enrichment of a given targeted protein from complex sample either in vivo or ex vivo.
在本出版物中，描述了一种新型的固相微萃取（SPME）涂层，该涂层用 DNA 适配体官能化，用于从稀释的人血浆中选择性富集低丰度蛋白质。这种方法基于将适配体配体共价固定在由亲水性聚合物聚丙烯腈-马来酸共聚物（PANCMA）在不锈钢棒上制成的电纺微纤维上。用人α-凝血酶作为模型蛋白，通过开发的 Apt-SPME 探针进行选择性提取，并通过液相色谱/串联质谱（LC-MS/MS）进行检测。SPME 探针对凝血酶的选择性捕获具有高选择性、良好的结合能力、高稳定性和良好的重复性。该蛋白质选择性探针用于直接从 20 倍稀释的人血浆样品中提取凝血酶，无需任何其他纯化。Apt-SPME 方法与 LC-MS/MS 结合，在稀释的人血浆中提供了 0.5-50 nM 的良好线性动态范围，相关系数良好（R(2)=0.9923），该方法的检测限为 0.30 nM。最后，成功地将 Apt-SPME 与 LC-MS/MS 方法用于临床人血浆样品中凝血酶的测定。该方法的一个缺点是与标准溶液相比，在未稀释的人血浆中的效率降低。然而，这种新的基于适配体亲和力的 SPME 探针为从复杂样品中选择性富集特定靶向蛋白质（无论是在体内还是体外）开辟了可能性。
