Pfizer Global Research and Development, Pfizer Inc, Ann Arbor, MI, USA.
Proteomics. 2010 Apr;10(7):1416-25. doi: 10.1002/pmic.200900431.
Efforts to discover protein biomarkers in plasma are hampered by the high abundance of few proteins, which interfere with the detection of low-abundant proteins. Different commercially available protein-partitioning products were tested for their ability to lower the detection limit of proteins in 2-D gels. Immuno-depletion using polyclonal antibodies raised against the proteins of highest abundance (Seppro IgY14 System) was compared with a two-step immuno-depletion strategy, where depletion with the Seppro IgY14 column was followed by depletion with the Seppro IgY-SuperMix system. The third strategy tested was protein pre-fractionation using the ProteoMiner kit, where proteins compete for binding sites on bead-bound peptide hexamers with different binding properties. The pre-fractionated protein samples were analyzed using 2-DE, which revealed stunning differences in protein patterns. However, detectable protein spots in the different plasma fractions contained exclusively high-abundant proteins normally present in plasma at concentrations between 1 microg and 40 mg/mL.
在血浆中发现蛋白质生物标志物的努力受到少数几种高丰度蛋白质的阻碍，这些蛋白质会干扰低丰度蛋白质的检测。测试了不同市售的蛋白质分离产品，以评估它们降低 2-D 凝胶中蛋白质检测下限的能力。使用针对丰度最高的蛋白质（Seppro IgY14 系统）制备的多克隆抗体进行免疫耗竭与两步免疫耗竭策略进行了比较，其中先用 Seppro IgY14 柱进行耗竭，然后再用 Seppro IgY-SuperMix 系统进行耗竭。测试的第三种策略是使用 ProteoMiner 试剂盒进行蛋白质预分级，其中蛋白质在珠上结合的肽六聚体上竞争结合位点，而肽六聚体具有不同的结合特性。使用 2-DE 分析预分级的蛋白质样品，结果显示蛋白质图谱存在显著差异。然而，不同血浆级分中可检测到的蛋白质斑点仅包含通常存在于血浆中的高丰度蛋白质，其浓度在 1μg 至 40mg/mL 之间。
