Enrichment of low-abundant serum proteins by albumin/immunoglobulin G immunoaffinity depletion under partly denaturing conditions
Enrichment of low-abundant serum proteins by albumin/immunoglobulin G immunoaffinity depletion under partly denaturing conditions
复制标题
DOI：
10.1002/elps.200500167
复制
发表时间：
2005-07-01
期刊：
影响因子：
2.9
通讯作者：
Huber, LA
中科院分区：
文献类型：
--
作者：
Huang, HL;Stasyk, T;Huber, LA
我们提出了一种简单的亲和力耗尽方案，以去除两种最丰富的血清蛋白，白蛋白和免疫球蛋白G （IgG）。在自然条件下，白蛋白/IgG被有效去除，二维电泳（2-DE）显示多种蛋白富集。此外，通过添加5%或20%乙腈（ACN）建立部分变性条件，以破坏低分子量（LMW）蛋白与载体蛋白白蛋白/IgG的结合。2-DE结果表明，与自然条件相比，变性条件下检测到的LMW蛋白总数增加。有趣的是，血清中5% ACN的存在比20% ACN的情况下显示出更好的LMW蛋白富集。在5% ACN条件下，选取2-DE凝胶中随机分布的7个白蛋白/IgG缺失血清点，用质谱法进行鉴定。与自然条件相比，变性条件下LMW蛋白5个斑点的强度增加。鉴定出的7个斑点中的3个（血清淀粉样蛋白P、维生素d结合蛋白和转甲状腺素）属于一组含量相对较低的蛋白质，仅占所有血清蛋白的1%。本文提出的方法提高了血清蛋白质组的分辨率，增加了二维凝胶上可见斑点的数量，并允许检测和质谱鉴定LMW蛋白和低丰度蛋白。
We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albumin/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the presence of 5% ACN in serum revealed better enrichment of LMW proteins when compared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and transthyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance.