Multidimensional liquid chromatography separation of intact proteins by chromatographic focusing and reversed phase of the human serum proteome - Optimization and protein database
Multidimensional liquid chromatography separation of intact proteins by chromatographic focusing and reversed phase of the human serum proteome - Optimization and protein database
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DOI：
10.1074/mcp.t500019-mcp200
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发表时间：
2006-01-01
影响因子：
7
通讯作者：
Van Eyk, JE
中科院分区：
文献类型：
--
作者：
Sheng, S;Chen, D;Van Eyk, JE
在生物标志物的发现中，可以通过优化蛋白质分离方法以及选择性地去除免疫球蛋白和白蛋白等丰度较高的蛋白质来检测血清蛋白质组中丰度较低的蛋白质。蛋白质组分离领域的一个相对较新的领域是Beckman Coulter的商业仪器PF2D，它使用层析聚焦在第一维分离蛋白质，然后在第二维在线反相色谱分离蛋白质，从而根据等电点和疏水性分离完整的蛋白质。在这项研究中，评估和优化的血清分离(未耗尽的血清和白蛋白-IgG耗尽的血清)是由PF2D。在传统方法和优化方法下，在不同的样品制备方案下，为健康人的血清建立蛋白质数据库。在第一维运行缓冲液中存在20%异丙醇的情况下，使用PF2D分离双耗尽血清，使我们能够毫不含糊地鉴定150种非冗余血清蛋白(不包括所有免疫球蛋白和白蛋白，至少有两个肽与可接受的吉祥物得分匹配)，其中81种以前没有在血清中鉴定过。其中，大量的细胞蛋白被鉴定为骨骼肌的特异性亚型，如骨骼肌肌钙蛋白T的快速抽动亚型、肌球蛋白碱性轻链1和肌浆/内质网钙ATPase。在健康个体血清中检测到特定的骨骼肌蛋白质异构体反映了发生在骨骼肌中的生理周转，这将影响使用通用的“细胞”蛋白质作为生物标志物的能力，而不进一步表征存在的精确异构体或翻译后修饰。
In biomarker discovery, the detection of proteins with low abundance in the serum proteome can be achieved by optimization of protein separation methods as well as selective depletion of the higher abundance proteins such as immunoglobins (e.g. IgG) and albumin. A relative newcomer to the proteomic separation arena is the commercial instrument PF2D from Beckman Coulter that separates proteins in the first dimension using chromatofocusing followed in line by reversed phase chromatography in the second dimension, thereby separating intact proteins based on pI and hydrophobicity. In this study, assessment and optimization of serum separation ( undepleted serum and albumin-IgG-depleted serum) by the PF2D is presented. Protein databases were created for serum obtained from a healthy individual under traditional and optimized methods and under different sample preparation protocols. Separation of the doubly depleted serum using the PF2D with 20% isopropanol present in the first dimension running buffer allowed us to unambiguously identify 150 non-redundant serum proteins ( excluding all immunoglobulin and albumin, a minimum of two peptide matches with acceptable Mascot score) in which 81 have not been identified previously in serum. Among them, numerous cellular proteins were identified to be specifically the skeletal muscle isoform, such as skeletal muscle fast twitch isoforms of troponin T, myosin alkali light chain 1, and sarcoplasmic/endoplasmic reticulum calcium ATPase. The detection of specific skeletal muscle protein isoforms in the serum from healthy individuals reflects the physiological turnover that occurs in skeletal muscle, which will have an impact on the ability to use generic "cellular" proteins as biomarkers without further characterization of the precise isoforms or post-translational modifications present.