Analysis of albumin-associated peptides and proteins from ovarian cancer patients
Analysis of albumin-associated peptides and proteins from ovarian cancer patients
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DOI：
10.1373/clinchem.2005.052944
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发表时间：
2005-10-01
影响因子：
9.3
通讯作者：
Liotta, LA
中科院分区：
文献类型：
--
作者：
Lowenthal, MS;Mehta, AI;Liotta, LA
背景资料：白蛋白结合低分子量分子，包括蛋白质和肽，然后获得更长的半衰期，从而保护结合的物质免于肾脏清除。我们开发了一种实验方法来分离天然状态的白蛋白，然后识别[质谱（MS）测序]相应的结合低分子量分子。我们用这种方法来分析汇集的血清从人类疾病的研究集（高风险的人没有癌症，n = 40; I期卵巢癌，n = 30; III期卵巢癌，n = 40），以证明这种方法作为一个发现methods.Methods的可行性：白蛋白被分离的固相亲和捕获在本地的结合和洗涤条件下。捕获的白蛋白相关的蛋白和肽进行分离，凝胶电泳，并进行迭代MS测序微毛细管反相串联MS。选择白蛋白结合的蛋白片段在人血清中通过Western blotting和免疫competition.Results确认：在总的，1208个单独的蛋白序列预测从所有3池。预测的序列主要是来自具有不同生物学功能的蛋白质的片段。超过三分之一的这些片段被确定的多个肽序列，并确定的物种的一半以上的体内裂解产物的亲本蛋白质。预计有700种血清肽或蛋白质在以前的血清数据库中没有报道。通过Western印迹和肽免疫竞争，在血液中免疫学证实了可能与癌症相关的较大分子的几个蛋白水解片段。BRCA 2，一个390 kDa的低丰度的核蛋白与癌症易感性，在血清中表示为一系列的特定片段绑定到albumin.Conclusion：载体蛋白收获提供了丰富的候选肽和蛋白质的来源与潜在的不同的组织和细胞的起源，可能反映重要的疾病相关的信息。(c)2005年美国临床化学协会。
Background: Albumin binds low-molecular-weight molecules, including proteins and peptides, which then acquire its longer half-life, thereby protecting the bound species from kidney clearance. We developed an experimental method to isolate albumin in its native state and to then identify [mass spectrometry (MS) sequencing] the corresponding bound low-molecular-weight molecules. We used this method to analyze pooled sera from a human disease study set (high-risk persons without cancer, n = 40; stage I ovarian cancer, n = 30; stage III ovarian cancer, n = 40) to demonstrate the feasibility of this approach as a discovery method.Methods: Albumin was isolated by solid-phase affinity capture under native binding and washing conditions. Captured albumin-associated proteins and peptides were separated by gel electrophoresis and subjected to iterative MS sequencing by microcapillary reversed-phase tandem MS. Selected albumin-bound protein fragments were confirmed in human sera by Western blotting and immunocompetition.Results: In total, 1208 individual protein sequences were predicted from all 3 pools. The predicted sequences were largely fragments derived from proteins with diverse biological functions. More than one third of these fragments were identified by multiple peptide sequences, and more than one half of the identified species were in vivo cleavage products of parent proteins. An estimated 700 serum peptides or proteins were predicted that had not been reported in previous serum databases. Several proteolytic fragments of larger molecules that may be cancer-related were confirmed immunologically in blood by Western blotting and peptide immunocompetition. BRCA2, a 390-kDa low-abundance nuclear protein linked to cancer susceptibility, was represented in sera as a series of specific fragments bound to albumin.Conclusion: Carrier-protein harvesting provides a rich source of candidate peptides and proteins with potential diverse tissue and cellular origins that may reflect important disease-related information. (c) 2005 American Association for Clinical Chemistry.