A novel approach and protocol for discovering extremely low-abundance proteins in serum
A novel approach and protocol for discovering extremely low-abundance proteins in serum
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DOI：
10.1002/pmic.200500774
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发表时间：
2006-09-01
期刊：
影响因子：
3.4
通讯作者：
Hirano, Hisashi
中科院分区：
文献类型：
--
作者：
Tanaka, Yoshinori;Akiyama, Hideo;Hirano, Hisashi
血清（血浆）的蛋白质组分析已成为确定早期疾病诊断和药物发现所必需的生物标志物的主要方法。然而，这些生物标志物的测定在分析上具有挑战性，因为血清蛋白/肽的动态浓度范围非常宽（超过 10 个数量级）。因此，在蛋白质组分析之前降低样品复杂性至关重要，特别是在分析低丰度蛋白质生物标志物时。在这里，我们展示了一种对人血清进行蛋白质组分析的新方法，该方法使用最初开发的血清蛋白分离装置和顺序连接的 3-D-LC-MS/MS 系统。我们的中空纤维膜血清预处理装置可以在1小时内自动高效去除高分子量蛋白质并浓缩低分子量蛋白质/肽。使用这种独特的设备对健康人体血清进行四次独立分析，然后使用 3-D-LC-MS/MS 成功生成 12000-13000 MS/MS 谱图，并检测到约 1800 个蛋白质（> 95% 可靠性）和 2300 个蛋白质（> 80% 可靠性）。我们相信，本文报道的独特的血清预处理装置和蛋白质组分析方案因其高通量（每个样品分析 3.7 天）和从血清或血浆样品中寻找低丰度蛋白质的高性能，可能成为搜索生理生物标志物的强大工具。
The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10 orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing low-abundance protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a sequentially linked 3-D-LC-MS/MS system. Our hollow-fiber-membrane-based serum pretreatment device can efficiently deplete high-molecular weight proteins and concentrate low-molecular weight proteins/peptides automatically within 1 h. Four independent analyses of healthy human sera pretreated using this unique device, followed by the 3-D-LC-MS/MS successfully produced 12000-13000 MS/MS spectra and hit around 1800 proteins (> 95% reliability) and 2300 proteins (> 80% reliability). We believe that the unique serum pretreatment device and proteomic analysis protocol reported here could be a powerful tool for searching physiological biomarkers by its high throughput (3.7 days per one sample analysis) and high performance of finding low abundant proteins from serum or plasma samples.