High-Yield Peptide-Extraction Method for the Discovery of Subnanomolar Biomarkers from Small Serum Samples
High-Yield Peptide-Extraction Method for the Discovery of Subnanomolar Biomarkers from Small Serum Samples
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DOI：
10.1021/pr9008018
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发表时间：
2010-04-01
影响因子：
4.4
通讯作者：
Kodera, Yoshio
中科院分区：
文献类型：
--
作者：
Kawashima, Yusuke;Fukutomi, Toshiyuki;Kodera, Yoshio
血清蛋白/肽反映了人类的生理或病理状态，是发现疾病生物标志物的一个有吸引力的目标。然而，高丰度蛋白的存在和血清蛋白/肽的大动态范围使得任何低丰度蛋白/肽的定量分析都具有挑战性。此外，由于载体蛋白的结合，分析肽（包括蛋白质的裂解片段）是困难的。在这里，我们开发了一种差分增溶（DS）方法来提取血清中的低分子量蛋白质/肽，与典型的肽提取方法（如有机溶剂沉淀法和超滤法）相比，该方法具有良好的重复性和产率。利用DS方法结合反相高效液相色谱分离和MALDI-TOF-MS，我们对1 μ L血清样品中的1500多个多肽进行了高质量的比较分析，包括亚纳摩尔范围的低丰度多肽和含有许多与载体蛋白（如白蛋白）结合的多肽。我们应用这种方法，成功地发现了四种新的结肠癌生物标志物候选物，这些候选物以前都没有在血清中观察到过，其中一种是可能起源于肿瘤细胞的蛋白zyxin片段。我们的结果表明，基于DS方法的血清肽分析将极大地有助于发现新的低丰度生物标志物。
Serum proteins/peptides reflect physiological or pathological states in humans and are an attractive target for the discovery of disease biomarkers. However, the existence of high-abundance proteins and the large dynamic range of serum proteins/peptides make any quantitative analysis of low-abundance proteins/peptides challenging. Furthermore, analyses of peptides, including the cleaved fragments of proteins, are difficult because of carrier protein binding. Here, we developed a differential solubilization (DS) method to extract low-molecular-weight proteins/peptides in serum with good reproducibility and yield as compared to typical peptide-extraction methods such as organic solvent precipitation and ultrafiltration. Using the DS method combined with reverse-phase HPLC fractionation followed by MALDI-TOF-MS, we performed high-quality comparative analyses of more than 1500 peptides from 1 mu L of serum samples, including low-abundance peptides in the subnanomolar range and containing many peptides bound to carrier proteins such as albumin. We applied this method and successfully discovered four new biomarker candidates of colon cancer, none of which have previously been observed in serum and one of which is a fragment of the protein zyxin that possibly originated from tumor cells. Our results indicate that serum peptide analyses based on the DS method should greatly contribute to the discovery of novel low-abundance biomarkers.