Development of novel serum peptidomic strategy and its application in the discovery of tumor marker peptides

血清蛋白反映人的生理或病理状态，是发现疾病生物标志物的一个有吸引力的选择。然而，血清蛋白的大动态范围使得任何定量分析都非常具有挑战性，因为大量这些低丰度蛋白通常被少数高丰度蛋白所掩盖。此外，由于载体蛋白的结合，分析肽，包括这些蛋白质的裂解片段，是困难的。我们建立了一种新的策略（称为k -法），用于从血清中提取肽，与传统的有机溶剂沉淀和超滤提取方法相比，具有高效率和高重复性。采用k -法提取低分子量蛋白/肽，采用反相高效液相色谱进行分离。随后用MALDI-TOF MS对分离的60个部分进行比较分析，使我们能够观察到总共超过1000个多肽。为了检验我们方法的可重复性，我们评估了来自健康志愿者的四种血清。超过90%的峰值通常在四种血清中的三种血清中观察到，几乎在所有血清中观察到相同的强度。我们的研究结果表明，高重复性的提取和分离使我们能够将90%以上的这些肽作为比较分析的目标。当考虑到人血清的个体差异和通常看到的血清肽的不稳定特性时，数据的高质量令人惊讶。因此，k -法为我们提供了提取与血清中载体蛋白结合的相对稳定的肽的能力。数据稳定的另一个因素是反相高效液相色谱的高重现性。然后，我们将这种方法应用于8名结肠癌患者和8名健康志愿者的血清。在四分之三的血清中通常观察到超过1000个肽。使用MALDI-TOF MS/MS，我们发现并鉴定了4个潜在的生物标志物，p值小于0.005。其中一种多肽的浓度范围为ng/mL，被鉴定为细胞蛋白的裂解片段，此前有报道称该片段在癌症组织中增加。这些结果表明，我们的新方法有可能在新的生物标志物发现中发挥突出作用。少

Serum proteins reflect physiological or pathological states in humans and are an attractive option for use in the discovery of disease biomarkers. However, the large dynamic range of serum proteins makes any quantitative analysis very challenging because a large number of these low abundance proteins are often masked by a few high abundance proteins. Furthermore, analyses of peptides, including the cleaved fragment of these proteins, are difficult because of carrier protein binding.We have established a novel strategy (call the K-method) for extracting peptides from serum with high efficiency and high reproducibility, as compared to the typical peptide extraction method that uses organic solvent precipitation and ultrafiltration. Using the K-method, we extracted low molecular weight proteins/peptides, followed by fractionation by RP-HPLC. The 60 fractions that were separated subsequently underwent comparative analysis by using MALDI-TOF MS, enabling us to observe a total of more than o … More ne thousand peptides. To examine the reproducibility of our method, we evaluated four sera from healthy volunteers. More than 90% of the peaks were commonly observed in three of the four sera, with almost the same intensities seen in all sera. Our results suggest that high reproducible extraction and fractionation enables us to treat more than 90% of these peptides as targets of comparative analysis. The high quality of the data was surprising when one considers the individual variations of human serum and the unstable properties of the serum peptides that are normally seen. Therefore, the K-method provides us with the ability to extract relatively stable peptides that bind to carrier proteins in serum. Another factor responsible for the stable data was the high reproducibility of the RP-HPLC.We then applied this method to the sera from eight colon cancer patients and eight healthy volunteers. More than 1,000 peptides were commonly observed in three-fourths of the sera. Using MALDI-TOF MS/MS, we discovered and identified four potential biomarkers, which had p values less than 0.005. One of the peptides, the concentration range of ng/mL, was identified as a cleaved fragment of a cellular protein that has previously been reported to increase in cancer tissue. These results indicate that our new method has the potential to play a prominent role in novel biomarker discoveries. Less