| Budget Amount *help |
¥17,010,000 (Direct Cost: ¥15,000,000、Indirect Cost: ¥2,010,000)
Fiscal Year 2007: ¥8,710,000 (Direct Cost: ¥6,700,000、Indirect Cost: ¥2,010,000)
Fiscal Year 2006: ¥8,300,000 (Direct Cost: ¥8,300,000)
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| Research Abstract |
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
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