Development of a novel extraction method of serum/plasma peptides and its application in the discovery of tumor marker peptides for colon cancer

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 low abundance proteins are often masked by a few high abundance proteins. Furthermore, analyses of peptides, including the cleaved fragment of proteins, are difficult because of carrier protein binding.
We have established a novel strategy, called 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 from 0.01 mL of serum, 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 1,500 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. Our results suggest that high reproducible extraction and fractionation enables us to treat more than 90% of these peptides as targets of comparative analysis. These results indicate that the reproducibility of the peptide extraction and RP-HPLC fractionation is extremely high.
We applied this method to the sera from eight colon cancer patients and eight healthy volunteers. 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.