title: High-yield peptide-extraction method for the discovery of subnanomolar biomarkers from small serum samples.
abstract: 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 microL 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.
authors.affiliation: Laboratory of Biomolecular Dynamics, Department of Physics, Kitasato University School of Science, Sagamihara, Kanagawa, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Kawashima
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Fukutomi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tomonaga
authors.forename: Hiroki
authors.initials: H
authors.lastname: Takahashi
authors.forename: Fumio
authors.initials: F
authors.lastname: Nomura
authors.forename: Tadakazu
authors.initials: T
authors.lastname: Maeda
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kodera
mesh_terms: D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000097:blood* / Q000737:chemistry; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D015179:Colorectal Neoplasms / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D010449:Peptide Mapping; D010455:Peptides / Q000097:blood* / Q000737:chemistry; D015203:Reproducibility of Results; D018401:Sample Size; D012680:Sensitivity and Specificity
chemical_list: D014408:Biomarkers, Tumor; D010455:Peptides