title: Establishment of a Strategy for the Discovery and Verification of Low-Abundance Biomarker Peptides in Plasma Using Two Types of Stable-Isotope Tags.
abstract: Serum and plasma contain thousands of different proteins and peptides, which can provide valuable information about the numerous processes that take place within the body. However, detailed analysis of proteins and peptides in serum and plasma remains challenging due to the presence of many high-abundance proteins, the large dynamic range of protein and peptide concentrations, the extensive complexity caused by posttranslational modifications, and considerable individual variability. In particular, detailed analysis and identification of native peptides is extremely difficult due to the tremendous variety of cleavage possibilities and posttranslational modifications, which results in extremely high complexity. Therefore, widely ranging searches based on peptide identification are difficult. Herein, we describe the highly accurate and sensitive quantitative analysis of over 2,500 peptides with the concentration limit of about 10 pM. The strategy combined isobaric tag labeling, amine-reactive 6-plex tandem mass tag labeling, and a modified differential solubilization method for high-yield peptide extraction [Saito, T. et al. J. Electrophoresis 2013 57: 1-9]. Using this strategy, we quantitatively analyzed six pooled plasma samples (three pre-surgery and three post-surgery) to discover potential candidate biomarker peptides of renal cell carcinoma. The concentrations of 27 peptides were found to be altered following surgery. A preliminary validation study was conducted using about 80 plasma samples to demonstrate the possibility that even unidentified potential candidate biomarker peptides can be verified using the isotope tag/dimethyl labeling method. We also discuss technical consideration and potential of this strategy for facilitating native peptide research.
authors.affiliation: Laboratory of Biophysics, Department of Physics, Kitasato University School of Science; Center for Disease Proteomics, Kitasato University School of Science.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kodera
authors.affiliation: Laboratory of Biophysics, Department of Physics, Kitasato University School of Science.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Hido
authors.affiliation: Laboratory of Biophysics, Department of Physics, Kitasato University School of Science.
authors.forename: Rika
authors.initials: R
authors.lastname: Kato
authors.affiliation: Laboratory of Biophysics, Department of Physics, Kitasato University School of Science.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Saito
authors.affiliation: Laboratory of Biophysics, Department of Physics, Kitasato University School of Science; Center for Disease Proteomics, Kitasato University School of Science.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Kawashima
authors.affiliation: Department of Urology, Kitasato University School of Medicine.
authors.forename: Satoru
authors.initials: S
authors.lastname: Minamida
authors.affiliation: Center for Disease Proteomics, Kitasato University School of Science; Department of Urology, Kitasato University School of Medicine.
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Matsumoto
authors.affiliation: Department of Urology, Kitasato University School of Medicine.
authors.forename: Masatsugu
authors.initials: M
authors.lastname: Iwamura
keywords: biomarker peptide; isobaric tag; isotope tag; plasma; renal cell carcinoma
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