title: Combined use of a solid-phase hexapeptide ligand library with liquid chromatography and two-dimensional difference gel electrophoresis for intact plasma proteomics.
abstract: The intact plasma proteome is of great interest in biomarker studies because intact proteins reflect posttranslational protein processing such as phosphorylation that may correspond to disease status. We examined the utility of a solid-phase hexapeptide ligand library in combination with conventional plasma proteomics modalities for comprehensive profiling of intact plasma proteins. Plasma proteins were sequentially fractionated using depletion columns for albumin and immunoglobulin, and separated using an anion-exchange column. Proteins in each fraction were treated with a solid-phase hexapeptide ligand library and compared to those without treatment. Two-dimensional difference gel electrophoresis demonstrated an increased number of protein spots in the treated samples. Mass spectrometric studies of these protein spots with unique intensity in the treated samples resulted in the identification of high- and medium-abundance proteins. Our results demonstrated the possible utility of a solid-phase hexapeptide ligand library to reveal greater number of intact plasma proteins. The characteristics of proteins with unique affinity to the library remain to be clarified by more extensive mass spectrometric protein identification, and optimized protocols should be established for large-scale plasma biomarker studies.
authors.affiliation: Division of Pharmacoproteomics, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Hagiwara
authors.forename: Yumi
authors.initials: Y
authors.lastname: Saito
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tomonaga
authors.forename: Yasufumi
authors.initials: Y
authors.lastname: Murakami
authors.forename: Tadashi
authors.initials: T
authors.lastname: Kondo