title: A novel approach and protocol for discovering extremely low-abundance proteins in serum.
abstract: The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10 orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing low-abundance protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a sequentially linked 3-D-LC-MS/MS system. Our hollow-fiber-membrane-based serum pretreatment device can efficiently deplete high-molecular weight proteins and concentrate low-molecular weight proteins/peptides automatically within 1 h. Four independent analyses of healthy human sera pretreated using this unique device, followed by the 3-D-LC-MS/MS successfully produced 12 000-13 000 MS/MS spectra and hit around 1800 proteins (>95% reliability) and 2300 proteins (>80% reliability). We believe that the unique serum pretreatment device and proteomic analysis protocol reported here could be a powerful tool for searching physiological biomarkers by its high throughput (3.7 days per one sample analysis) and high performance of finding low abundant proteins from serum or plasma samples.
authors.affiliation: Toray Industries, Inc., New Frontiers Research Laboratories, Kamakura, Kanagawa, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Hideo
authors.initials: H
authors.lastname: Akiyama
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Kuroda
authors.forename: Giman
authors.initials: G
authors.lastname: Jung
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Tanahashi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sugaya
authors.forename: Jun
authors.initials: J
authors.lastname: Utsumi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawasaki
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hirano
mesh_terms: D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002853:Chromatography, Liquid; D006801:Humans; D013058:Mass Spectrometry; D008970:Molecular Weight; D012680:Sensitivity and Specificity
chemical_list: D001798:Blood Proteins