title: Clinical-scale high-throughput human plasma proteome analysis: lung adenocarcinoma.
abstract: Clinical proteomics requires the stable and reproducible analysis of a large number of human samples. We report a high-throughput comprehensive protein profiling system comprising a fully automated, on-line, two-dimensional microflow liquid chromatography/tandem mass spectrometry (2-D microLC-MS/MS) system for use in clinical proteomics. A linear ion-trap mass spectrometer (ITMS) also known as a 2-D ITMS instrument, which is characterized by high scan speed, was incorporated into the microLC-MS/MS system in order to obtain highly improved sensitivity and resolution in MS/MS acquisition. This system was used to evaluate bovine serum albumin and human 26S proteasome. Application of these high-throughput microLC conditions and the 2-D ITMS resulted in a 10-fold increase in sensitivity in protein identification. Additionally, peptide fragments from the 26S proteasome were identified three-fold more efficiently than by the conventional 3-D ITMS instrument. In this study, the 2-D microLC-MS/MS system that uses linear 2-D ITMS has been applied for the plasma proteome analysis of a few samples from healthy individuals and lung adenocarcinoma patients. Using the 2-D and 1-D microLC-MS/MS analyses, approximately 250 and 100 different proteins were detected, respectively, in each HSA- and IgG-depleted sample, which corresponds to only 0.4 microL of blood plasma. Automatic operation enabled the completion of a single run of the entire 1-D and 2-D microLC-MS/MS analyses within 11 h. Investigation of the data extracted from the protein identification datasets of both healthy and adenocarcinoma groups revealed that several of the group-specific proteins could be candidate protein disease markers expressed in the human blood plasma. Consequently, it was demonstrated that this high-throughput microLC-MS/MS protein profiling system would be practically applicable to the discovery of protein disease markers, which is the primary objective in clinical plasma proteome projects.
authors.affiliation: Clinical Proteome Center, Tokyo Medical University, Shinjuku, Tokyo, Japan.
authors.forename: Kiyonaga
authors.initials: K
authors.lastname: Fujii
authors.forename: Tomoyo
authors.initials: T
authors.lastname: Nakano
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Kanazawa
authors.forename: Shingo
authors.initials: S
authors.lastname: Akimoto
authors.forename: Takashi
authors.initials: T
authors.lastname: Hirano
authors.forename: Harubumi
authors.initials: H
authors.lastname: Kato
authors.forename: Toshihide
authors.initials: T
authors.lastname: Nishimura
mesh_terms: D000230:Adenocarcinoma / Q000097:blood*; D000418:Albumins / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood*; D013058:Mass Spectrometry; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D013997:Time Factors
chemical_list: D000418:Albumins; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease