title: Analysis of human liver proteome using replicate shotgun strategy.
abstract: In this study, a liquid-based shotgun strategy was used to comprehensively identify the expression of human liver proteome. Proteins were extracted from human liver tissue and digested in-solution. The tryptic digest mixture was desalted and separated by off-line strong cation exchange (SCX) chromatography with a 60-min elution. The MS/MS spectra were acquired in data-dependent mode after an RP chromatographic separation combined with linear IT MS analysis. To obtain the most comprehensive human liver proteome, each SCX fraction was run six times in RPLC MS/MS manner. Finally, more than 6,000,000 MS/MS spectra were collected. Using a relatively strict filter criteria, 24,311 proteins (48.42% of the predicted human proteome from human International Protein Index (IPI) protein database 3.07) corresponding to 13,150 nonredundant proteins were successfully identified, in which 7001 proteins (53.24%) were identified by two or more peptides, which could be considered as a high-confident dataset. Among the 6149 proteins (46.76%) identified by single peptide, 3812 proteins (61.99%) were detected more than twice in six repeated runs. Comparative analysis between different runs shows that the overlap of identified proteins between any two runs ranged from 25 to 44%. Of the nonredundant proteins identified, 8919 proteins (67.83%) were detected more than twice and 4231 proteins (32.17%) were detected only once in six RPLC MS/MS runs. The Gene Ontology annotation shows that the identified proteins come from various subcellular components. In addition, a large number of low abundant proteins were identified. The dynamic range of the approach reached at least nine orders of magnitude by estimating the concentration of proteins.
authors.affiliation: Department of Genomics and Proteomics, Beijing Institute of Radiation Medicine, Beijing, PR China.
authors.forename: Ming
authors.initials: M
authors.lastname: Chen
authors.forename: Wantao
authors.initials: W
authors.lastname: Ying
authors.forename: Yanping
authors.initials: Y
authors.lastname: Song
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.forename: Bing
authors.initials: B
authors.lastname: Yang
authors.forename: Songfeng
authors.initials: S
authors.lastname: Wu
authors.forename: Ying
authors.initials: Y
authors.lastname: Jiang
authors.forename: Yun
authors.initials: Y
authors.lastname: Cai
authors.forename: Fuchu
authors.initials: F
authors.lastname: He
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Qian
mesh_terms: D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D020543:Proteome