A dataset of human liver proteins identified by protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry.
Proteins from human liver carcinoma Huh7 cells, representing transformed liver cells, and cultured primary human fetal hepatocytes (HFH) and human HH4 hepatocytes, representing nontransformed liver cells, were extracted and processed for proteome analysis. Proteins from stimulated cells (interferon-alpha treatment for the Huh7 and HFH cells and induction of hepatitis C virus [HCV] proteins for the HH4 cells) and corresponding control cells were labeled with light and heavy cleavable ICAT reagents, respectively. The labeled samples were combined, trypsinized, and subject to cation-exchange and avidin-affinity chromatographies. The resulting cysteine-containing peptides were analyzed by microcapillary LC-MS/MS. The MS/MS spectra were initially analyzed by searching the human International Protein Index database using the SEQUEST software (1). Subsequently, new statistical algorithms were applied to the collective SEQUEST search results of each experiment. First, the PeptideProphet software (2) was applied to discriminate true assignments of MS/MS spectra to peptide sequences from false assignments, to assign a probability value for each identified peptide, and to compute the sensitivity and error rate for the assignment of spectra to sequences in each experiment. Second, the ProteinProphet software (3) was used to infer the protein identifications and to compute probabilities that a protein had been correctly identified, based on the available peptide sequence evidence. The resulting protein lists were filtered by a ProteinProphet probability score p > or = 0.5, which corresponded to an error rate of less than 5%. A total of 1,296, 1,430, and 1,476 proteins or related protein groups were identified in three subdatasets from the Huh7, HFH, and HH4 cells, respectively. In total, these subdatasets contained 2,486 unique protein identifications from human liver cells. An increase of the threshold to p > or = 0.9 (corresponding to an error rate of less than 1%) resulted in 2,159 unique protein identifications (1,146, 1,235, and 1,318 for the Huh7, HFH, and HH4 cells, respectively).
The Institute for Systems Biology, 1441 North 34th Street, Seattle, WA 98103, USA. wyan@systemsbiology.org
Wei
W
Yan
Hookeun
H
Lee
Eric W
EW
Deutsch
Catherine A
CA
Lazaro
Weiliang
W
Tang
Eric
E
Chen
Nelson
N
Fausto
Michael G
MG
Katze
Ruedi
R
Aebersold
D000465:Algorithms; D000595:Amino Acid Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry; D016208:Databases, Factual; D022781:Hepatocytes / Q000737:chemistry*; D006801:Humans; D007554:Isotopes / Q000737:chemistry*; D008099:Liver / Q000737:chemistry*; D008113:Liver Neoplasms / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D020543:Proteome / Q000032:analysis; D040901:Proteomics; D012984:Software
D007554:Isotopes; D011506:Proteins; D020543:Proteome; D003545:Cysteine