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Submitted on June 14, 2004
Institute for Systems Biology, Seattle, WA 98103
Corresponding Author: wyan{at}systemsbiology.org
Proteins from human liver carcinoma Huh7 cells, representing a transformed, and cultured primary human fetal hepatocytes (HFH) and human HH4 hepatocytes, representing non-transformed liver cells, were extracted and processed for proteome analysis. Proteins from stimulated cells [Interferon-a 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-tandem mass spectrometry (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=0.5 which corresponded to an error rate of less than 5%. A total of 1296, 1430, and 1476 proteins or related protein groups were identified in three sub-datasets from the Huh7, HFH, and HH4 cells, respectively. In total, these sub-datasets contained 2486 unique protein identifications from human liver cells. An increase of the threshold to P=0.9 (corresponding to an error rate of less than 1%) resulted in 2159 unique protein identifications (1146, 1235, and 1318 for the Huh7, HFH, and HH4 cells, respectively).
Revised on July 21, 2004
Accepted on July 21, 2004
A dataset of human liver proteins identified by protein profiling via isotope coded affinity tag (ICAT) and tandem mass spectrometry
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