Proteome Profile of Human Breast Cancer Tissue Generated by LC−ESI−MS/MS Combined with Sequential Protein Precipitation and Solubilization
journal contributionposted on 01.08.2008, 00:00 by Yan Gong, Nan Wang, Fang Wu, Carol E. Cass, Sambasivarao Damaraju, John R. Mackey, Liang Li
Comprehensive proteome profiling of breast cancer tissue samples is challenging, as the tissue samples contain many proteins with varying concentrations and modifications. We report an effective sample preparation strategy combined with liquid chromatography (LC) electrospray ionization (ESI) quadrupole time-of-flight (QTOF) MS/MS for proteome analysis of human breast cancer tissue. The complexity of the breast cancer tissue proteome was reduced by using protein precipitation from a tissue extract, followed by sequential protein solubilization in solvents of different solubilizing strength. The individual fractions of protein mixtures or subproteomes were subjected to trypsin digestion and the resultant peptides were separated by strong cation exchange (SCX) chromatography, followed by reversed-phase capillary LC combined with high resolution and high accuracy ESI-QTOF MS/MS. This approach identified 14407 unique peptides from 3749 different proteins based on peptide matches with scores above the threshold scores at the 95% confidence level in MASCOT database search of the acquired MS/MS spectra. The false positive rate of peptide matches was determined to be 0.95% by using the target-decoy sequence search strategy. On the basis of gene ontology categorization, the identified proteins represented a wide variety of biological functions, cellular processes, and cellular locations.
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sample preparation strategyprotein mixturesprotein precipitationSCXpeptidebreast cancer tissue samplesQTOFcation exchangesolubilizing strengththreshold scoresSolubilizationComprehensive proteomebreast cancer tissue proteomeproteome analysisMSgene ontology categorizationProteome ProfileHuman Breast Cancer Tissue Generatedtissue samplesbreast cancer tissueSequential Protein Precipitationtrypsin digestionelectrospray ionizationMASCOT database searchLCESIsequential protein solubilization