Proteome Profile of Human Breast Cancer Tissue Generated by LC−ESI−MS/MS Combined with Sequential Protein Precipitation and Solubilization Yan Gong Nan Wang Fang Wu Carol E. Cass Sambasivarao Damaraju John R. Mackey Liang Li 10.1021/pr800229j.s002 https://acs.figshare.com/articles/journal_contribution/Proteome_Profile_of_Human_Breast_Cancer_Tissue_Generated_by_LC_ESI_MS_MS_Combined_with_Sequential_Protein_Precipitation_and_Solubilization/2924515 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. 2008-08-01 00:00:00 sample preparation strategy protein mixtures protein precipitation SCX peptide breast cancer tissue samples QTOF cation exchange solubilizing strength threshold scores SolubilizationComprehensive proteome breast cancer tissue proteome proteome analysis MS gene ontology categorization Proteome Profile Human Breast Cancer Tissue Generated tissue samples breast cancer tissue Sequential Protein Precipitation trypsin digestion electrospray ionization MASCOT database search LC ESI sequential protein solubilization