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the spectral count was employed to perform a quantitative analysis of desps in mcf-7/dox and mcf-7/doxh. the spectral count represents the number of observed ms/ms spectra for a protein from two independent experiments, which is used to eliminate the biases brought by experiment variation. then, the desps were compared by the log 2 transformed spectral count.
all data files were created by searching ms/ms spectra against the human international protein index protein sequence database (ipi.human.v3.63.fasta, 84118 entries), by using the turbosequest program in the bioworks 3.3 software suite, with a precursor-ion mass tolerance of 2.0 amu and fragment-ion mass tolerance of 1.0 amu. trypsin was set as the protease with two missed cleavage sites allowed. carbamidomethylation (+57.02150 da) was searched as a fixed modification on cysteine, representing alkylation with iodoacetamide, while oxidized methionine (+15.99492 da) was searched as a variable modification. the searched peptides and proteins were validated by peptideprophet and proteinprophet in the trans-proteomic pipeline (tpp, v.4.2) using default parameters. proteins with proteinprophet p value greater than 0.9 and with no less than two kinds of unique peptides were considered as true identifications. a randomized database of the ipi.fasta was used as a decoy database to calculate the false discovery rate (fdr) of protein identification. the fdr was calculated by the ratio of the number of matches to the randomized database to the combined number of matches to the ipi.fasta and its randomized derivative. fdr for proteinprophet p0.9 was less than 1%. proteins containing the same peptides were grouped, and the protein containing the highest probability in each group was remained.
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