In the few questionable results (sequence coverage <20% and identification p-values between 0.001 and 0.05), since we could not make MS/MS analysis, we applied additional criteria to accept protein identification: (1) the search engine assignment of peptide ions must explain most of the non-background signal (2) the p I and molecular weight of the identified protein must be consistent with the location in the 2D gel (3) the mass error of peptide fragments must follow an internally consistent pattern as for proteins with highly significant identifications (see Additional file 3 ).
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