The aim of our study was to analyze the proteomic pattern of human macrophages obtained over a 4 year period from blood donors. The purpose was to simulate a long-term clinical study to assess the application of 2-D DIGE technique for differential proteomic analysis of these scarce samples. Bioinformatic analysis of 2-D DIGE gels of 19 different cultures of macrophages assessed whether they did or did not contain at least specific five spots identified by MS as being or containing bovine deoxyribonuclease I (DNase I). Bovine DNase I was used during sample treatment to remove nucleic acids from protein extracts. Macrophages were classified in two groups, which appeared to be differentiated by the completeness of DNase I treatment. Further detailed analysis revealed a different proteomic pattern of macrophage protein samples according to the completeness of this treatment. The major group of proteins affected, accounting for one third of the differentially expressed proteins, included proteins involved in cell motion and actin cytoskeleton reorganization. The use of DNase I for the removal of nucleic acids from protein samples must be avoided in proteomic studies since it can generate bias in the analysis of protein expression patterns.
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