Modifications of lipid-based gene delivery systems to improve efficacy is a continued effort in gene therapy research. Current advances include targeting specificity, controlled release and small molecule incorporation. While showing great progress, additional advances are necessary to increase transfection efficiency and decrease inflammatory toxicity. Previously, we demonstrated that sequentially injecting liposome, followed by DNA resulted in high transfection levels, but also significantly decreased the inflammatory response typically associated with lipoplex delivery. Here we attempt to elucidate the role of serum proteins in vector efficacy. Both lipoplexes and sequential complexes formed in the presence of mouse serum were examined by 2D gel electrophoresis. Unique or abundant proteins in sequential complexes were investigated for potential anti-inflammatory or target specific activity. Several serum proteins significantly reduced inflammation, while increasing the levels of transgene activity up to threefold compared to lipoplex. Furthermore, the same proteins did not decrease cytokine levels when added to preformed lipoplex. The results indicate sequential complexes (i.e., liposome mixed with protein, then DNA) and lipoplex are fundamentally different. These results suggest that a combination of protein content and DNA placement within the structure is responsible for the significantly improved efficacy and decreased inflammatory toxicity of these modified non-viral vectors.