Multiprotein Complex Analysis for Serum Proteomics by 2D BN/SDS Gel Electrophoresis

[Introduction] Serum is the most generally informative proteome from a medical standpoint and at the same time, its complexity and enormous dynamic range make serum the most difficult specimen to be dealt with. As low molecular weight proteins may be excreted through glomerular filtration system in a very short time range, targeting multiprotein complex in serum may be an attractive way of elucidating pathogenesis of various diseases. Here we established method for multiprotein complex analysis in serum using two dimensional Blue Native / SDS Gel electrophoresis (2D BN/SDS) and evaluated its diagniostic potentials by applying into animal models. [Methods] We collect murine serum samples from two experimental models; 1) Acute brain ischemia model and 2) Experimental Autoimmune Encephalomyelitis model (EAE). Both experiments were performed on SJL/J mouse background. Blood sampling was performed from orbital plexus in both experiments. Serum was obtained in each time point. One microliter of serum was run on a 2D BN/SDS gels. For the first dimension, 5–15% gradient gels cast on the Bio-Rad protean minigel system and were run overnight at 4 °C. Then the second dimension was run on a 15% gel with SDS condition. Proteins were stained with CBB reagents and stored at 4 °C before analysis. The data analysis was conducted by computational gel evaluating system and protein/peptide identification was performed by MALDI-TOF-MS. [Results and Discussion] We have generated a standard protocol for diagnostic 2D BN/SDS PAGE for serum samples. Applying this method, we have mapped all the peptides/proteins on this diagnostic 2D gels as a basic database of mouse serum. In the next step, abundance of each peptides/proteins was plotted according to its time course for both of the animal studies and have determined its annotation. As a result, multiprotein complex in serum shows a dynamic change on this diagnostic 2D gels and this was beautifully moved in a time course dependent manner. Especially, hemoglobin- haptoglobin (Hb-Hpt) complex revealed most dynamic change in both an acute brain ischemia and an experimental autoimmune encephalomyelitis models. By contrast, the complement system was shifted in terms of the complex formation in the later part of an acute brain ischemic model. This method can be applied to human serum proteomics.