Proteomic Analysis Of The Plasma Of The Septic Mouse Induced By Cecal Ligation And Puncture

Background Sepsis and resulting multiple system organ dysfunction are the leading cause of death in critically ill patients in the world. After numerous unsuccessful trials of anti-inflammatory agents in patients with sepsis, investigators doubted that mortality could be decreased. Although it is generally appreciated that rampant, deregulated inflammatory pathways play a major role, a comprehensive understanding of the pathophysiology mechanism has remained elusive. Plasma contains not only the classical plasma proteins, but also all tissue proteins (as leakage markers) plus very numerous distinct immunoglobulin sequences. By comparing the plasma proteome between sepsis animals and controls, it may be possible to identify proteins that play important roles in the disease process and thus to study the pathophysiology mechanism of sepsis.Objective To evaluate whether treatment of plasma samples with DEAE-Cibacron Blue 3GA before 2-DE analysis can be used to remove albumin in order to increase the detection sensitivity of proteins present in low abundance and to identify proteins that play a role in the sepsis induced by cecal ligation and puncture by comparing the plasma proteome between operated mouse and the control and thus to study the pathophysiology mechanism of sepsis. Materials and Methods 1. Serum samples of mouse were treated with DEAE- Cibacron Blue 3GA and then subjected to SDS-PAGE and 2-DE. Protein spots were visualized using the silver staining method. Computer analysis of 2-DE and SDS-PAGE imaging was carried out usingImageMaster Platium software. 2. Male Kunming mice were subjected to cecal ligation and puncture (CLP group) or sham operation (control group). Blood was drawn at 4h, 24h after treatment, respectively. Image spots of the 2-DE gel were initially detected, matched and then manually edited. Five gel images of plasma samples in the CLP group were averaged and then compared with average gel of the control at the same time point. Only those significantly different spots (three-fold increase or decrease) were selected for analysis by mass spectrometry.Results 1. Eighty percents of albumin was removed when treated with DEAE-Cibacron Blue 3GA. Compared with results without treatment , the stained spots of 2-DE were significantly increased from 211±19 to 392±11. Forty minutes of DEAE-Cibacron Blue 3GA treatment resulted in enhanced visualization of 13 protein spots by three-fold, 6 by five-fold, 6 by ten-fold and 5 by twenty- fold. 2. Haptoglobin was overexpressed 24h after operation than that of 4h after operation. But there was not any difference between the CLP group and the control. 3. Comparison of the 2-DE images between the CLP group and the control 24h after operation showed that 39 distinct protein sports were overexpressed in plasma of CLP group, 18 of which were unique in CLP group. 4. Thirty-three of the 39 protein sports were identified by mass spectrometry. They collapsed into 8 distinct proteins: Complement C3, Serum amyloid P-component(SAP), kininogen, leucine-rich α2- glycoprotein, α1-acid glycoprotein, αl-antitrypsin, transferrin and hemopexin. Conclusion 1. Treatment of plasma samples with DEAE-Cibacron Blue 3GA before 2-DE analysis can be used to remove albumin in order to increase the detection sensitivity of proteins present in low abundance. 2. The increased expression of haptoglobin is associated with the trauma due to operation but not...