Sepsis and septic shock are one of the main causes of death in intensive care units, with mortality rate up to 50%. This high mortality rate has raised awareness about sepsis and septic shock worldwide and many efforts are being made towards the discovery of biomarkers and new therapies to prevent and treat this life-threatening ...»»»»
Sepsis and septic shock are one of the main causes of death in intensive care units, with mortality rate up to 50%. This high mortality rate has raised awareness about sepsis and septic shock worldwide and many efforts are being made towards the discovery of biomarkers and new therapies to prevent and treat this life-threatening illness. Sepsis and septic shock have been associated with dysregulation of proteolysis in the organism. Peptidomics analysis of blood plasma is a suitable tool to identify peptides and proteins related to these diseases, which could help to better understand the role of proteolysis in the progression of the disease. Being plasma one of the most complex biological samples, the aim of this study was to optimize a sample cleanup method suitable for high throughput plasma peptide extraction compatible with nanoLC-MS/MS which allows the study of proteolysis in sepsis and septic shock. Up to nine methods were tested and the reproducibility of the two best performing ones was evaluated in three technical replicates analysis. The best performing method was a combination of ultrafiltration with 10KDa filter followed by two solid phase extraction steps: C18-like chromatography in 96 well plate format followed by strong cationic exchange (SCX) chromatography in micro-column format. Using this method more than 400 peptides were identified (ID). Neither system clogging nor matrix effect was observed, when applied for the analysis of 15 plasma samples from septic shock patients (Day 1 ICU entry & Day 7 after entry) and sepsis patients as a control (SC). Proteolysis dysregulation during sepsis and septic shock was evaluated and the highest proteolysis level was observed in SC replicates with 404 peptide IDs (SD = 81) and the lowest was observed in septic shock at DAY 7 with 175 peptide IDs (SD = 121). Moreover, two potential biomarker candidates were detected after Progenesis QI quantitative analysis (p-value < 0.05). Serum Amyloid A2 peptides were found to be 4 times more abundant in Septic Shock DAY 1 compared to DAY 7 and could be candidates for prognosis of septic shock are. Hepcidin peptides were found to be 9 times more abundant in DAY 1 than in SC and could be candidates for Septic Shock. Further studies are needed in order to confirm the two biomarker candidates as well as to fully understand the dysregulation of proteolysis and its relation with septic shock and sepsis.^^^^
