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Contents Follicular fluid provides the microenvironment within which somatic cells proliferate and differentiate, and the oocyte matures. It contains a number of soluble factors implicated in various stages of follicular development, most of them being functionally unknown. The presence of several high-abundance proteins, mainly originating from the blood circulation, is a major challenge of follicular fluid proteomic analysis, as these proteins can mask or decrease the visualization of follicle-specific proteins. In this study, we evaluated the efficiency of two immunodepletion columns (ProteomeLab (TM) IgY-HSA and MARS-6) on follicular fluids of human, porcine and canine prior to 2D-PAGE. Our results showed that both columns were suitable to remove some of the high-abundance proteins present in human and canine follicular fluid. In conclusion, we demonstrated that the immunodepletion strategy enables the detection of new protein spots, increases resolution and highly improves the intensity of low-abundance proteins by 2D-PAGE.

Sepsis, defined as the systemic host response to infection, is not a single disease but a complex and heterogenous process. Effective therapies and their evaluation have been hampered by insufficient characterization of the underlying pathogenesis and in consequence limited ability to stratify patients into more homogenous subgroups. As no single or any combination of biomarkers at present can reliable be used for prediction there is an immense and vast growing interest in protein profiling. The proteomic analysis of plasma samples represents a challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins in addition to the desired biomarkers of interest that are typically low in abundance. The detection of possible biomarkers therefore requires a specific depletion of these high abundance proteins and/or optimized multidimensional protein fractionation methods based on charge, size, hydrophobicity or any combination therefore. The IgY-PF2D-nanoLC-MS/MS strategy applied in this study combines immunoaffinity subtraction chromatography (IgY-12) for the removal of high abundance plasma proteins with two-dimensional protein fractionation (PF2D) for separation and nano LC-MS/MS for identification of proteins. In this study, plasma samples from patients with sepsis and healthy controls were examined. More than 120 adult patients with manifest sepsis, severe sepsis and septic shock were included. Citrated plasma samples from five male patients with a clinically similar cause of sepsis and underlying disease were used for this part of the study. The plasma samples were taken from three survivors and two non-survivors at the first (diagnosis of sepsis) and last day (relocation or death) of their stay in the intensive care unit (ICU) respectively. Citrated plasma samples from three age matched healthy male individuals were used as reference. A total of 1,800 fractions were analyzed and 233 distinct proteins were identified with. 132 proteins from healthy male individuals, identified by double and/or triple determination with high reliability, were regarded as the reference protein pool. Only significantly up-regulated peaks present in all of the sepsis samples were selected for MS analysis, resulting in the identification of 17 distinct proteins present only in septic patients. Whereas most of these proteins are known to be involved in inflammatory response, some are considerably novel and exploring their roles in association with the pathophysiology of sepsis may lead to the identification of new biomarkers for sepsis.