The interest for the development of new methods and the application of hyphenated techniques for complex biological samples characterization is growing, nowadays, more and more. This work faces the application and setup of hyphenated techniques with two aims: a) to identify a new biomarker that can be associated to PSA (Prostate Specific Antigen) for prostate cancer (ADK) diagnosis, b) to develop a hyphenated technique for glycosphingolipids analysis. In clinical proteomics, the identification

more »

... f new biomarkers is hampered by the discrepancy between the quantitative range of detection of instruments and the concentration range of proteins and peptides in biological fluids. Since the quantitative range of detection of instruments cannot be changed, the critical point is to reduce the dynamic range in examined biological fluids. In serum, this can be obtained by the elimination of high abundant proteins thanks to a selective immunodepletion. The serum low abundant protein fraction from 30 ADK patients and 30 controls was analyzed by MALDI profiling. The applied methodology was able to detect differentially abundant peaks in spectra of ADK vs no ADK controls. To transfer the obtained results in clinical diagnostic, the identification of species revealed by MALDI profiles is mandatory for two main reasons: data validation through the use of immunometric techniques and methodological development for a large scale test. Thanks to the hyphenation between chromatografic techniques, such as gel filtration on a HPLC and reverse phase on a nanoLC, and MALDI mass spectrometry, we were able to identify the highly changed 2021 m/z peak, that corresponds to C3f peptide and that derives from the proteolyses of Complement C3b. To confirm its increment in ADK patients with low PSA values an immunoblotting experiment was conducted. The results were validated on a second group of patients and controls. The increase of C3f expression could be related to a deregulation of C3 complement, cleft to iC3b, that inhibits the selffeeding cycle of the alternative pathway hampering the association of C3b to factor B. Therefore C3f peptide could be used in association to PSA serum value to decrease the number of "false negative". An important aspect of the biomedical research and, indirectly, of clinical diagnostic is represented by a class of signal molecules that are important in a number of diseases, including neurological and cardiovascular disorders, cancer, lipid storage diseases, and the metabolic syndrome. Despite the increasing clinical interest, glycosphingolipids (GSLs) analysis cannot benefit by high-throughput techniques that allow an easy identification and quantitation of these species, characterized by similar molecular weights. Actually, their characterization is based on radioactive counts after metabolic radiolabeling, or on staining with proper dyes that doesn't allow a precise quantitation. New approaches including LC-MS are largely used but they are time consuming and operator dependent. We develop the direct hyphenation HPTLC-MALDI, to obtain a precise qualitative and quantitative analysis of the glycosphingolipids profile from murine myoblasts, and to distinguish every fatty acid chain variant for each GSL. An important aspect is that the methodological setup was coupled to biological samples analysis and results were compared to those obtained through the use of radioactive precursors and densitometric analysis on GSLs extracted from wild type and NEU3 overexpressing murine myoblasts. The proposed hyphenation is of great applicability to the glycosphingolipids characterization because it reduces the