Clinical proteomics aims for the identification and characterization of proteins, which are important from the medical point of view. Apart of raw cancer tissue, different body fluids are used as the source of proteins, but the most popular are blood plasma or serum and in some cases also cerebrospinal fluid. The proteins found may serve as biomarkers, prognostic proteins or targets for newly developed drugs. For the analysis of these proteins commonly known techniques, such as electrophoresis and mass spectrometry are in use. In case of mass spectrometry for profiling and identification of proteins both traditional and tandem mass spectrometers are applicable. There are two general mass spectrometry-based approaches in diagnostic proteomics. The first one supports identification of proteins in samples of interest, which are unique or significantly differ in comparison with control samples. In this case multidimensional fractionation of proteins combined with identification by tandem mass spectrometry is usually performed. In last years so called pattern diagnostic proteomics became very popular, mainly because of its productivity and ease of use. This approach is based on comparison of different sample profiles acquired with MALDI-TOF (or SELDI) mass spectrometers. The patterns of differentiated proteins are studied, but identification of proteins is not needed, although can be performed when necessary. In our project we concentrate on pattern proteomic analysis of blood serum in diagnosis of breast cancer. Protein profiles of samples obtained from patients suffering from breast cancer are compared with control samples for new potential biomarker discovery. The point of our study is the analysis of large amount of samples, which are specifically prepared for analysis. In this presentation preliminary results, mainly studies on preparation of good quality spectra and control samples preparation techniques are presented. While in many studies the depletion of blood plasma from high abundant proteins prior to further analysis is routinely executed to concentrate the protein fractions of interest, recent studies have shown that these practices may result in removing potentially important diagnostic information carried with transporter proteins within the blood such as albumin for instance. These proteins are known to bind physiologically important species such as hormones, cytokines, and fragments of proteins derived from action of different proteinases. For our purposes we decided to look for the indirect solution and to fractionate the blood plasma without removal of small proteins and peptides by gentle denaturation of samples. We also examined the methods to improve the reproducibility of analyses as it is very important factor when doing semi-quantitative proteomics. The optimal parameters of MALDI spectra acquisition were tested to improve the quality of analyses.

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