Proteome/Proteomic analysis serum samples

Bjorhall, K., Miliotis, T., Davidsson, P. (2005). Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples. Proteomics 5, 307-317. 

Without sample preparation, the analysis of a dilute serum sample by CE with ultraviolet (UV) detection leads to the separation and detection of the major serum proteins, y-globulin, transferrin (Tf), P-lipoproteins, haptoglobin, a2-macroglobulin, a i-antitrypsin (AAT), a i-lipoproteins, hiunan semm albumin (HSA), pre-albumin, and complements [32], as shown in Figure 22.4. These proteins often mask other important analytes that are usually more clinically relevant than the major proteins. In fact, it has been widely mentioned that global approaches to proteome analysis detect less than 0.1% of the protein species present in a sample, which span a range of 10 orders of magnitude between the most abundant and the less abundant proteins [33]. 

The sample materials from which proteins for proteomics studies may be extracted include fresh or snap-frozen cells from varied sources such as biological fluids, (serum, urine, plasma) and solid tissues such as biopsy specimens. Moreover, proteins isolated from ethanol-fixed paraffin-embedded tissues can be utilized for MS analysis.2 Protocols for the identification of proteins from formalin-fixed paraffin-embedded (FFPE) tissues have been recently developed.3 4 FFPE materials are the most common forms of biopsy archives utilized worldwide, and represent an important advancement for the large-scale interrogation of proteins in archival patient-derived materials. Finally, laser capture microdissected tissues have been successfully used for MS analysis.45... 

Any proteomic study starts with the collection of proteins from biological samples such as cell culture media, cultured cells, serum, or any biological fluid, and a variety of animal tissues. The first step is to obtain a protein sample under conditions of least protein degradation. This involves use of various protease inhibitors that stop the protein degradation. The use of protease inhibitors depends on the type of sample and the analytical technique used in the subsequent analysis. The selection of protease inhibitors used is critical since many protease inhibitors and detergents used in the preparation of tissue homogenates can interfere with mass spectrometry (MS)... 

The use of two-dimensional gel electrophoresis for differential analysis in proteomics was revolutionized by the introduction of 2-D fluorescence difference gel electrophoresis (2-D DIGE). This fluorescence-based technique allows the use of multiplexed samples and an internal standard that virtually eliminates gel-to-gel variability, resulting in increased confidence that differences found between samples are due to real induced changes, rather than inherent biological variation or experimental variability. 2-D DIGE has quickly become the gold standard for gel-based proteomics for studying tissues, as weU as cell culture and bodily fluids such as serum or plasma. 

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