Body_Fluids

Body Fluids

Body fluids such as plasma, tear, and saliva, are expected to be excellent sources of protein biomarkers in the human body, since they contact a variety of tissues that can be affected by diseases like cancer. Furthermore, there is a need to characterize the protein components of these fluids with in-depth methods in both healthy and disease conditions.

On our group, we have been applying state-of-art mass spectrometry to catalogue proteins present on several body fluids as the urine, cerebrospinal fluid (CSF), seminal fluid, tear fluid, and others. Peripheral body fluids (i.e., which contact only specific tissues and not the whole system as the plasma) are attractive targets because a lower dilution of proteins derived from adjacent tissue is expected, thus increasing the chance that potential markers have detectable levels on these fluids, when compared to its level on plasma.

At the current stage, the main objective of our studies is to generate data from healthy donors that rely on high-stringent identification criteria, resulting in high-confidence protein hits, with a false positive rate lower than 1 in 10000. Our routine workflow involves the fractionation of the sample though SDS-PAGE, then in-situ digestion of excised gel pieces of the electrophoretic run followed by LC-MS/MS. Our results showed that, with this technology, we can improve the number of described proteins on previous studies on several fold. All identification data is stored on the Max-Planck Unified Database (MAPU, http://proteome.biochem.mpg.de/), being available for the scientific community after publication.

Our future perspective is to use our current knowledge as an initial screening to be used as reference on subsequent studies, involving the analysis of samples originated from disease conditions.

In a large group of proteomics approaches the obvious advantages of subcellular fractionation are often ignored. Although it is possible to perform a global proteome profiling using non-fractionated (whole lysates) or roughly fractionated but not purified cell components, these approaches are limited to identification of almost exclusively highly abundant proteins. Further reduction of protein complexity by applying different types of protein and peptide fractionation might enable identification of some proteins present in minute amounts. Unfortunately, this mutidimensional separation approach usually leads to a generation of high number of fractions that have to be analyzed individually which requires extensive analytical capabilities in terms of instrumentation and time. Even circumventing these technical limitations, large differences between different proteins in individual fractions could make it impossible to profile and map low abundant proteins.

Subcellular fractionation offers an efficient way to reduce sample complexity but also can provide information on intra-cellular distribution of proteins and or their forms. Most types of organelles can be efficiently purified or enriched in this way. Combination of differential and gradient centrifugation enables separation of various organelles by their size and buoyant density. The extent of enrichment of a particular organelle can be quantitatively monitored by measuring enzymatic activities accompanying them, the so- called marker activity. Mitochondria prepared in this way were used for a comprehensive study on mitochondrial and plasma membranes.