Biomarkers discovery by peptide and protein profiling in biological fluids based on functionalized magnetic beads purification and mass spectrometry.

Proteomics aims for the full identification and quantification of all expressed proteins in any organism. This is however an extremely tedious task since one gene often accounts for multiple proteins due to gene splicing and processing of proteins, such as the addition of post-translational modifications. Moreover, the concentration range of occurring proteins varies more than a factor of one million. For these reasons, protein profiling was considered a promising technique in the early days of proteomics. Ideally a protein profile can be observed in one single measurement. In various clinical studies profiling methods have been successful in the detection of proteome variations as a consequence of an altered homeostasis. Proteins that are differentially expressed as a consequence of a disease are very useful in medical science as they can be used as new biomarkers for the diagnosis, prognosis and as possible therapeutic targets. In order to find such proteins or biomarkers two different kinds of biological material have been used: tissue samples and body fluids. Tissues are obtained from biopsies, from stable cell lines or cell cultures, or from subcellular fractions. Despite their large usage tissues suffer from several disadvantages. Tissue samples are difficult to obtain and are comprised of several different type of cells. Standardization of the methods to obtain subcellular fraction that affects its preparation and purity is a challenge not yet solved. The difference between a cell culture and its corresponding wild type present in the body limits the translation of information derived from the first to the latter. On the contrary body fluids do not suffer from these limitations inherent to tissue samples. Fluids are very easily accessible with non- or very low-invasive methods at relatively low cost. They perfuse all the organs in the body carrying secreted protein from tissues. Therefore the protein profile of the biological fluids can reflect the status of the body. Among biological fluids serum, plasma and urine are the most analyzed samples but also cerebrospinal fluid (CSF), saliva, amniotic fluids have been used. Moreover classical methods to investigate the tissue proteome, aiming at biomarker discovery, are generally based on two-dimensional electrophoresis (2DE) and are not suitable for clinical chemistry lab requirements in which large sample cohorts have to be analyzed in a short time. This addresses another great potential of body fluids profiling: the analysis can be carried out high-throughput without sacrificing robustness and quality of the method. In fact 2DE is a laborious process that is difficult to automate. It still suffers from several technical limitations in terms of repeatability and reproducibility even though progress has been made using three different fluorescent labels that enables simultaneous migration of three samples on the same gel (e.g proteins extracted from control and disease, and the internal standard).Since the beginning of the 1990ties, when this new term (proteomics) was coined, a lot of progress has been made. Among them, several strategies to search these biomarkers in biological fluids have been developed in order to try to tackle some of the limitations of the current methods. Nowadays, mass spectrometry (MS) is the method of choice for the analysis of proteins, and as a consequence the field is now often referred to as MS-based proteomics. Direct analysis of the biological fluids with mass spectrometry is a challenging approach due to the sample complexity. To carry out a repeatable and robust mass spectrometric analysis of proteins in body fluids a suitable clean-up procedure is required in which salts and detergents are removed. The presence of salts can suppress the ionization in the mass spectrometer and chromatographic profiles may be influenced by from tailing due to co-elution of contaminants1. Therefore a pre-fractionation of the fluids is essential in order to increase the number of proteins that can be detected within a single MS-experiment, thus facilitating the discovery of new markers. Moreover, the fractionation of the biological fluids will also enrich low abundant proteins in fractions. These approaches lead to build the protein profile of the different biological fluids. Variations observed in patient profiles of body fluids compared to those of controls can be used to find the best pattern of signals that allows to discriminate two populations or to stratify the patients according to tumour stage or to the response to the therapy. One the major advantages of this strategy is that no pre-knowledge of the identity of signals selected for the cluster is needed to allow their use as biomarkers2. A specific agent to capture proteins enriches the sample and thus contributes to sensitivity enhancement. In general, protein separation techniques are based on different protein physical properties, such as size, isoelectric point, solubility and affinity. Materials known from different chromatographic platforms are coupled to the surface of a carrier in order to obtain peptides and proteins. One of the first approaches to pre-fractionate the body fluid proteome using an activated surface was the Surface-Enhanced Laser Desorption/Ionization (SELDI) technique. The SELDI technique for protein profiling is probably the most known and widely used approach in which biological fluids are applied directly to a target plate that is later introduced into a mass spectrometer. After removing unbound material to the modified surface of the SELDI chip, the molecular weight of the captured proteins on the target plate is determined using a time-of-flight (TOF) mass analyzer3. In this way the body fluid protein profile for the studied population is obtained. This technology is not free of criticism. In particular not very good reproducibility of the results due to drift, noise or the use of different lots of chips are reported. Moreover the direct identification of these markers cannot be carried out using the SELDI-TOF system. Their identity has to be determined with different analytical approaches. Promising alternatives to this technology are based on magnetic beads with a functionalized or activated surface or on miniaturized chromatographic systems that allow off-line fractionation of the proteome present in the fluids before MS analysis. The combination of magnetic bead purification and matrix-assisted laser desorption ionization (MALDI) TOF-MS has been shown a powerful alternative to the SELDI-platform: the active surface of magnetic beads is much larger, resulting in a higher binding capacity, and identification of captured peptides and protein is possible through the use of a more advanced TOF mass analyzer. Moreover, only a small part of the eluted peptide/proteins fractions are used for the protein profile and the remaining sample can be to use to identify markers with other MS-approaches (e.g. MALDI-TOF/TOF or LC-ESI-MS/MS) without the need of additional purification. This review is mainly focussed on the pre-fractionation based on magnetic beads and their applications.