One of the powerful multidimensional separation methods in proteomics is ion-exchange chromatography (IEC) in the first dimension. Reversed-phase liquid chromatography (RPLC) most often in the second dimension is due to its compatibility with the downstream mass spectrometry (sample concentration, desalting properties, and used volatile solvents). IEC is very suitable for the separation of proteins and peptides based on their differences on overall charges. IEC’s stationary phase is either anion or cation exchanger, prepared by immobilization of positively or negatively charged

functional groups learn more on the surface of chromatographic column, respectively. Proteins or peptide separation occurs by linear change of the mobile-phase composition (salt concentration or pH) that decreases the interactions with the stationary phase

and finally eluted [17]. For neuroproteomic studies, Gao et al. [26] have described a method for the 2-D differential display of proteins of inflicted vs. non inflicted pediatric TBI cerebrospinal fluid (CSF) study. Also, Kobeissy et al. [27] have used a mixed cation- and anion-exchange chromatography (CAX) and 1-D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) approach for differential protein separation, differentially expressed protein bands are excised and trypsinized followed by nanoLC and ESI-MS/MS protein identification. With this method, 59 proteins were identified as potential biomarker candidates

(Fig. 1). Protein marker candidates identified Bafilomycin A1 chemical structure include MAP-2, ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1), collapsin response element-2 Monoiodotyrosine (CRMP-2), synaptotagmin and alpha II-spectrin breakdown products UCH-L1 was one of these proteins that was subsequently confirmed to be a good translational biomarker for TBI. Liu et al. [28] first validated that UCH-L1 marker is not only differentially expressed in rat brain tissue, but also in biofluids following brain injury in rodents. CSF is important here as it is proximal to the injured organ, and thus likely to have these candidate markers in high concentrations. Indeed that was the case for UCH-L1 – which is elevated not only in the rat model of TBI (controlled cortical impact; CCI) but also in the rat model of ischemic stroke (using both quantitative immunblotting and sandwich ELISA method) [28]. Secondly with the aid of the two antibody-based sandwich ELISA, they were able to identify elevation of UCH-L1 in serum in both injury models as well. Subsequently, UCH-L1 protein was found to be elevated in human CSF and serum samples in both adult and in pediatric TBI [29], [30] and [31] (Table 1). Others have also used 2-D separation followed by MS/MS to identify candidate protein alterations for SCI [16], [32], [33] and [34]. For example, Yan et al.