Among 59 proteins, two novel proteins: glutathione S-transferase P (GSTP1),

peroxiredoxin-1 (PRDX1) were found to be elevated in blood samples from stroke patients [44] and [45]. The team of Sanchez used human postmortem CSF as a model of global brain insult and identified two markers. PARK7 and nucleoside diphosphate kinase A (NDKA) that are subsequently validated to be candidate plasma markers for stroke in CSF and in plasma [46]. Lastly, Cuadrado et al. analyzed the human brain Protease Inhibitor Library supplier proteome following ischemic stroke and identified 39 proteins by 2D-gel electrophoresis/MALDI-based proteomics. Among those that are confirmed by immunblotting in the brain parenchyma are dihydropyrimidinase-related protein 2 (CRMP-2), vesicle-fusing ATPase (N-ethylmaleimide-sensitive fusion protein; NSF) and Rho GDP-dissociation inhibitor 1 (Rho-GDI alpha) [47]. For potentially plasma markers that can differentiate ischemic from hemorrhagic stroke: S100B plasma levels were increased in intracerebral hemorrhage (ICH),

whereas sRAGE levels were decreased in ICH as compared to Ischemic stroke thus INCB024360 cell line S100B/RAGE pathway might be promising markers in this regard [48] (Table 1). For clinical utility purposes, it is often important to not only identify what marker is present in a clinical sample, but how much of the candidate marker is present. This is particularly important in biofluid samples such as CSF and serum/plasma. Sandwich ELISA is the most classic quantitative detection method for proteins. However, it requires two high affinity antibodies that are compatible with each other (non-competing) to the same target proteins, and the assay constructed is compatible with the matrix environment without high background. Alternatively, if a target protein can be identified and quantified

by a mass spectrometry-based aminophylline method, it can be a powerful approach. There are two basic approaches for quantification: relative quantification (samples are differentially labeled then, the peak intensity ratio between heavy and light peptides is measured to compare protein abundance) and absolute quantification (a known amount of isotope-labeled standard is mixed with the analyte, the absolute amount of the analyte is calculated from the ratio of ion intensities). Many labeling methods have been developed, including chemical, isobaric, and metabolic labeling techniques. The isotope-coded affinity tags (ICAT) is a chemical labeling method [49] and [50], in which the Cys residues in proteins is coupled with a compound containing stable isotope (light and heavy) that is used for labeling of different samples. Both samples are then combined and subjected to protease digestion followed by affinity-purification of Cys-containing peptides. Another in vitro labeling method is Isobaric tagging with a molecular tag that has a distinct added mass.