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A new 6-plex isobaric mass tagging technology is presented, and proof of principle studies are carried out using standard protein mixtures and human cerebrospinal fluid (CSF) samples. The Tandem Mass Tags (TMT) comprise a set of structurally identical tags which label peptides on free amino-terminus and epsilon-amino functions of lysine residues. During MS/MS fragmentation, quantification information is obtained through the losses of the reporter ions. After evaluation of the relative quantification with the 6-plex version of the TMT on a model protein mixture at various concentrations, the quantification of proteins in CSF samples was performed using shotgun methods. Human postmortem (PM) CSF was taken as a model of massive brain injury and comparison was carried out with antemortem (AM) CSF. After immunoaffinity depletion, triplicates of AM and PM CSF pooled samples were reduced, alkylated, digested by trypsin, and labeled, respectively, with the six isobaric variants of the TMT (with reporter ions from m/z = 126.1 to 131.1 Th). The samples were pooled and fractionated by SCX chromatography. After RP-LC separation, peptides were identified and quantified by MS/MS analysis with MALDI TOF/TOF and ESI-Q-TOF. The concentration of 78 identified proteins was shown to be clearly increased in PM CSF samples compared to AM. Some of these proteins, like GFAP, protein S100B, and PARK7, have been previously described as brain damage biomarkers, supporting the PM CSF as a valid model of brain insult. ELISA for these proteins confirmed their elevated concentration in PM CSF. This work demonstrates the validity and robustness of the tandem mass tag (TMT) approach for quantitative MS-based proteomics.

Following any form of brain insult, proteins are released from damaged tissues into the cerebrospinal fluid (CSF). This body fluid is therefore an ideal sample to use in the search for biomarkers of neurodegenerative disorders and brain damage. In this study, we used human post-mortem CSF as a model of massive brain injury and cell death for the identification of such protein markers. Pooled post-mortem CSF samples were analyzed using a protocol that combined immunoaffinity depletion of abundant CSF proteins, off-gel electrophoresis, SDS-PAGE and protein identification by LC-MS/MS. A total of 299 proteins were identified, of which 172 proteins were not previously described to be present in CSF. Of these 172 proteins, more than 75% have been described as intracellular proteins suggesting that they were released from damaged cells. Immunoblots of a number of proteins were performed on individual post-mortem CSF samples and confirmed elevated concentrations in post-mortem CSF compared to ante-mortem CSF. Interestingly, among the proteins specifically identified in the post-mortem CSF, several have been previously described as biochemical markers of brain damage.