Thymosin beta 4 is differentially expressed in the cerebrospinal fluid of Creutzfeldt-Jakob disease patients: a MALDI-TOF MS protein profiling study

MALDI-TOF protein profiling analysis permits the detection of peptides and small proteins in complex protein mixtures with great accuracy. We applied this analysis to cerebrospinal fluid (C S F) from 15 patients affected by Creutzfeldt-Jakob disease (CJD). We compared the levels of the normalized ion signals of 11 sporadic and 4 genetic CJD forms with those from ten healthy control subjects and eight non-CJD relapsing-remitting multiple sclerosis patients. In so doing, we detected 61 differentially expressed ion signals in CJD samples compared to controls. Among the 61 signals, 3 signals had significantly increased levels with high statistical significance (p <0.0001) and were located at 3238.3 m/z, 4963.7 m/z, and 8565.3 m/z. We characterized the 5.0 and 8.6 kDa proteins as thymosin beta 4 N-acetylated and free ubiquitin, respectively, while the 3.2-kDa peptide remained uncharacterized. Although elevated ubiquitin levels have previously been described in CJD, we have demonstrated for the first time the involvement of thymosin beta 4 in a neurodegenerative disease. To support the validity of thymosin beta 4 levels obtained by MALDI-TOF analysis, an independent enzyme immunoassay analysis was performed. Moreover, a validation cohort consisting of CSF from three CJD patients, five healthy subjects, and six non-CJD relapsing-remitting multiple sclerosis patients was analyzed in a similar way, yielding superimposable results. We propose that thymosin beta 4 is a potential new candidate marker for the ante mortem diagnosis of CJD disease.