Thymosin beta 4 is differentially expressed in thecerebrospinal 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 proteinmixtures with great accuracy.We applied this analysis to cerebrospinal fluid (CSF) from 15 patients affected by Creutzfeldt-Jakob disease (CJD).We compared the levels of the normalized ion signals of 11 sporadic and 4 geneticCJDformswith those fromten 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 b4 Nacetylated and free ubiquitin, respectively, while the 3.2-kDa peptide remained uncharacterized. Although elevated ubiquitin levels have previously been described in CJD, we havedemonstrated for the first time the involvement of thymosin b4 in a neurodegenerative disease. To support the validity of thymosin b4 levels obtained byMALDI-TOFanalysis, 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 b4 is a potential new candidate marker for the antemortemdiagnosis of CJD disease.