Identification of biomarkers indicating cellular changes after treatment of neuronal cells with the C3 exoenzyme from Clostridium botulinum using the iTRAQ protocol and LC-MS/MS analysis.

Proteomic approaches are used to identify biomarkers, to monitor pathological changes inside of cells and for a better diseases diagnosis. Comparable changes in protein homeostasis also occur in differentiating cells and proteomic techniques should be suitable to identify biomarkers that indicate different steps of cellular development. The C3 exoenzyme from Clostridium botulinum (C3bot) inactivates Rho GTPases and induces morphological cellular changes like cell rounding and neurite outgrowth [G. Ahnert-Hilger, M. Höltje, G. Grosse, G. Pickert, C. Mucke, B. Nixdorf-Bergweiler, P. Boquet, F. Hofmann, I. Just, J. Neurochem. 90 (2004) 9]. To investigate these observations further a comparative proteomic approach has been chosen to elucidate C3bot effects in the neuroblastoma cell line model SH-SY5Y. The screening method applied for biomarker detection was based on the stable isotope approach isobaric tagging for relative and absolute quantification (iTRAQ). Proteins of C3bot-treated and untreated cells were digested and peptides were labeled by the iTRAQ reagent, combined, and separated by means of a two-dimensional nano-HPLC system. Peptide analysis was performed in a MALDI-TOF/TOF mass spectrometer. Identification and quantification of peptides and their corresponding proteins were accomplished by MS/MS spectra analysis. Overall, five replicate measurements identified 355 different proteins of which 235 were accessible for quantification. C3bot altered the concentration of 55 proteins (at least 1.3-fold) and several proteins were identified as possible biomarker candidates that indicate C3bot-induced cellular changes.