Figure 2.

iTRAQ-based mass spectrometry to identify tau protein–protein interactions modulated by aa 18–28. A, production of recombinant tau proteins. GST-tagged tauFL and tauΔ18–28 were produced in E. coli. Samples of washed and eluted protein were separated by SDS-PAGE and visualized by Coomassie Brilliant Blue. B, experimental setup for quantitative comparison of tau protein–protein interactions in dependence of the 11-aa motif in tau's N-terminal region using 8-plex iTRAQ. Technical replicates of GST pulldown with GST-tagged tauFL and tauΔ18–28 were subjected to tryptic digest and iTRAQ labeled (labels 113, 114, 115, 116, 117, 118, 119, and 121). Peptides were detected by mass spectrometry (Triple TOF 5600⁺ mass spectrometer, SCIEX). Cross-comparison ratios of iTRAQ-labeled peptides were determined by ProteinPilot version 4.0 software.