Fig. 4.

Amounts of GPI-anchored proteins are reduced in CSF of patients with ALS. A Schematic outlining the experimental strategy to identify GPI-anchored proteins in patient CSF. Forty CSF samples from 20 control and 20 ALS patients were analyzed for global proteome analysis. Master pool was prepared by pooling equal volumes of CSF from 40 samples, aliquoted into 4 parts and added to one of the 11-plex channels for the normalization between 4 batches of 11-plex TMT experiments. After enzyme digestion of CSF proteins, the resulting peptides were labeled with TMT reagent, followed by pooling the TMT-labeled samples. Subsequently, the pooled peptides were pre-fractionated using basic pH RPLC and each fraction was analyzed by LC–MS/MS. Acquired mass spectra were searched against a reference protein database and statistical analysis was conducted to identify differentially expressed proteins. B After normalizing protein abundance values from 40 samples, GPI-anchored proteins were selected, and statistical analysis for the selected proteins was conducted. The proteins outside q-value cutoff lines (q-value 0.1) are differentially expressed proteins