Fig. 1

Ceruloplasmin in PD patients CSF shows structural changes, ⁹⁶²NGR-motif deamidation and gain of integrin-binding function. a) Structural changes. Western blot analysis after limited trypsin proteolysis (Try, +) showed higher sensitivity of the endogenous ceruloplasmin from PD’s CSF compared to Cp from healthy subjects (H-CSF). b) Deamidationof the ⁹⁶²NGR-motif. CSF samples (70 μl each) from healthy subjects (H, n = 13) and PD patients (PD, n = 10) were digested with trypsin and directly analysed by quantitative parallel reaction monitoring mass spectrometry. Data are reported as peak-intensity of the peptide containing the ⁹⁶²NGR-motif as it is (MHAINGR) and deamidated (MHAIDGR), normalized for the ceruloplasmin internal reference peptide (IRP), namely LISVDTEHSNIYLQNGPDR, which yielded the lowest median CV for both ⁹⁶²NGR- and ⁹⁶²DGR-containing peptides. Five technical replicates for each sample were run and use for the quantification. c) Binding to αvβ6 of endogenous ceruloplasmin from CSF of healthy subjects (H-CSF) or PD patients (PD-CSF) revealed by ELISA at time zero or after 9 days aging. Two independent experiments in triplicate were performed using CSFs of different subjects in each experiment (total subjects for each group n = 8). In B and C data were analyzed by student’s t test; means with standard error, calculated using pooled data from different experiments, are indicated (** = p < 0.01)