Figure 1 Peptidomic workflow for nano-liquid chromatography coupled to electrospray ionization and tandem mass spectrometry (nano-LC-ESI-MS/MS) for the analysis of purified Tau protein, rat differentiated primary culture and mouse brain lysate. Subjecting the proteins to an in vitro calpain-1 digestion results in both high molecular weight (HMW) protein fragments and low molecular weight (LMW) proteolytic peptides production. HMW fragments were reduced with 10% β-mercaptoethanol (βME) and 8× sample buffer. The LMW fragments were subjected to C18 zip-tip and analyzed using reverse-phase- ultra-high-pressure chromatography (RP-UHPLC) followed by nano-LC-ESI-MS/MS with a data-dependent acquisition (DDA). The HMW fragments were validated by western blotting. Panel (A) shows the workflow followed for proteolysis of recombinant purified tau-441 (non-phosphorylated and phosphorylated). Panel (B) shows the isolation of cortex fraction of transgenic human tau (htau) mouse brain. Panel (C) shows the protocol followed for the treatment of rat cerebrocortical primary neuronal culture with neurotoxic agents. The neurotoxic agents were used includes okadaic acid (OA), staurosporine (STS), A23187, and maitotoxin (MTX).

Figure 2 Identification of high molecular weight calpain-mediated tau proteolytic peptides by immunoblotting. (A) (recombinant tau), (B) (recombinant phospho-tau), and (C) (mouse brain lysate) are Western blots displaying intact tau and high-molecular-weight tau breakdown products (HMW-tau-BDPs) treated with different calpain-1 concentrations (1:100, 1:50, 1:25, and 1:10). (D–F) are quantification graphs of the intact tau (63–65K) and tau-BDP (40K/38K) from tau, p-tau, and mouse brain lysate, respectively. Total tau antibody (DA9, amino acids (a.a.): 102–145) was used. Densitometric quantification of the intact and tau-BDP was performed using image-J. Data are presented as ± standard error of the mean (SEM) for n = 3. Statistical analysis was performed with one-way analysis of variance (ANOVA). For multiple comparisons, one-way ANOVA followed by the Bonferroni’s post-hoc test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns: non-significant.

Figure 3 Characterization of low molecular weight calpain-mediated tau proteolytic peptides by mass spectrometry. (A–C) panels show tau (P10636-8) sequence with colored residues representing the peptides identified from recombinant tau, recombinant p-tau, and mouse brain lysate tau, respectively. The underlined residues represent the overlapping sequence between two proteolytic peptides. Amino acid residues in black color represent an unidentified sequence. (D–F) are schematic representations of tau, p-tau, and mouse brain htau peptides generated from calpain-1 digestion, respectively. The core peptides are shown in a colored rectangular box, while the variability of the different lengths of peptides is represented as error bars. These error bars were calculated from all biological replicates peptides that were high in confidence, XCorr value of 3, and minimum PSM value of 20 from Sequest HT search engine of proteome discoverer 2.2. Peptides found in control were omitted. Brackets on y-axis between letters represent adjacent amino acids that are not part of the original sequence but might be necessary for calpain cleavage. (G–I) are top-scored peptides based on peptide sequence matches (PSMs) from tau, p-tau, and mouse brain lysate samples, respectively. The y-axis represents the count number of each peptide, and x-axis represents mass over charge ratio (m/z) for non-phosphorylated-tau-441. For each selected peptide, at least 20 PSM and an XCorr value of 3 was used from proteome discoverer 2.2 software to present high confidence identifications.

Figure 4 Tau proteolytic peptides heatmap profile from samples digested by calpain. (A–C) shows mass spectrometric intensities peptide profile of calpain-mediated tau, p-tau, and mouse brain, respectively (accession#: P10636-8). At each amino acid residue along the protein, the height of the green bars is proportional to the count of peptides overlapping this position. The intensity of the color (green) is proportional to the sum of the peptide intensities overlapping this position. The left side of the plots shows the dilution of calpain used for each condition.