Human tissues of prefrontal cortical
regions were provided by the
Brain and Body Donation Program at Banner Sun Health Research Institute.
The AD case with short post-mortem interval (<3 h) was clinically
and pathologically characterized in accordance with established criteria.32 (link) This study was approved by Banner Sun Health
Research Institute. Adult rat brains were purchased from Pel Freez
Biologicals, and rat brain peptides were prepared as previously described.33 (link) The cerebral cortex of AD brain was homogenized
in 100 μL of lysis buffer (0.1 M Tris, pH 8.5, 8 M urea, 0.15%
sodium deoxycholate) at 4 °C using 0.5 mm glass beads for 5 min
in a Bullet Blender instrument (Next Advance).34 (link),35 (link) The entire cell lysate without clarification of the insoluble materials
was digested with Lys-C (Wako, 200:1 by weight) at room temperature
for 0.5 h in the lysis buffer, followed by trypsin digestion (Promega,
200:1 by weight) in 2 M urea, 0.1 M Tris-HCl, pH 8.5 at room temperature
overnight. The peptides were then acidified with 0.15% TFA, precleared
by centrifugation, desalted with Sep-Pak C18 SPE column (Waters),
and eluted with 40% acetonitrile (ACN) plus 0.1%TFA. The eluent was
dried and stored at −80 °C for further usage.15 (link) Protein quantification was carried out by short
SDS-gel-based staining and BCA method.31 (link)
regions were provided by the
Brain and Body Donation Program at Banner Sun Health Research Institute.
The AD case with short post-mortem interval (<3 h) was clinically
and pathologically characterized in accordance with established criteria.32 (link) This study was approved by Banner Sun Health
Research Institute. Adult rat brains were purchased from Pel Freez
Biologicals, and rat brain peptides were prepared as previously described.33 (link) The cerebral cortex of AD brain was homogenized
in 100 μL of lysis buffer (0.1 M Tris, pH 8.5, 8 M urea, 0.15%
sodium deoxycholate) at 4 °C using 0.5 mm glass beads for 5 min
in a Bullet Blender instrument (Next Advance).34 (link),35 (link) The entire cell lysate without clarification of the insoluble materials
was digested with Lys-C (Wako, 200:1 by weight) at room temperature
for 0.5 h in the lysis buffer, followed by trypsin digestion (Promega,
200:1 by weight) in 2 M urea, 0.1 M Tris-HCl, pH 8.5 at room temperature
overnight. The peptides were then acidified with 0.15% TFA, precleared
by centrifugation, desalted with Sep-Pak C18 SPE column (Waters),
and eluted with 40% acetonitrile (ACN) plus 0.1%TFA. The eluent was
dried and stored at −80 °C for further usage.15 (link) Protein quantification was carried out by short
SDS-gel-based staining and BCA method.31 (link)
