Supplemental Method 1a
Blue native-polyacrylamide gel electrophoresis (BN-PAGE)
Membrane pellets from the 41% sucrose gradient ultracentrifugation fraction
were solubilised in extraction buffer (1.5 M 6-aminocaproic acid, 300 mM Bis–
Tris, pH 7.0) and 10% Triton X-100 (stock solution was added at a ratio of 1:4
to achieve a final concentration of 2% Triton X-100) and vortexed every 10 min
for 1 h. Following solubilisation, the samples were cleared by centrifugation at
20 000 x g for 60 min at 4°C. The protein concentration was estimated using the
BCA protein assay kit (Pierce, Rockford, IL, USA), where 50 μg of the
membrane protein preparation was applied onto the gels. Next, 16 µL of BN
PAGE loading buffer [5% (w/v) Coomassie G250 in 750 mM 6-aminocaproic
acid] was mixed with 100 µL of the membrane protein preparation and loaded
onto the gel. BN-PAGE was performed in a PROTEAN II xi Cell (BioRad,
Germany) using a 4% stacking gel and 5–18% running gel. The BN-PAGE gel
buffer contained 500 mM 6-aminocaproic acid and 50 mM Bis–Tris, pH 7.0; the
cathode buffer contained 50 mM tricine, 15 mM Bis–Tris and 0.05% (w/v)
Coomassie G250, pH 7.0; and the anode buffer contained 50 mM Bis–Tris, pH
7.0. The voltage was set to 50 V for 1 h and 75 V for 6 h and was then
sequentially increased to 400 V (maximum current 15 mA/gel, maximum
voltage 500 V) until the dye front reached the bottom of the gel(Ghafari et al.
2012a). Native high-molecular-mass markers were purchased from Invitrogen
(Carlsbad, CA, USA).
Supplemental Method 1b
In-gel digestion of proteins and peptides
Spots selected from the SDS gel recognised by antibodies against the dopamine
D1 receptor subunit were placed into a 1.5-mL Eppendorf tube. The gel pieces
were washed with 50 mM ammonium bicarbonate and then washed twice with
washing buffer (50% 100 mM ammonium bicarbonate/50% acetonitrile) for 30
min each with vortexing. Acetonitrile (100%; 100 µL) was added to the tube to
completely cover the gel pieces and the mixture was incubated for 10 min. The
gel pieces were completely dried using a SpeedVac concentrator. Reduction of
the cysteine residues was performed using a 10 mM dithiothreitol (DTT)
solution in 100 mM ammonium bicarbonate pH 8.6 for 60 min at 56°C. After
the DTT solution was discarded, the same volume of 55 mM iodoacetamide
(IAA) solution in 100 mM ammonium bicarbonate buffer pH 8.6 was added and
the sample was incubated in darkness for 45 min at 25°C to achieve alkylation
of the cysteine residues. The IAA solution was replaced with washing buffer
(50% 100 mM ammonium bicarbonate/50% acetonitrile) and washed twice for
15 min with vortexing. The gel pieces were washed and dried in 100%
acetonitrile (ACN) followed by drying in a SpeedVac. The dried gel pieces were
re-hydrated in a 12.5 ng/µL trypsin (Promega, Germany) solution that was
reconstituted with 25 mM ammonium bicarbonate or 12.5 ng/µL chymotrypsin
(Roche, Germany) solution buffered in 25 mM ammonium bicarbonate. The gel
pieces were incubated for 16 h (overnight) at 37°C (trypsin) or 25°C
(chymotrypsin). The supernatant was then transferred into new 0.5-mL tubes,
and the peptides were extracted with 50 µL of 0.5% formic acid/20%
acetonitrile for 20 min in a sonication bath. This step was repeated two times.
Samples in the extraction buffer were pooled into 0.5-mL tubes and evaporated
in a SpeedVac concentrator. The volume was reduced to approximately 20 µL,
and 20 µL of HPLC grade water (Sigma, Germany) was added(Ghafari et al.
2012b).
Supplemental Method 1c
Mass spectrometry
MASCOT searches were performed using MASCOT 2.2.06 (Matrix Science,
London, UK) against the latest UniProtKB database for protein identification.
The searching parameters were set as follows: enzyme selected as trypsin or
chymotrypsin with three and five maximum missing cleavage sites, respectively;
species taxonomy was limited to human; a mass tolerance of 5 ppm was used for
peptide tolerance; 20 mmu was used for MS/MS tolerance; the ion score cutoff
was lower than 15; fixed modification of carbamidomethyl (C) and variable
modification of oxidation (M) were used and deamidation (N, Q) and
phosphorylation (S, T, Y) were applied. Positive protein identifications were
made based on a significant MOWSE score. After protein identification, an
error-tolerant search was performed to detect non-specific cleavage and
unassigned modifications. Returned protein identification information was
manually inspected and filtered to obtain confirmed protein identification lists.
Higher sequence coverage was obtained using Modiro® software with the
following parameters: the selected enzymes used exhibited three and five
maximum missing cleavage sites, a peptide mass tolerance of 0.2 Da was used
for peptide tolerance, 0.2 Da was used for the fragment mass tolerance and
modification 1 of carbamidomethyl(C) and modification 2 of methionine
oxidation was applied. Positive protein identification was first based on the
spectra, and subsequently, each identified peptide was considered significant
based on the ion charge status of the peptide, b- and y-ion fragmentation quality,
ion score (>200) and significance scores (>80). Protein identifications were
manually inspected and filtered to obtain the confirmed protein identification
lists (Ghafari et al. 2012b).
Ghafari M, Falsafi S.K, Hoeger H , Lubec G (2012a) Hippocampal levels of GluR1 and
GluR2 complexes are modulated by training in the Multiple T-maze in C57BL/6J
mice. Brain Struct Funct 217(2), 353-62
Ghafari M , Hoger H, Keihan Falsafi S, Russo-Schlaff, N, Pollak A, Lubec G (2012b) Mass
spectrometrical identification of hippocampal NMDA receptor subunits NR1, NR2AD and five novel phosphorylation sites on NR2A and NR2B. J Proteome Res 11(3),
1891-6