Speedvac

FAM-Aβ_(1–42) peptides (Anaspec, AS-23525) were dissolved in trifluoroacetic acid (Sigma, T6508), lyophilized in a SpeedVac, and resuspended in PBS/DMSO solution at 37 °C overnight to a final concentration of 2 μg/μl. FAM-Aβ aggregates (500 nl) were stereotaxically injected into hippocampi of control and MG^4E mice at P14 using the following coordinates (relative to the bregma): AP (anterior posterior), −2.25 mm; ML (medial lateral), ±1.80 mm; DV (dorsal ventral), −2.0 mm. Mice were perfused 18–19 h after FAM-Aβ injection.

SDS-PAGE gel spots were excised and subjected to in-gel trypsin digestion as follows. Protein-containing gel slices were destained in 50% methanol (Fisher, St. Louis, MO, USA), 100 mM NH₄HCO₃ (Sigma-Aldrich, Dallas, TX, USA), followed by reduction in 10 mM Tris(2-carboxyethyl)phosphine (Pierce, Rockford, IL, USA) and alkylation in 55 mM iodoacetamide (Sigma-Aldrich). Gel slices were then dehydrated in acetonitrile, followed by addition of 100 ng porcine trypsin (Promega, Madison, WI, USA) in 100 mM NH₄HCO₃ (Sigma-Aldrich) and incubated at 37 °C for 12–16 h. The trypsin solution was extracted and the gel slices were dehydrated in a SpeedVac (Holbrook, NY, USA). Chymotrypsin in 100 mM Tris-HCl, pH 7.8 containing 10 mM CaCl₂ was added to gels. Gels were incubated at 37 °C for 4–6 h. Following the second enzyme digestion, peptides were extracted with 5% formic acid in 50% acetonitrile and dried in a SpeedVac. The peptide products were reconstituted with 0.1% formic acid, 2% acetonitrile, followed by MS/MS analysis using an LTQ XL mass spectrometer (Thermo, San Diego, CA, USA). Proteins and modifications are identified from MS/MS spectra by database searching Mascot (a trademark of Matrix Science, Inc., Boston, MA, USA) and SEQUEST using search engines. The analysis was performed by L Li (Institute of Molecular Medicine, the University of Texas Health Center, Houston, TX, USA).

Lipid A was precipitated from GMMA using mild acid hydrolysis with 1% acetic acid for 2 h at 100 °C (35 ). Samples were centrifuged at 14,000 × g for 15 min; the pellets were resuspended in water and washed twice with water. The pellets were dried overnight using a SpeedVac and resuspended in chloroform/methanol 4:1 and mixed with an equal volume of Super DHB solution (Sigma). 2 μl of the mixture were loaded to the target plate (MTP 384 target plate ground steel BC, Bruker Daltonics) and analyzed by Ultraflex MALDI-TOF (Bruker Daltonics) in reflectron ion-negative mode. A peptide calibration standard (Bruker Daltonics), mixed with the Super DHB solution, was included in each analysis. For MS/MS analysis of lipid A, main peaks from the linear mode analysis were selected for collision-induced dissociation, and the resulting fragments were detected by MALDI TOF-TOF in ion negative mode. For each sample, spectra represent the integration of the analysis of 20 different areas of the spot by 50 single laser shots. The m/z rations were determined by Flex Analysis software in comparison with the peptide standard.

Two hundred micrograms of each sample was reduced by adding dithiothreitol to a final concentration of 100 mM and incubated at 95 °C for 5 min. After cooled, 200 μL UT buffer (8 M urea and 150 mM Tris-HCl, pH 8.0) was added and mixed, and then transferred into an ultrafiltration tube (10-kDa cutoff, Sartorius, Goettingen, Germany) for centrifuging at 14 000 × g for 15 min. The sample was subsequently alkylated by adding 100 μL iodoacetamide solution (50 mM iodoacetamide in UT buffer), followed by incubation for 30 min at room temperature in the dark and centrifugation at 14 000 × g for 10 min. Two wash steps with 100 μL UT buffer and 100 μL 25 mM ammonium bicarbonate solution were performed, with centrifugation at 14 000 × g for 10 min. Finally, the sample was digested by adding 40 μL trypsin (Promega, Madison, WI, USA) buffer (4 μg trypsin in 25 mM ammonium bicarbonate buffer) and incubated at 37 °C for 16–18 h. The filter unit was transferred to a new tube and centrifuged at 14 000 × g for 10 min. The digested peptides were collected as a filtrate, and peptide concentration was quantified using a Nanodrop 2000 spectrophotometer (Wilmington, DE, USA) at OD₂₈₀27 (link). Samples were desalted using a C18 solid phase extraction column (66872-U, Sigma, USA), dried in a SpeedVac, and stored at −80 °C.

Amyloid beta (Aβ1–42) oligomers were produced in a similar manner to that described in (Ryan et al, 2010 (link)). Briefly, Aβ1–42 (A9810 Sigma) was suspended in hexafluoroisopropanol (HFIP, Sigma‐Aldrich) to 1 mM. HFIP was then dried with a nitrogen stream and then lyophilized using a SpeedVac. Samples could then be stored for further use at −20°C. Peptide films were resuspended in DMSO to 5 mM. Samples were then sonicated for 10 min, diluted to 200 μM with ice‐cold PBS + 0.05% SDS and vortexed for 30 s. Aggregation was allowed to proceed for 24 h at 4°C. The solution was further diluted with PBS to 100 μM and incubated for 2 weeks at 4°C.