Nanoflow hplc 2d system

The hippocampal cytoplasmic fraction from G72Tg, WT and ¹⁵N-labeled CD1 mice was collected as previously described [9 (link)]. ¹⁵N-labeled CD1 mouse hippocampal tissue was used as internal standard so as to compare the unlabeled G72Tg and WT mice. The experimental design for the quantitative proteomics analysis is as previously described (Figure 1 in [9 (link)]). The Bradford assay (BioRad Laboratories, Hercules, CA, USA) was used to quantitate total protein amount. For each G72Tg/WT pair, the cytoplasmic fractions of G72Tg and WT hippocampi were mixed 1:1 (w/w) with the cytoplasmic fraction of the same ¹⁵N-labeled CD1 mouse reference based on protein amount. Proteomics sample preparation for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in gel digestion and peptide extraction was performed as previously described [9 (link),10 (link)]. Peptide extracts were lyophilized, re-dissolved in 0.1% formic acid, filtered and analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) by a nanoflow HPLC-2D system (Eksigent, Dublin, CA, USA) which was coupled online to an LTQ Orbitrap XL™ Hybrid FT Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany), as previously described [9 (link)]. Detailed MS parameters were as previously described [12 (link)].

Tryptic peptides were then dissolved in 0.1% formic acid and analyzed with a nanoflow HPLC-2D system (Eksigent, Dublin, CA, USA) coupled on-line to an LTQ-Orbitrap XL^TM mass spectrometer. Samples were on-line desalted for 10 min with 0.1% formic acid at 3 μl/min (Zorbax-C18 (5 μm) guard column, 300 μm × 5 mm; Agilent Technologies, Santa Clara, CA, USA) and separated via RP-C18 (Dr. Maisch, Germany, 3 μm) chromatography (in-house packed Pico-frit column, 75 μm × 15 cm, New Objective, Woburn, MA, USA). Peptides were eluted with a gradient of 95% acetonitrile/0.1% formic acid from 10 to 45% over 93 min at a flow rate of 200 nl/min. Column effluents were directly infused into the mass spectrometer via a nano-electrospray ion source (Thermo Fisher Scientific). The mass spectrometer was operated in positive mode applying a data-dependent scan switch between MS and MS/MS acquisition. Full scans were recorded in the Orbitrap mass analyzer (profile mode, m/z 380–1600, resolution R = 60,000 at m/z 400). The MS/MS analyses of the five most intense peptide ions for each scan were recorded in the LTQ mass analyzer in centroid mode.

Mouse hippocampus was homogenized in a buffer containing 2M NaCl, 10 mM HEPES/NaOH, 1 mM EDTA and protease inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitors (Sigma, St. Louis, MO, USA). Homogenates were sonicated with an ultra-sonicator (Branson, Danbury, CT, USA) and centrifuged (16100 g, 20 min, 4 °C). The protein concentration was determined by Bradford assay.
Protein extracts were mixed with equal amounts of ¹⁵N-labeled DBA/2 mouse hippocampal protein extract24 . Fourty μg of ¹⁴N- ¹⁵N hippocampal protein mixture was separated in a 10% SDS-PAGE gel and stained with Coomassie Brilliant Blue R-250 (BioRad, Hercules, CA, USA) overnight. After destaining and cutting the gel lane into slices, tryptic peptides were produced and extracted as previously described42 (link). Extracted peptides were analyzed by liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) using a nanoflow HPLC-2D system (Eksigent, Dublin, California) coupled online to an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Protein identification and quantitation were performed as described previously22 (link).