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Choroid plexus proteome with and without LPS
Choroid plexus was isolated and pooled from the 5 mice after the perfusion with bromo phenol blue from 0 h and 6 h LPS treated mice. Proteins were isolated and trypsin digested and labelled with the NHS-porp (0h –LPS and 6h +LPS). Pre-fractionated using RP-HPLC and analysed on the Qexactive.
Sample Processing Protocol
For preparation of the choroid plexus cell lysate, CP was pooled from the five mice, and lysed in laemmli sample buffer (2% SDS, 10% glycerol, 120 mM tris-cl pH-6.8, and 0.02% bromophenol blue) with protease inhibitor (Roche). Samples were centrifuged at high speed to remove the debris and the supernatant was used for the further analysis. These samples were briefly separated by SDS-PAGE loaded in 4-12% precast gradient gel (Criterion XT, Biorad) and stained with coomassie blue (SimplyBlue™, Life technologies). Stained protein bands were carefully excised under sterile conditions and carefully washed with 100 µl of the sterile water was added, briefly vortexed and incubated at room temperature for 15 minutes, supernatant was discarded after centrifugation. Then 100 µl of 50% acetonitrile was added and briefly vortexed, supernatant was discarded after 15 minutes of incubation. Next, bands were washed with 100 µl of the 100% acetonitrile, briefly vortex and incubated for 15 minutes at room temperature and supernatant was discarded after centrifugation. These bands were completely dried in speedvac. In-gel digestion was done by using trypsin enzyme (Promega; V5280; Trypsin Gold, Mass Spectrometry grade) and incubated at 37°C overnight. Next day samples were briefly centrifuged and supernatant was transferred to new tube, pH was adjusted to two using formic acid to inactivate the trypsin. Then the peptide mixture was labeled with freshly prepared N-hydroxy succinimide propionate (PBS samples were labeled with light isotope (12C3) and LPS treated samples were labeled with heavy isotope (13C3) of N-hydroxy succinimide propionate). Excess labeling was quenched by add adding 40mM of glycine and the pH was adjusted to two using formic acid. Light and heavy labeled samples were mixed in 1:1 ratio and the quality of the labeling was checked before pre-fractionation. These peptides were pre-fractionated using RP-HPLC (C18-HD; 3µM, 12 cm column with 250µM diameter), 100 fractions of one minute each were collected that were further pooled to make 20 final fractions for LC-MS/MS analysis.
Data Processing Protocol
The mass spectrometer was operated in data-dependent, positive ionization mode, automatically switching between MS and MS/MS acquisition for the 10 most abundant peaks in a given MS spectrum. The source voltage was 3.4 kV and the capillary temperature was 275°C. One MS1 scan (m/z 400−2000, AGC target 3 × 106 ions, maximum ion injection time 80 ms) acquired at a resolution of 70,000 (at 200 m/z) was followed by up to 10 tandem MS scans (resolution 17,500 at 200 m/z) of the most intense ions fulfilling predefined selection criteria (AGC target 5 × 104 ions, maximum ion injection time 60 ms, isolation window 2 Da, fixed first mass 140 m/z, spectrum data type: centroid, underfill ratio 2%, intensity threshold 1.7xE4, exclusion of unassigned, 1, 5-8, >8 charged precursors, peptide match preferred, exclude isotopes on, dynamic exclusion time 20 s). The HCD collision energy was set to 25% Normalized Collision Energy and the polydimethylcyclosiloxane background ion at 445.12002 Da was used for internal calibration (lock mass). From the MS/MS data in each LC-MS/MS run, Mascot Generic Files were created using the Mascot Distiller software (version 126.96.36.199, Matrix Science, www.matrixscience.com/Distiller). While generating these peak lists, grouping of spectra was allowed in Mascot Distiller with a maximal intermediate retention time of 30 s, and a maximum intermediate scan count of 5 was used where possible. Grouping was done with 0.005 Da precursor tolerance. A peak list was only generated when the MS/MS spectrum contained more than 10 peaks. There was no de-isotoping and the relative signal to noise limit was set at 2. These peak lists were then searched using the Mascot search engine (MatrixScience, www.matrixscience.com) using the Mascot Daemon interface (version 2.4, Matrix Science). Spectra were searched against the SwissProt restricted to Mus musculus taxonomy. Variable modifications were set to methionine oxidation, pyro-glutamate formation of amino terminal glutamine, and acetylation of the protein N-terminus. Mass tolerance on precursor ions was set to 10 ppm (with Mascot’s C13 option set to 1), and on fragment ions to 20 mmu. The instrument setting was put on ESI-QUAD. Enzyme was set to trypsin, allowing for 1 missed cleavage. Only peptides that were ranked first and scored above the Mascot significance score threshold set at 95% confidence were withheld. The 95% threshold corresponds to a chance of 1 in 100 for an identified peptide to be a false positive (approximately corresponding to 1% FDR).
Sriram Balusu, VIB-Medical protein research
Kris Gevaert, VIB Department of Medical Protein Research, UGent Proteome Analysis and Bioinformatics Unit A. Baertsoenkaai 3 B9000 Gent Belgium ( lab head )
Balusu S, Van Wonterghem E, De Rycke R, Raemdonck K, Stremersch S, Gevaert K, Brkic M, Demeestere D, Vanhooren V, Hendrix A, Libert C, Vandenbroucke RE. Identification of a novel mechanism of blood-brain communication during peripheral inflammation via choroid plexus-derived extracellular vesicles. EMBO Mol Med. 2016 Sep 5. pii: e201606271 PubMed: 27596437
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