Computational protocol: Cell-Intrinsic Control of Interneuron Migration Drives Cortical Morphogenesis

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Protocol publication

[…] For differential proteome analysis of ganglionic eminences obtained from WT and CCP1cKO mice, we used a proteomics shotgun approach in combination with post-metabolic peptide labeling using NHS esters of 12C3- or 13C3-propionate. In brief, four ganglionic eminences per condition were pooled and pulverized in liquid nitrogen, and the resulting tissue powders were suspended in lysis buffer containing CHAPS (50 mM sodium phosphate pH 7.5, 100 mM NaCl, 0.8% wt/vol CHAPS) with protease inhibitors added (Complete Protease Inhibitor Cocktail, Roche). Tissue suspensions from each condition were separately subjected to three rounds of freeze-thaw lysis and cleared by centrifugation at 16,000 g for 5 min at 4°C. Sample protein concentrations were determined for each condition using the Bio-Rad DC Protein Assay Kit (Biorad), and the amount of clear lysate containing 300 μg of proteins (approximately 0.5 mL of cleared lysate) were processed as follows. To denature proteins, guanidinium hydrochloride was added to each lysate to a final concentration of 4 M. Each sample was desalted in 20 mM triethylammonium bicarbonate pH 8.0 over a NAP-5 desalting column (GE Healthcare). Then, each protein mixture was digested overnight at 37°C with sequencing-grade endoproteinase LysC (endoLys-C, Promega) at an enzyme/substrate ratio of 1/400 (w/w). Afterward, the resulting peptides were labeled post-metabolically using isotopic variants of N-hydroxysuccinimide (NHS)-propionate (i.e., NHS ester of 12C3-propionate for the CCP1 cKO mice sample and 13C3-propionate for the WT mice). The propionylation reagents were quenched by the addition of 40 mM glycine and the peptide mixture heated for 60 min at 100°C to reverse possible O-propionylation of Ser, Thr and Tyr. Equal amounts of CCP1 WT and cKO peptide sample were mixed, and following oxidation of methionines, peptides were fractionated via RP-HPLC in 30 s intervals, as described previously. Following peptide fractionation, peptide fractions eluting 12 min apart were pooled to reduce the number of LC-MS/MS runs (24 per analysis).The obtained peptide mixtures were introduced into an LC-MS/MS system, an Ultimate 3000 RSLC nano-LC (Thermo Fischer Scientific) in-line connected to a Q-exactive (Thermo Fisher Scientific), for MS analysis. Peptides were loaded on a trapping column (made in-house; 100 μm inner diameter x 20 mm, 5 μm beads, C18 Reprosil-HD, Dr. Maisch). After flushing from the trapping column, the sample was loaded on a reverse-phase column (made in-house; 75 μm inner diameter x 150 mm, 3 μm beads, C18 Reprosil-HD, Dr. Maisch) packed in the needle (PicoFrit SELF/P PicoTip emitter, PF360-75-15-N-5, New Objective). Peptides were loaded with solvent A (0.1% trifluoroacetic acid, 2% acetonitrile in water) and were separated with a linear gradient from 2% solvent A’ (0.1% formic acid in water) to 55% solvent B (0.1% formic acid, 80% acetonitrile in water) at a flow rate of 300 nL/min, followed by a wash reaching 100% solvent B for 15 min.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. In the Q-exactive, full-scan MS spectra (m/z 375–1500) were acquired in the Orbitrap at a target value of 1E6 with maximum ion injection time of 80 ms, and a resolution of 60,000 at 200 m/z. The 10 most intense ions fulfilling a predefined criterion (AGC target 1E5 ions, maximum ion injection time of 60 ms, isolation window of 1.5 m/z, fixed first mass of 145 m/z, spectrum data type: centroid, underfill ratio 2%, intensity threshold 1.3E4, exclusion of unassigned, singly charged precursors, peptide match preferred, exclude isotopes on, dynamic exclusion time of 12 s) were subjected to tandem MS scans at a resolution 15,000 (at 200 m/z).From the MS/MS data in each LC-MS/MS run, Mascot Generic Files were created using Mascot Distiller software (version 2.5.1, Matrix Science). In the generation of these peak lists, grouping of spectra was allowed in Mascot Distiller with a maximum intermediate retention time of 30 s, and a maximum intermediate scan count of 5 was used where possible. Grouping was done with a 0.005-Da precursor tolerance. A peak list was only generated when the MS/MS spectrum contained more than 10 peaks. There was no deisotoping, and the relative signal-to-noise limit was set at 2. These peak lists were then searched with the Mascot search engine (Matrix Science) using the Mascot Daemon interface (version 2.5.1, Matrix Science, Spectra were searched against the Swiss-Prot database with taxonomy set to Mus musculus (release 2015_05). Two different type of searches were performed: in one of them, the enzyme was set to semi-endoLys-C. In both cases, enzyme settings allowed for one missed cleavage, nonetheless allowing for cleavage when lysine was followed by proline. Variable modifications were set to pyroglutamate formation of N-terminal glutamine and acetylation of the N terminus. Methionine oxidation was set as a fixed modification. Determination of the light (12C3-propionyl) and heavy (13C3-propionyl) labeled peptides for further quantification was enabled using the quantitation option in Mascot. The mass tolerance on precursor ions was 10 ppm (with Mascot’s C13 option set to 1), and that on fragment ions was 20 mmu. The peptide charge was set at 2+ and 3+, and the instrument specification was set as ESI-QUAD. Only peptides that were ranked one, have a minimum amino acid length of eight and scored above the threshold score, set at 99% confidence, were withheld. The false discovery rate was calculated for every search and was always found to be lower than 1.5%. [...] Total RNA was extracted from GFP FACS-sorted cells from CCP1 WT and cKO GEs and purified on columns (Quiagen RNeasy mini kit). ARN quality was controlled with an Agilent 2100 Bioanalyzer. DNA libraries were prepared after depletion of ribosomal RNAs. Paired-end sequencing was done on NextSeq 500 Illumina. Sequences alignment was done on mouse genome using the Tuxedo workflow, using Bowtie2. RNaseq data can be found at GEO: GSE104090. [...] Gene expression analysis was performed using Cuffdiff program. Differences of gene expression in interneurons from WT or cKO CCP1 mouse embryos was calculated from log2fold change of fragments per kilobase of exon per million reads (FPKM) values. See and . [...] Identified peptides were quantified using Mascot Distiller Tool, version (Matrix Science in the precursor mode. All data management was done in ms-lims and data integration was performed using R ( embedded in KNIME. The results of analyses are shown as the peptide ratio of the light (L)-labeled (CCP1cKO mice) versus the heavy (H)-labeled sample (CCP1 WT mice). Robust statistics was applied to the base-2 logarithm values of the peptide ratios accepted as valid by Mascot Distiller. The median of the peptide ratio distributions of both experiments was corrected to zero. Further, peptide ratios being set as FALSE were verified by individual inspection. Protein ratios were then calculated by taking the median of the peptide ratios that identified that protein. To identify significantly altered proteins, robust statistics was applied, proteins falling within the 95% confidence interval and that were identified by at least two peptides were considered not to be affected by Ccp1 conditional knock-out. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium ( via the PRIDE partner repository with the dataset identifier PRIDE: PXD006397. […]

Pipeline specifications

Software tools Tuxedo, Bowtie2, Cufflinks
Application RNA-seq analysis