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Quantitative proteomic profiling reveals novel region-specific markers in the adult mouse brain

ABSTRACT: Here we present the data obtained from a high precision 18O-labeling strategy used to quantitatively map membrane proteins isolated from myelin-enriched fractions purified from distinct brain regions of an adult mouse. To this end, we present an updated catalog of myelin-associated proteins found in the vertebrate CNS with relevance for investigations of myelin disorders. We also present a number of regiospecific protein markers of the brain, many of which remain unannotated in current online brain mapping databases. Data analysis: Tandem mass spectra were extracted from .RAW files and searched using the SEQUEST- PVM v.27 (rev.9) database program against a Mouse protein database downloaded as FASTA-formatted sequences from EBI-IPI (database version 3.72) which contains 56957 entries where priority was given to UniProt identifiers, as well as reverse decoy sequences to empirically assess the false identification rate. Mass tolerances for precursor (MS) and product ions (MS/MS) were set to 3 and 0 m/z, respectively. Two SEQUEST searches were performed per labeling experiment setting the enzyme selectivity to trypsin while allowing one missed cleavage. In both searches, protein modifications included fixed cysteine carbamidomethylation (57 Da) whilst the 18O-specific search included a static C-terminal modification mass of 4 Da (to account for tryptic peptides fully incorporating two 18O atoms). This improved the identification of unlabeled/labeled peptide pairs without exceeding the individual search mass tolerances. Following the database searches, the utility program DTASelect was invoked to assemble SEQUEST identifications and in each case, create an exhaustive list of the most significant peptide matches to the MS/MS spectra.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: L Dagley

PROVIDER: PXD000327 | Pride | 2013-11-22

REPOSITORIES: Pride

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Quantitative proteomic profiling reveals novel region-specific markers in the adult mouse brain.

Dagley Laura F LF White Carl A CA Liao Yang Y Shi Wei W Smyth Gordon K GK Orian Jacqueline M JM Emili Andrew A Purcell Anthony W AW

Proteomics 20140106 2-3

Despite major advances in neuroscience, a comprehensive understanding of the structural and functional components of the adult brain compartments remains to be fully elucidated at a quantitative molecular level. Indeed, over half of the soluble- and membrane-annotated proteins are currently unmapped within online digital brain atlases. In this study, two complementary approaches were used to assess the unique repertoire of proteins enriched within select regions of the adult mouse CNS, including ...[more]

PMID: 24259518

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Project description:Wild type yeast cells were metabolically labeled in SILAC medium. The heavy (H)-SILAC-labeled cells were then continuously exposed to 0.01% MMS to induce replication stress, and the light (L)-SILAC-labeled cells were mock-exposed. After 3 hours, heavy and light cells were harvested and lysed. To ensure reproducibility, the entire experiment was repeated, and the labels were swapped such that the (L)-SILAC-labeled wild type yeast cells were exposed to 0.01% MMS for 3 hours, and the (H)-SILAC-labeled wild type yeast cells were mock-exposed. Lysates from heavy and light cells were mixed 1:1 by protein mass, reduced and treated with iodoacetamide and then digested with trypsin. 5 mg of each protein lysate was fractionated by high-pH reverse phase (RP) liquid chromatography into 12 samples. For proteome analysis, 2% of each fraction was dried down and re-suspended for LC-MS/MS analysis. The remaining 98% was processed to enrich for phosphopeptides using immobilized metal affinity chromatography (IMAC), dried down, and re-suspended in 0.1% FA, 3% ACN for LC-MS/MS analysis. Global and phosphopeptide-enriched samples were analyzed by LC-MS/MS on a Thermo LTQ-Orbitrap Velos mass spectrometer. Raw MS/MS spectra were searched against version 3.69 of the Yeast International Protein Index (IPI) sequence database using three independent search engines (MaxQuant/Andromeda, Spectrum Mill, and xTandem). All searches were performed with the tryptic enzyme constraint set for up to two missed cleavages, oxidized methionine set as a variable modification, and carbamidomethylated cysteine set as a static modification. For MaxQuant, the peptide MH+ mass tolerances were set at 20 ppm. For X!Tandem, the peptide MH+ mass tolerances were set at ±2.0 Da with post-search filtering of the precursor mass to 50 ppm and the fragment MH+ mass tolerances were set at ±0.5 Da. For Spectrum Mill, peptide MH+ mass tolerances were set at 20 ppm and fragment MH+ mass tolerances were set at ±0.7 Da. The overall FDR was set at ≤3% based on a decoy database search. Phosphosite localization probabilities are reported in the MaxQuant results. Any site with a probability greater than 0.8 was considered to be localized. Quantification of the Heavy:Light ratios was performed using MaxQuant software, with a minimum ratio count of 2 and using unique + razor peptides for quantification.

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Dataset Information

Quantitative proteomic profiling reveals novel region-specific markers in the adult mouse brain

Publications

Quantitative proteomic profiling reveals novel region-specific markers in the adult mouse brain.

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