PLOS ONE
Ontology highlight
ABSTRACT: To identify the molecular changes underlying this decreased ability for MECP2-mutant NPCs to differentiate into glia, we applied stable isotope labeling by amino acids in cell culture (SILAC) with quantitative multidimensional protein identification technology (MudPIT) mass spectrometry (MS)-based shotgun proteomics. By metabolically incorporating heavy isotopically labeled amino acids into the synthesized proteins, one culture produces a heavy version of each protein, allowing the mass spectrometer to discriminate between heavy labeled proteins from one culture and unlabeled light proteins from a second culture. By mixing the light and heavy extracts in a 1:1 ratio, we quantitatively compared their proteomes. In order to narrow our focus to only the proteins most perturbed in both MECP2 mutant populations, we used two distinct analytical paradigms, both of which involved determining ratios of light to heavy proteins to calculate the Mutant / WT ratio. In the Ratio of Ratios (RoR) paradigm, quantified proteins are normalized using a common internal standard that can accurately correct for incomplete labeling and other instrument-based biases. In the Label Swap (LS) paradigm, we generated two ratios for each protein from four samples. In this way, we focused on those proteins that were significantly altered in both MECP2-mutant son / paternal control comparisons.
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: John R. Yates III
PROVIDER: MSV000083090 | MassIVE | Thu Nov 01 14:11:00 GMT 2018
REPOSITORIES: MassIVE
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