Methodological considerations of label-free neuroproteomics: A case study involving a rat model of chronic methamphetamine exposure




Sahyouni, Fatima
Prokai, David
Kobeissy, Firas H.
Gold, Mark S.
Cadet, Jean L.
Prokai, Laszlo


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Purpose: This presentation is aimed at evaluating label-free quantitative neuroproteomics methodologies to reveal biological pathways associated with chronic methamphetamine exposure in rats.Methods: Striatal samples were harvested from drug-treated and naïve animals (adult male Sprague-Dawley rats). After a urea-based protein extraction and sample preparation following routine proteomics workflows, data-dependent LC-MS/MS analyses were run on a hybrid linear ion trap–FTMS instrument. Parameters evaluated in the context of detecting statistically significant changes in protein expression elicited by methamphetamine in the rat striatum included the choice of database search engines (Mascot and SEQUEST) and the method by which label-free quantitative information was obtained (spectral counting, MS/MS total ion currents from identified spectra, and precursor-ion abundance). Identifications were validated using the Scaffold software suite, whose label-free quantification and statistical modules were used for processing data files and organizing results. Additionally, bioinformatics was performed using Ingenuity Pathway Analysis (IPA).Results: We queried our mass spectrometry-based proteomics data using the Mascot and SEQUEST search algorithms separately and conjointly for complementary protein identifications. For label-free quantitative analysis, identified proteins were revealed as differentially expressed following evaluation of quantitative method, along with the use of appropriate statistical analysis (G- and t-tests, pConclusions: Although our study focused on methodical aspects of quantitative label-free shotgun neuroproteomics initially, IPA of our results not only captured processes already recognized to be impacted by methamphetamine exposure (dopamine and serotonin biosynthesis, oxidative stress, cell signaling, etc), but also revealed protein associations hitherto unknown at proteome level. Thus, the latter would be worthy of future pursuit in the framework of mechanistic interpretations and as potential neurobiological indicators or consequences of methamphetamine abuse.