Multiplexed Isobaric Tag‐Based Profiling of Seven Murine Tissues Following In Vivo Nicotine Treatment Using a Minimalistic Proteomics Strategy

Abstract

Nicotine is a major addictive compound in tobacco and a component of smoking-related products, such as e-cigarettes. Once internalized, nicotine can perturb many cellular pathways and can induce alterations in proteins across different cell types, however the mechanisms thereof remain undetermined. We hypothesize that both tissue-specific and global protein abundance alterations result from nicotine exposure. As such, we present the first proteome analysis of multiple tissues from nicotine-exposed mice. We treat mice via oral administration of nicotine at 200μg/mL in drinking water for 21 days. We investigate 7 tissues (brain, heart, kidney, liver, lung, pancreas, and spleen) from treated (n=5) and untreated control (n=5) mice. For each tissue, a TMT10-plex experiment (5 versus 5) was assembled. We apply a minimalistic proteomics strategy was employed using TMT reagents efficiently and fractionating by centrifugation-based reversed-phase columns to streamline sample preparation. Combined, we quantified over 11,000 non-redundant proteins from over 138,000 different peptides in 7 TMT10-plex experiments. Between 7 and 126 proteins are significantly altered in tissues from nicotine-exposed mice. Among these proteins, only 11 are altered in two or more tissues, many classified as from extracellular exosomes or involved in lipid metabolism. Our data showcase the vast extent of nicotine exposure across murine tissue.

Streamlined Tandem Mass Tag (SL-TMT) Protocol: An Efficient Strategy for Quantitative (Phospho)proteome Profiling Using Tandem Mass Tag-Synchronous Precursor Selection-MS3

“…For this full proteome analysis, a total of 96 fractions were collected and concatenated so that every 24 th fraction was pooled (i.e., samples in wells A1, C1, E1, and G1 were combined) and only alternating pooled fractions (a total of 12) were analyzed (13). Alternatively, if <300 μg of total peptide was to be fractionated, comparable proteome depth may be achieved using Pierce high-pH reversed-phase spin columns according to manufacturer’s instructions (23). Each peptide fraction was analyzed using a 2.5 hr SPS-MS3 method (Figure 1I).…”

Quantitative Proteome Analysis of Brain Subregions and Spinal Cord from Experimental Autoimmune Encephalomyelitis Mice by TMT‐Based Mass Spectrometry

“…Before TMT labeling, the protein concentration of each sample was measured with the tryptophan assay. TMT labeling was then performed with several modifications to the manufacturer's protocol . Briefly, the TMT reagent (0.8 mg) was dissolved in acetonitrile, and 10 µL of the reagent was added to 30 µL of peptides along with ACN to give a final concentration of 30% v/v.…”

Proteomic Profiling of Extracellular Vesicles Isolated From Cerebrospinal Fluid of Former National Football League Players at Risk for Chronic Traumatic Encephalopathy

“…To generate EV protein profiling of former NFL players’ CSF, we analyzed EV proteins isolated from the former NFL players and CTRL cohorts by NanoLC-MS/MS using TMT-based labeling (Paulo et al, 2018). We identified a total of 429 unique proteins across both cohorts (Supplementary Tables S2, S3).…”