BACKGROUND: The intestinal epithelium plays a significant role in human health by functioning as a barrier to prevent the influx of luminal antigens that cause inflammation. Dysfunction of the epithelial barrier may lead to poor digestive or systemic health conditions and is associated with multiple chronic conditions, including celiac disease and inflammatory bowel disease. In healthy individuals, barrier dysfunction can be triggered by environmental cues, such as oxidative stress caused by prolonged exposure of reactive oxygen species (ROS). These increased oxidative stress levels result in excessive amounts of pro-inflammatory mediators which exacerbate barrier dysfunction. Recently, we showed that a proprietary cationic bovine whey protein extract was able to reduce ROS production in an in vitro intestinal epithelial cell model (Caco-2).
AIM: To better understand the mechanisms-of-action, we investigated the effects of this whey extract on the whole intracellular proteome of Caco-2 cells during oxidative stress.
METHODS: Eighteen independent cell monolayers were randomly divided into four groups. Two groups were treated with the whey extract for 24 hours. Following removal of the treatment, one non-treated group and one treated group were challenged with 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH) – a known inducer of intracellular ROS. After whole cell lysis and filter aided sample preparation (FASP) tryptic digestion, we applied ion-mobility-enhanced data-independent acquisition (DIA) label-free quantitative mass spectrometry (timsTOF pro2, Bruker Daltonics) to analyse the intracellular proteome of all individual samples. Various statistical approaches were used to study the proteome-wide responses upon AAPH-induced oxidative stress and the effect of whey extract treatment, by comparing the four groups: untreated unchallenged cells (negative control, n=4), untreated challenged cells (n=5), pre-treated unchallenged cells (n=3), and pre-treated challenged cells (n=4).
FINDINGS: From the raw datafiles we extracted approximately 150,000 features per sample, of which we identified over 84,600 peptides, that could be mapped to more than 7,500 non-redundant human proteins after combining all results. Protein pathway analysis provided more insight into the protein interactions and involved pathways during oxidative stress.
INTERPRETATION: By studying the full intracellular proteomes of an intestinal epithelial cell model with and without oxidative stress, we obtained a better understanding of the involved protein pathways and mechanism-of-action, which aids in investigating the inflammatory dampening effects of potential therapeutic compounds.