Chemical tools for isolation of endogenous claudins from carcinoma cells
thesisposted on 2022-03-29, 02:42 authored by Payal Barua
Claudins are a family of tetraspan transmembrane proteins that regulate the barrier function and ion selective paracellular permeability of the tight junction. Claudins have diverse roles in normal tissues as well as different cancers, and it is necessary to understand the regulation of endogenous claudins in different cellular environments. As low-abundance and hydrophobic proteins, detection and analysis of claudins has been a major challenge. Here a chemical method for tagging and enriching endogenous surface claudins is investigated for analysis by mass spectrometry. Model human colorectal carcinoma, HCT-116 cells, were surface labelled with sulfo-NHS-S-S-biotin, followed by NeutrAvidin affinity purification and then LC/MS/MS analysis. Various cell-lysis, labelling and elution conditions for optimized recovery of claudins were examined by western-blot analysis. Following the optimized labeling and affinity pull-down protocol, the biotinylated elute fraction was analysed in 1D liquid LC/MS/MS analysis. A total of 389 unique protein groups were identified including 58 proteins found in ≥2 samples. Although peptides of claudins still remain to be detected, 19 plasma membrane proteins were identified with the optimized protocol. This sets the stage for a future advanced workflow that combines membrane fractionation with surface biotinylation for MS based proteome-wide analysis of claudins.