Selenium (Se) functions as a cellular redox gatekeeper through its incorporation into
proteins as the 21st
amino acid,
selenocysteine (Sec). Supplementation of macrophages with exogenous Se (as
sodium selenite) downregulates
inflammation and intracellular oxidative stress by effectively restoring redox homeostasis upon challenge with bacterial
endotoxin lipopolysaccharide (LPS). Here, we examined the use of a standard Tandem Mass Tag (TMT)-labeling mass spectrometry-based proteomic workflow to quantitate and examine temporal regulation of
selenoproteins in such inflamed cells. Se-deficient murine primary bone marrow-derived macrophages (BMDMs) exposed to LPS in the presence or absence of
selenite treatment for various time periods (0-20 h) were used to analyze the selenoproteome expression using isobaric labeling and shotgun proteomic workflow. To overcome the challenge of identification of Sec
peptides, we used the identification of non-Sec containing
peptides downstream of Sec as a reliable evidence of ribosome readthrough indicating efficient decoding of Sec
codon. Results indicated a temporal regulation of the selenoproteome with a general increase in their expression in inflamed cells in a Se-dependent manner. Selenow, Gpx1, Msrb1, and Selenom were highly upregulated upon stimulation with LPS when compared to other
selenoproteins. Interestingly, Selenow appeared to be one amongst the highly regulated
selenoproteins in macrophages that was previously thought to be mainly restricted to myocytes. Collectively, TMT-labeling method of non-Sec
peptides offers a reliable method to quantitate and study temporal regulation of
selenoproteins; however, further optimization to include Sec-
peptides could make this strategy more robust and sensitive compared to other semi-quantitative or qualitative methods. Graphical Abstract.