Selenoproteome Identification in Inflamed Murine Primary Bone Marrow-Derived Macrophages by Nano-LC Orbitrap Fusion Tribrid Mass Spectrometry

Abstract

Selenium (Se) functions as a cellular redox gate keeper through its incorporation into proteins as the 21 st 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 spectrometrybased 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 hours) 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.

Selenium-dependent metabolic reprogramming during inflammation and resolution

“…2 C ), we identified 121 proteins that were differentially regulated at 0 h in Se-treated cells compared with unstimulated Se-deficient BMDMs (“naïve” cells). Although majority of the selenoproteins were increased as reported earlier upon exogenous Se addition ( 26 ), nuclear factor-erythroid 2-related factor 2 target genes, such as thioredoxin reductase1 (Txnrd1) and Txnrd2, and hemeoxygenase 1 (Hmox1) were also increased by Se supplementation followed by LPS stimulation for 8 h. In contrast, 510 proteins were differentially regulated upon stimulation with LPS for 20 h ( Fig. 2 C ).…”

“…Three-week-old male C57Bl/6 mice were purchased from Taconic Biosciences, Inc and maintained on either an AIN-76–based semipurified Se-deficient diet (<0.01 ppm; Se-D) or Se-supplemented diet (0.4 ppm as selenite; Se-S) purchased from Harlan Teklad, for 7 weeks before being used in the experiments. Femoral bone marrow from Se-D and Se-S mice were used as a source of BMDMs upon culture with no exogenous or 250-nM Se (as sodium selenite) addition, respectively, as described ( 26 ). Cells were cultured in biological triplicates for 7 days with or without Se (as sodium selenite; 250 nM) as described previously ( 19 , 26 ).…”

“…Femoral bone marrow from Se-D and Se-S mice were used as a source of BMDMs upon culture with no exogenous or 250-nM Se (as sodium selenite) addition, respectively, as described ( 26 ). Cells were cultured in biological triplicates for 7 days with or without Se (as sodium selenite; 250 nM) as described previously ( 19 , 26 ). On the eighth day, cells were stimulated with LPS (100 ng/ml; Sigma), where cells were treated with LPS for an hour followed by replacement with fresh culture media until harvest at 0, 4, 8, and 20 h after stimulation.…”

“…The peptide digest was labeled with TMT and fractionated (lot number SH253273, and sample to TMT channel information is provided in Table S1 ). The proteomic dataset was acquired on samples in biological triplicate using an UltiMate 3000 RSLCnano system coupled online to an Orbitrap Fusion MS (Thermo Fisher Scientific) as described previously ( 26 ). Briefly, peptides were resolved on an Acclaim PepMap C18 column (2 microns, 75 μm i.d.…”

“…Monocyte/macrophage-specific deletion of selenoproteins has led us to recognize the importance of the selenoproteome in the transition of M1- to M2-like phenotype and resolution as seen in experimental models of gut inflammation and hematologic malignancies ( 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ). Recent studies from our laboratory using tandem mass tag (TMT)-labeling method of nonselenocysteine peptides in murine bone marrow–derived macrophages (BMDMs) indicated a temporal regulation with a general increase in the selenoproteome upon LPS stimulation in a Se-dependent manner ( 26 ). Selenow, Gpx1, Msrb1, and Selenom were highly upregulated upon stimulation with LPS when compared with other selenoproteins.…”