The proteome wide, mass spectrometry based identification of protein C-termini is hampered by factors such as poor ionization efficiencies, low yielding labeling strategies, or the need for enrichment procedures. We present a bottom-up proteomics workflow to identify protein C-termini utilizing a combination of strong cation exchange chromatography, on-solid phase charge-reversal derivatization and LC–MS/MS analysis. Charge-reversal improved both MS and MS/MS spectra quality of peptides carrying nonbasic C-terminal residues, allowing the identification of a high number of noncanonical C-termini not identified in nonderivatized samples. Further, we could show that C-terminal 18O labeling introduced during proteolytic processing of the samples is not suitable to distinguish internal from C-terminal peptides. The presented workflow enables the simultaneous identification of proteins by internal peptides and additionally provides data for the C- and N-terminome. Applying the developed workflow for the analysis of a Saccharomyces cerevisiae proteome allowed the identification of 734 protein C-termini in three independent biological replicates, and additional 789 candidate C-termini identified in two or one of three biological replicates, respectively. The developed analytical workflow allowed us to chart the nature of the yeast C-terminome in unprecedented depth and provides an alternative methodology to assess C-terminal proteolytic protein processing.

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Bacteroides thetaiotaomicron is a gram negative bacterium within the human gut microbiome that metabolizes a wide range of dietary and mucosal polysaccharides. Here, we analyze the proteome response of B. thetaiotaomicron cultivated on two different carbon sources, glucose and sucrose. Two quantitative LC-MS based proteomics approaches, encompassing label free quantification and isobaric labeling by tandem mass tags were applied. The results obtained by both workflows were compared with respect to the number of identified and quantified proteins, peptides supporting identification and quantification, sequence coverage, and reproducibility. A total of 1719 and 1696 proteins, respectively, were quantified, covering 35 % of the predicted B. thetaiotaomicron proteome. The data show that B. thetaiotaomicron widely maintains its intracellular proteome upon change of the carbohydrates and that major changes are observed solely in the machinery necessary to make use of the carbon sources provided. With respect to the central role of carbohydrates on gut health these data contribute to the understanding of how different carbohydrates contribute to shape bacterial community in the gut microbiome. All proteomics raw data have been uploaded to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD033704.

Low serum levels of 1α, 25-dihydroxyvitamin D3 (VD3) are associated with a higher mortality in trauma patients with sepsis or ARDS. However, the molecular mechanisms behind this observation are not yet understood. VD3 is known to stimulate lung maturity, alveolar type II cell differentiation, or pulmonary surfactant synthesis and guides epithelial defense during infection. In this study, we investigated the impact of VD3 on the alveolar–capillary barrier in a co-culture model of alveolar epithelial cells and microvascular endothelial cells respectively in the individual cell types. After stimulation with bacterial LPS (lipopolysaccharide), gene expression of inflammatory cytokines, surfactant proteins, transport proteins, antimicrobial peptide, and doublecortin-like kinase 1 (DCLK1) were analyzed by real-time PCR, while corresponding proteins were evaluated by ELISA, immune-fluorescence, or Western blot. The effect of VD3 on the intracellular protein composition in H441 cells was analyzed by quantitative liquid chromatography-mass spectrometry-based proteomics. VD3 effectively protected the alveolar–capillary barrier against LPS treatment, as indicated by TEER measurement and morphological assessment. VD3 did not inhibit the IL-6 secretion by H441 and OEC but restricted the diffusion of IL-6 to the epithelial compartment. Further, VD3 could significantly suppress the surfactant protein A expression induced in the co-culture system by LPS treatment. VD3 induced high levels of the antimicrobial peptide LL-37, which counteracted effects by LPS and strengthened the barrier. Quantitative proteomics identified VD3-dependent protein abundance changes ranging from constitutional extracellular matrix components and surfactant-associated proteins to immune-regulatory molecules. DCLK1, as a newly described target molecule for VD3, was prominently stimulated by VD3 (10 nM) and seems to influence the alveolar–epithelial cell barrier and regeneration.