Correlation between Targeted qPCR Assays and Untargeted DNA Shotgun Metagenomic Sequencing for Assessing the Fecal Microbiota in Dogs

DNA shotgun sequencing is an untargeted approach for identifying changes in relative abundances, while qPCR allows reproducible quantification of specific bacteria. The canine dysbiosis index (DI) assesses the canine fecal microbiota by using a mathematical algorithm based on qPCR results. We evaluated the correlation between qPCR and shotgun sequencing using fecal samples from 296 dogs with different clinical phenotypes. While significant correlations were found between qPCR and sequencing, certain taxa were only detectable by qPCR and not by sequencing. Based on sequencing, less than 2% of bacterial species (17/1190) were consistently present in all healthy dogs (n = 76). Dogs with an abnormal DI had lower alpha-diversity compared to dogs with normal DI. Increases in the DI correctly predicted the gradual shifts in microbiota observed by sequencing: minor changes (R = 0.19, DI 2, DI > 5, and DI > 8, respectively), compared to dogs with a normal DI (DI < 0, all targets within the RI), as higher R-values indicated larger dissimilarities. In conclusion, the qPCR-based DI is an effective indicator of overall microbiota shifts observed by shotgun sequencing in dogs

Detection and Identification of Heme <i>c</i>‑Modified Peptides by Histidine Affinity Chromatography, High-Performance Liquid Chromatography–Mass Spectrometry, and Database Searching

Multiheme c-type cytochromes (proteins with covalently attached heme <i>c</i> moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme <i>c</i> modified peptides are typically not observed or, if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme <i>c</i> tryptic peptides from purified bovine heart cytochrome <i>c</i>, two bacterial decaheme cytochromes, and subjected these samples to LC–MS/MS analysis. Enriched bovine cytochrome <i>c</i> samples yielded 3- to 6-fold more confident peptide–spectrum matches to heme <i>c</i> containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from <i>Sideroxydans lithotrophicus</i> ES-1, heme <i>c</i> peptides for 4 of the 10 expected sites were observed by LC–MS/MS; following HAC fractionation, peptides covering 9 out of 10 sites were obtained. Heme <i>c</i> peptide spiked into <i>E. coli</i> lysates at mass ratios as low as 1 × 10^–4 was detected with good signal-to-noise after HAC and LC–MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme <i>c</i> peptides. The results suggest that accounting for the double thioether link between heme <i>c</i> and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide–spectrum matches of 20–100-fold for bovine cytochrome <i>c</i>

Detection and Identification of Heme <i>c</i>‑Modified Peptides by Histidine Affinity Chromatography, High-Performance Liquid Chromatography–Mass Spectrometry, and Database Searching

Multiheme c-type cytochromes (proteins with covalently attached heme <i>c</i> moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme <i>c</i> modified peptides are typically not observed or, if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme <i>c</i> tryptic peptides from purified bovine heart cytochrome <i>c</i>, two bacterial decaheme cytochromes, and subjected these samples to LC–MS/MS analysis. Enriched bovine cytochrome <i>c</i> samples yielded 3- to 6-fold more confident peptide–spectrum matches to heme <i>c</i> containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from <i>Sideroxydans lithotrophicus</i> ES-1, heme <i>c</i> peptides for 4 of the 10 expected sites were observed by LC–MS/MS; following HAC fractionation, peptides covering 9 out of 10 sites were obtained. Heme <i>c</i> peptide spiked into <i>E. coli</i> lysates at mass ratios as low as 1 × 10^–4 was detected with good signal-to-noise after HAC and LC–MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme <i>c</i> peptides. The results suggest that accounting for the double thioether link between heme <i>c</i> and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide–spectrum matches of 20–100-fold for bovine cytochrome <i>c</i>

S-glutathionyl-(chloro)hydroquinone reductases: a new class of glutathione transferases functioning as oxidoreductases

Glutathione transferases (GSTs) are best known for transferring glutathione (GSH) to hydrophobic organic compounds, making the conjugates more soluble. However, the omega-class GSTs of animals and the lambda-class GSTs and dehydroascorbate reductases (DHARs) of plants have little or no activity for GSH transfer. Instead, they catalyze GSH-dependent oxidoreductions. The lambda-class GSTs reduce disulfide bonds, the DHARs reduce the disulfide bonds and dehydroascorbate, and the omega-class GSTs can reduce more substrates, including disulfide bonds, dehydroascorbate, and dimethylarsinate. Glutathionyl-(chloro)hydroquinone reductases (GS-HQRs) are the newest class of GSTs that mainly catalyze oxidoreductions. Besides the activities of the other three classes, GS-HQRs also reduce GS-hydroquinones, including GS-trichloro-p-hydroquinone, GS-dichloro-p-hydroquinone, GS-2-hydroxy-p-hydroquinone, and GS-p-hydroquinone. They are conserved and widely distributed in bacteria, fungi, protozoa, and plants, but not in animals. The four classes are phylogenetically more related to each other than to other GSTs, and they share a Cys-Pro motif at the GSH-binding site. Hydroquinones are metabolic intermediates of certain aromatic compounds. They can be auto-oxidized by O(2) to benzoquinones, which spontaneously react with GSH to form GS-hydroquinones via Michael’s addition. GS-HQRs are expected to channel GS-hydroquinones, formed spontaneously or enzymatically, back to hydroquinones. When the released hydroquinones are intermediates of metabolic pathways, GS-HQRs play a maintenance role for the pathways. Further, the common presence of GS-HQRs in plants, green algae, cyanobacteria, and halobacteria suggest a beneficial role in the light-using organisms