Coenzyme A precursors flow from mother to zygote and from microbiome to host.

Coenzyme A (CoA) is essential for metabolism and protein acetylation. Current knowledge holds that each cell obtains CoA exclusively through biosynthesis via the canonical five-step pathway, starting with pantothenate uptake. However, recent studies have suggested the presence of additional CoA-generating mechanisms, indicating a more complex system for CoA homeostasis. Here, we uncovered pathways for CoA generation through inter-organismal flows of CoA precursors. Using traceable compounds and fruit flies with a genetic block in CoA biosynthesis, we demonstrate that progeny survive embryonal and early larval development by obtaining CoA precursors from maternal sources. Later in life, the microbiome can provide the essential CoA building blocks to the host, enabling continuation of normal development. A flow of stable, long-lasting CoA precursors between living organisms is revealed. This indicates the presence of complex strategies to maintain CoA homeostasis.

Chemical isotope labeling for quantitative proteomics.

Advancements in liquid chromatography and mass spectrometry over the last decades have led to a significant development in mass spectrometry-based proteome quantification approaches. An increasingly attractive strategy is multiplex isotope labeling, which significantly improves the accuracy, precision and throughput of quantitative proteomics in the data-dependent acquisition mode. Isotope labeling-based approaches can be classified into MS1-based and MS2-based quantification. In this review, we give an overview of approaches based on chemical isotope labeling and discuss their principles, benefits, and limitations with the goal to give insights into fundamental questions and provide a useful reference for choosing a method for quantitative proteomics. As a perspective, we discuss the current possibilities and limitations of multiplex, isotope labeling approaches for the data-independent acquisition mode, which is increasing in popularity.

Disruption of vitamin A homeostasis by the biocide tetrakis(hydroxymethyl) phosphonium sulphate in pregnant rabbits.

The biocide tetrakis(hydroxymethyl)phosphonium sulphate (THPS) and other members of the tetrakis(hydroxymethyl) phosphonium salts (THPX) family are associated with liver toxicity in several mammalian species and teratogenicity in rabbits. Malformations include skeletal changes and abnormalities in eye development and are very similar to those seen with vitamin A deficiency or excess. For this reason, it was hypothesized that teratogenicity of THPS(X) might be attributed to disturbances in retinol availability and/or metabolism as a result of maternal toxicity, for example, either due to insufficient dietary intake by the mothers or due to liver toxicity. Therefore, in the present study, liver toxicity and vitamin A homeostasis were studied in pregnant rabbits that were exposed to 13.8 or 46.0 mg/kg THPS during organogenesis and in precision-cut liver slices of rats and rabbits exposed to 0-70 µM THPS. Results show that in vivo exposure to THPS leads to a marked reduction of food intake, increased plasma concentrations of γ-glutamytransferase, degenerative changes in the liver and to changes in retinoid content in liver and plasma in the rabbits during organogenesis. In addition, THPS, both in vivo and ex vivo, caused a change in expression of proteins related to vitamin A metabolism and transport. Together, these observations could explain the birth defects observed in earlier teratogenicity studies.

N-Dealkylation of Amines.

N-dealkylation, the removal of an N-alkyl group from an amine, is an important chemical transformation which provides routes for the synthesis of a wide range of pharmaceuticals, agrochemicals, bulk and fine chemicals. N-dealkylation of amines is also an important in vivo metabolic pathway in the metabolism of xenobiotics. Identification and synthesis of drug metabolites such as N-dealkylated metabolites are necessary throughout all phases of drug development studies. In this review, different approaches for the N-dealkylation of amines including chemical, catalytic, electrochemical, photochemical and enzymatic methods will be discussed.

The Isotopic Ac-IP Tag Enables Multiplexed Proteome Quantification in Data-Independent Acquisition Mode.

Data-independent acquisition (DIA) is an increasingly used approach for quantitative proteomics. However, most current isotope labeling strategies are not suitable for DIA as they lead to more complex MS2 spectra or severe ratio distortion. As a result, DIA suffers from a lower throughput than data-dependent acquisition (DDA) due to a lower level of multiplexing. Herein, we synthesized an isotopically labeled acetyl-isoleucine-proline (Ac-IP) tag for multiplexed quantification in DIA. Differentially labeled peptides have distinct precursor ions carrying the quantitative information but identical MS2 spectra since the isotopically labeled Ac-Ile part leaves as a neutral loss upon collision-induced dissociation, while fragmentation of the peptide backbone generates regular fragment ions for identification. The Ac-IP-labeled samples can be analyzed using general DIA liquid chromatography-mass spectrometry settings, and the data obtained can be processed with established approaches. Relative quantification requires deconvolution of the isotope envelope of the respective precursor ions. Suitability of the Ac-IP tag is demonstrated with a triplex-labeled yeast proteome spiked with bovine serum albumin that was mixed at 10:5:1 ratios, resulting in measured ratios of 9.7:5.3:1.1.

Gut bacterial deamination of residual levodopa medication for Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by both motor and non-motor symptoms. Gastrointestinal tract dysfunction is one of the non-motor features, where constipation is reported as the most common gastrointestinal symptom. Aromatic bacterial metabolites are attracting considerable attention due to their impact on gut homeostasis and host's physiology. In particular, Clostridium sporogenes is a key contributor to the production of these bioactive metabolites in the human gut. RESULTS: Here, we show that C. sporogenes deaminates levodopa, the main treatment in Parkinson's disease, and identify the aromatic aminotransferase responsible for the initiation of the deamination pathway. The deaminated metabolite from levodopa, 3-(3,4-dihydroxyphenyl)propionic acid, elicits an inhibitory effect on ileal motility in an ex vivo model. We detected 3-(3,4-dihydroxyphenyl)propionic acid in fecal samples of Parkinson's disease patients on levodopa medication and found that this metabolite is actively produced by the gut microbiota in those stool samples. CONCLUSIONS: Levodopa is deaminated by the gut bacterium C. sporogenes producing a metabolite that inhibits ileal motility ex vivo. Overall, this study underpins the importance of the metabolic pathways of the gut microbiome involved in drug metabolism not only to preserve drug effectiveness, but also to avoid potential side effects of bacterial breakdown products of the unabsorbed residue of medication.

A Collision-Induced Dissociation Cleavable Isobaric Tag for Peptide Fragment Ion-Based Quantification in Proteomics.

Quantifying peptides based on unique peptide fragment ions avoids the issue of ratio distortion that is commonly observed for reporter ion-based quantification approaches. Herein, we present a collision-induced dissociation-cleavable, isobaric acetyl-isoleucine-proline-glycine (Ac-IPG) tag, which conserves the merits of quantifying peptides based on unique fragments while reducing the complexity of the b-ion series compared to conventional fragment ion-based quantification methods thus facilitating data processing. Multiplex labeling is based on selective N-terminal dimethylation followed by derivatization of the ε-amino group of the C-terminal Lys residue of LysC peptides with isobaric Ac-IPG tags having complementary isotope distributions on Pro-Gly and Ac-Ile. Upon fragmentation between Ile and Pro, the resulting y ions, with the neutral loss of Ac-Ile, can be distinguished between the different labeling channels based on different numbers of isotope labels on the Pro-Gly part and thus contain the information for relative quantification, while b ions of different labeling channels have the same m/z values. The proteome quantification capability of this method was demonstrated by triplex labeling of a yeast proteome spiked with bovine serum albumin (BSA) over a 10-fold dynamic range. With the yeast proteins as the background, BSA was detected at ratios of 1.14:5.06:9.78 when spiked at 1:5:10 ratios. The raw mass data is available on the ProteomeXchange with the identifier PXD 018790.

A Versatile Isobaric Tag Enables Proteome Quantification in Data-Dependent and Data-Independent Acquisition Modes.

Quantifying proteins based on peptide-coupled reporter ions is a multiplexed quantitative strategy in proteomics that alleviates the problem of ratio distortion caused by peptide cofragmentation, as commonly observed in other reporter-ion-based approaches, such as TMT and iTRAQ. Data-independent acquisition (DIA) is an attractive alternative to data-dependent acquisition (DDA) due to its better reproducibility. While multiplexed labeling is widely used in DDA, it is rarely used in DIA, presumably because current approaches lead to more complex MS2 spectra, severe ratio distortion, or to a reduction in quantification accuracy and precision. Herein, we present a versatile acetyl-alanine-glycine (Ac-AG) tag that conceals quantitative information in isobarically labeled peptides and reveals it upon tandem MS in the form of peptide-coupled reporter ions. Since the peptide-coupled reporter ion is precursor-specific while fragment ions of the peptide backbone originating from different labeling channels are identical, the Ac-AG tag is compatible with both DDA and DIA. By isolating the monoisotopic peak of the precursor ion in DDA, intensities of the peptide-coupled reporter ions represent the relative ratios between constituent samples, whereas in DIA, the ratio can be inferred after deconvoluting the peptide-coupled reporter ion isotopes. The proteome quantification capability of the Ac-AG tag was demonstrated by triplex labeling of a yeast proteome spiked with bovine serum albumin (BSA) over a 10-fold dynamic range. Within this complex proteomics background, BSA spiked at 1:5:10 ratios was detected at ratios of 1.00:4.87:10.13 in DDA and 1.16:5.20:9.64 in DIA.

Selective Maleylation-Directed Isobaric Peptide Termini Labeling for Accurate Proteome Quantification.

Isobaric peptide termini labeling (IPTL) is an attractive protein quantification method because it provides more accurate and reliable quantification information than traditional isobaric labeling methods (e.g., TMT and iTRAQ) by making use of the entire fragment-ion series instead of only a single reporter ion. The multiplexing capacity of published IPTL implementations is, however, limited to three. Here, we present a selective maleylation-directed isobaric peptide termini labeling (SMD-IPTL) approach for quantitative proteomics of LysC protein digestion. SMD-IPTL extends the multiplexing capacity to 4-plex with the potential for higher levels of multiplexing using commercially available 13C/15N labeled amino acids. SMD-IPTL is achieved in a one-pot reaction in three consecutive steps: (1) selective maleylation at the N-terminus; (2) labeling at the ε-NH2 group of the C-terminal Lys with isotopically labeled acetyl-alanine; (3) thiol Michael addition of an isotopically labeled acetyl-cysteine at the maleylated N-terminus. The isobarically labeled peptides are fragmented into sets of b- and y-ion clusters upon LC-MS/MS, which convey not only sequence information but also quantitative information for every labeling channel and avoid the issue of ratio distortion observed with reporter-ion-based approaches. We demonstrate the SMD-IPTL approach with a 4-plex labeled sample of bovine serum albumin (BSA) and yeast lysates mixed at different ratios. With the use of SMD-IPTL for labeling and a narrow precursor isolation window of 0.8 Th with an offset of -0.2 Th, accurate ratios were measured across a 10-fold mixing range of BSA in a background of yeast proteome. With the yeast proteins mixed at ratios of 1:5:1:5, BSA was detected at ratios of 0.94:2.46:4.70:9.92 when spiked at 1:2:5:10 ratios with an average standard deviation of peptide ratios of 0.34.

Avian yolk androgens are metabolized rather than taken up by the embryo during the first days of incubation.

Several studies show effects of yolk androgens in avian eggs on the phenotype of the offspring. Yolk hormone concentrations decline strongly within the first few days of incubation. Although early embryonic uptake of yolk androgens is suggested by the presence of radioactivity in the embryo when eggs are injected with radiolabelled androgens, these studies do not verify the chemical identity of radioactive compound(s), although it is known that these androgens can be metabolized substantially. By using stable isotope-labelled testosterone and androstenedione in combination with mass spectrometry, enabling verification of the exact molecular identity of labelled compounds in the embryo, we found that after 5 days of incubation the androgens were not taken up by the embryo. However, their concentrations in the entire yolk albumen homogenates declined strongly, even when corrected for dilution by albumen and water. Our results indicate metabolism of maternal androgens, very likely to 5ß-androstane-3α,17ß-diol, etiocholanolone and their conjugated forms. The results imply that the effects of increased exposure of the embryo to maternal androgens take place either before this early conversion or are mediated by these metabolites with an as yet unknown function, opening new avenues for understanding hormone-mediated maternal effects in vertebrates.

Targeted Proteomics to Study Mitochondrial Biology.

Targeted mass spectrometry in the selected or parallel reaction monitoring (SRM or PRM) mode is a widely used methodology to quantify proteins based on so-called signature or proteotypic peptides. SRM has the advantage of being able to quantify a range of proteins in a single analysis, for example, to measure the level of enzymes comprising a biochemical pathway. In this chapter, we will detail how to set up an SRM assay on the example of the mitochondrial protein succinate dehydrogenase [ubiquinone] flavoprotein subunit (mouse UniProt-code Q8K2B3). First, we will outline the in silico assay design including the choice of peptides based on a range of properties. We will further delineate different quantification strategies and introduce the reader to LC-MS assay development including the selection of the optimal peptide charge state and fragment ions as well as a discussion of the dynamic range of detection. The chapter will close with an application from the area of mitochondrial biology related to the quantification of a set of proteins isolated from mouse liver mitochondria in a study on mitochondrial respiratory flux decline in aging mouse muscle.

Bioconjugation of Supramolecular Metallacages to Integrin Ligands for Targeted Delivery of Cisplatin.

Cisplatin occupies a crucial role in the treatment of various malignant tumors. However, its efficacy and applicability are heavily restricted by severe systemic toxicities and drug resistance. Our study exploits the active targeting of supramolecular metallacages to enhance the activity of cisplatin in cancer cells while reducing its toxicity. Thus, Pd2L4 cages (L = ligand) have been conjugated to four integrin ligands with different binding affinity and selectivity. Cage formation and encapsulation of cisplatin was proven by NMR spectroscopy. Upon encapsulation, cisplatin showed increased cytotoxicity in vitro, in melanoma A375 cells overexpressing αvß3 integrins. Moreover, ex vivo studies in tissue slices indicated reduced toxicity toward healthy liver and kidney tissues for cage-encapsulated cisplatin. Analysis of metal content by ICP-MS demonstrated that the encapsulated drug is less accumulated in these organs compared to the "free" cisplatin.

One- vs two-phase extraction: re-evaluation of sample preparation procedures for untargeted lipidomics in plasma samples.

Lipidomics is a rapidly developing field in modern biomedical research. While LC-MS systems are able to detect most of the known lipid classes in a biological matrix, there is no single technique able to extract all of them simultaneously. In comparison with two-phase extractions, one-phase extraction systems are of particular interest, since they decrease the complexity of the experimental procedure. By using an untargeted lipidomics approach, we explored the differences/similarities between the most commonly used two-phase extraction systems (Folch, Bligh and Dyer, and MTBE) and one of the more recently introduced one-phase extraction systems for lipid analysis based on the MMC solvent mixture (MeOH/MTBE/CHCl3). The four extraction methods were evaluated and thoroughly compared against a pooled extract that qualitatively and quantitatively represents the average of the combined extractions. Our results show that the lipid profile obtained with the MMC system displayed the highest similarity to the pooled extract, indicating that it was most representative of the lipidome in the original sample. Furthermore, it showed better extraction efficiencies for moderate and highly apolar lipid species in comparison with the Folch, Bligh and Dyer, and MTBE extraction systems. Finally, the technical simplicity of the MMC procedure makes this solvent system highly suitable for automated, untargeted lipidomics analysis.

Specific Affinity Enrichment of Electrochemically Cleaved Peptides Based on Cu(II)-Mediated Spirolactone Tagging.

Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Electrochemical oxidation yielding specific cleavage C-terminal to tyrosine (Tyr) and tryptophan (Trp) residues provides a potential alternative to enzymatic digestion and a possibility for further chemical labeling by introducing reactive spirolactone moieties. However, spirolactone-containing peptides suffer from low stability due to hydrolysis and intramolecular side reactions. We found that Cu(II) ions stabilize the spirolactone and prevent intramolecular side reactions during chemical labeling, allowing efficient chemical tagging with a reduced excess of labeling reagent without intramolecular side reactions. On the basis of this reaction, we developed an analytical procedure combining electrochemical digestion, Cu(II)-mediated spirolactone biotinylation, and enrichment by avidin affinity chromatography with mass spectrometry. The method was optimized with the tripeptide LWL and subsequently applied to chicken egg white lysozyme, in which one biotinylated electrochemistry (EC)-cleaved peptide was identified after affinity enrichment. This proof-of-principle shows that specific enrichment of electrochemically cleaved spirolactone-containing peptides can be used for protein identification and notably that inclusion of Cu(II) ions is essential for stabilizing spirolactones for subsequent biotinylation.

Living on the edge: substrate competition explains loss of robustness in mitochondrial fatty-acid oxidation disorders.

BACKGROUND: Defects in genes involved in mitochondrial fatty-acid oxidation (mFAO) reduce the ability of patients to cope with metabolic challenges. mFAO enzymes accept multiple substrates of different chain length, leading to molecular competition among the substrates. Here, we combined computational modeling with quantitative mouse and patient data to investigate whether substrate competition affects pathway robustness in mFAO disorders. RESULTS: First, we used comprehensive biochemical analyses of wild-type mice and mice deficient for medium-chain acyl-CoA dehydrogenase (MCAD) to parameterize a detailed computational model of mFAO. Model simulations predicted that MCAD deficiency would have no effect on the pathway flux at low concentrations of the mFAO substrate palmitoyl-CoA. However, high concentrations of palmitoyl-CoA would induce a decline in flux and an accumulation of intermediate metabolites. We proved computationally that the predicted overload behavior was due to substrate competition in the pathway. Second, to study the clinical relevance of this mechanism, we used patients' metabolite profiles and generated a humanized version of the computational model. While molecular competition did not affect the plasma metabolite profiles during MCAD deficiency, it was a key factor in explaining the characteristic acylcarnitine profiles of multiple acyl-CoA dehydrogenase deficient patients. The patient-specific computational models allowed us to predict the severity of the disease phenotype, providing a proof of principle for the systems medicine approach. CONCLUSION: We conclude that substrate competition is at the basis of the physiology seen in patients with mFAO disorders, a finding that may explain why these patients run a risk of a life-threatening metabolic catastrophe.

Translational Targeted Proteomics Profiling of Mitochondrial Energy Metabolic Pathways in Mouse and Human Samples.

Absolute measurements of protein abundance are important in the understanding of biological processes and the precise computational modeling of biological pathways. We developed targeted LC-MS/MS assays in the selected reaction monitoring (SRM) mode to quantify over 50 mitochondrial proteins in a single run. The targeted proteins cover the tricarboxylic acid cycle, fatty acid ß-oxidation, oxidative phosphorylation, and the detoxification of reactive oxygen species. Assays used isotopically labeled concatemers as internal standards designed to target murine mitochondrial proteins and their human orthologues. Most assays were also suitable to quantify the corresponding protein orthologues in rats. After exclusion of peptides that did not pass the selection criteria, we arrived at SRM assays for 55 mouse, 52 human, and 51 rat proteins. These assays were optimized in isolated mitochondrial fractions from mouse and rat liver and cultured human fibroblasts and in total liver extracts from mouse, rat, and human. The developed proteomics approach is suitable for the quantification of proteins in the mitochondrial energy metabolic pathways in mice, rats, and humans as a basis for translational research. Initial data show that the assays have great potential for elucidating the adaptive response of human patients to mutations in mitochondrial proteins in a clinical setting.

Efficient and Selective Chemical Labeling of Electrochemically Generated Peptides Based on Spirolactone Chemistry.

Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Cleavage of the peptide bond following electrochemical oxidation of Tyr or Trp results in a spirolactone moiety at the newly formed C-terminus offering a handle for chemical labeling. In this work, we developed a highly efficient and selective chemical labeling approach based on spirolactone chemistry. Electrochemically generated peptide-spirolactones readily undergo an intramolecular rearrangement yielding isomeric diketopiperazines precluding further chemical labeling. A strategy was established to prevent intramolecular arrangement by acetylating the N-terminal amino group prior to electrochemical oxidation and cleavage allowing the complete and selective chemical labeling of the tripeptide LWL and the decapeptide ACTH 1-10 with amine-containing reagents. As examples, we show the successful introduction of a fluorescent label and biotin for detection or affinity enrichment. Electrochemical digestion of peptides and proteins followed by efficient chemical labeling constitutes a new, powerful tool in protein chemistry and protein analysis.

Electrochemical Protein Cleavage in a Microfluidic Cell with Integrated Boron Doped Diamond Electrodes.

Specific electrochemical cleavage of peptide bonds at the C-terminal side of tyrosine and tryptophan generates peptides amenable to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for protein identification. To this end we developed a microfluidic electrochemical cell of 160 nL volume that combines a cell geometry optimized for a high electrochemical conversion efficiency (>95%) with an integrated boron doped diamond (BDD) working electrode offering a wide potential window in aqueous solution and reduced adsorption of peptides and proteins. Efficient cleavage of the proteins bovine insulin and chicken egg white lysozyme was observed at 4 out of 4 and 7 out of 9 of the predicted cleavage sites, respectively. Chicken egg white lysozyme was identified based on 5 electrochemically generated peptides using a proteomics database searching algorithm. These results show that electrochemical peptide bond cleavage in a microfluidic cell is a novel, fully instrumental approach toward protein analysis and eventually proteomics studies in conjunction with mass spectrometry.

Microbial Flavoprotein Monooxygenases as Mimics of Mammalian Flavin-Containing Monooxygenases for the Enantioselective Preparation of Drug Metabolites.

Mammalian flavin-containing monooxygenases, which are difficult to obtain and study, play a major role in detoxifying various xenobiotics. To provide alternative biocatalytic tools to generate flavin-containing monooxygenases (FMO)-derived drug metabolites, a collection of microbial flavoprotein monooxygenases, sequence-related to human FMOs, was tested for their ability to oxidize a set of xenobiotic compounds. For all tested xenobiotics [nicotine, lidocaine, 3-(methylthio)aniline, albendazole, and fenbendazole], one or more monooxygenases were identified capable of converting the target compound. Chiral liquid chromatography with tandem mass spectrometry analyses of the conversions of 3-(methylthio)aniline, albendazole, and fenbendazole revealed that the respective sulfoxides are formed in good to excellent enantiomeric excess (e.e.) by several of the tested monooxygenases. Intriguingly, depending on the chosen microbial monooxygenase, either the (R)- or (S)-sulfoxide was formed. For example, when using a monooxygenase from Rhodococcus jostii the (S)-sulfoxide of albendazole (ricobendazole) was obtained with a 95% e.e. whereas a fungal monooxygenase yielded the respective (R)-sulfoxide in 57% e.e. For nicotine and lidocaine, monooxygenases could be identified that convert the amines into their respective N-oxides. This study shows that recombinantly expressed microbial monooxygenases represent a valuable toolbox of mammalian FMO mimics that can be exploited for the production of FMO-associated xenobiotic metabolites.

Functional and structural characterization of an unusual cofactor-independent oxygenase.

The vast majority of characterized oxygenases use bound cofactors to activate molecular oxygen to carry out oxidation chemistry. Here, we show that an enzyme of unknown activity, RhCC from Rhodococcus jostii RHA1, functions as an oxygenase, using 4-hydroxyphenylenolpyruvate as a substrate. This unique and complex reaction yields 3-hydroxy-3-(4-hydroxyphenyl)-pyruvate, 4-hydroxybenzaldehyde, and oxalic acid as major products. Incubations with H2(18)O, (18)O2, and a substrate analogue suggest that this enzymatic oxygenation reaction likely involves a peroxide anion intermediate. Analysis of sequence similarity and the crystal structure of RhCC (solved at 1.78 Å resolution) reveal that this enzyme belongs to the tautomerase superfamily. Members of this superfamily typically catalyze tautomerization, dehalogenation, or decarboxylation reactions rather than oxygenation reactions. The structure shows the absence of cofactors, establishing RhCC as a rare example of a redox-metal- and coenzyme-free oxygenase. This sets the stage to study the mechanistic details of cofactor-independent oxygen activation in the unusual context of the tautomerase superfamily.