Search

Lung implantable devices have been widely adopted as mechanical interventions for a wide variety of pulmonary pathologies. Despite successful initial treatment, long-term efficacy can often be impacted by fibrotic or granulation tissue formation at the implant sites. This study aimed to explore the lung-device interface by identifying the adhered proteome on lung devices explanted from patients with severe emphysema. In this study, scanning electron microscopy is used to visualize the adhesion of cells and proteins to silicone and nitinol surfaces of explanted endobronchial valves. By applying high-resolution mass-spectrometry, the surface proteome of eight explanted valves is characterized, identifying 263 unique protein species to be mutually adsorbed on the valves. This subset is subjected to gene enrichment analysis, matched with known databases and further validated using immunohistochemistry. Enrichment analyses reveal dominant clusters of functionally-related ontology terms associated with coagulation, pattern recognition receptor signaling, immune responses, cytoskeleton organization, cell adhesion and migration. Matching results show that extracellular matrix proteins and damage-associated molecular patterns are cardinal in the formation of the surface proteome. This is the first study investigating the composition of the adhered proteome on explanted lung devices, setting the groundwork for hypothesis generation and further exploration. STATEMENT OF SIGNIFICANCE: This is the first study investigating the composition of the adhered proteome on explanted lung devices. Lung implantable devices have been widely adopted as mechanical interventions for pulmonary pathologies. Despite successful initial treatment, long-term efficacy can often be impacted by fibrotic or granulation tissue formation around the implant sites. We identified the adhered proteome on explanted lung devices using several techniques. We identified 263 unique protein species to be mutually adsorbed on explanted lung devices. Pathway analyses revealed that these proteins are associated with coagulation, pattern recognition receptor signaling, immune responses, cytoskeleton organization, cell adhesion and migration. Furthermore, we identified that especially extracellular matrix proteins and damage-associated molecular patterns were cardinal in the formation of the surface proteome.

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.

Here, we present multifunctional fluorescent nanodiamonds (FNDs) for simultaneous drug delivery and free radical detection. For this purpose, we modified FNDs containing nitrogen vacancy (NV) centers with a diazoxide derivative. We found that our particles enter cells more easily and are able to deliver this cancer drug into HeLa cells. The particles were characterized by infrared spectroscopy, dynamic light scattering, and secondary electron microscopy. Compared to the free drug, we observe a sustained release over 72 h rather than 12 h for the free drug. Apart from releasing the drug, with these particles, we can measure the drug's effect on free radical generation directly. This has the advantage that the response is measured locally, where the drug is released. These FNDs change their optical properties based on their magnetic surrounding. More specifically, we make use of a technique called relaxometry to detect spin noise from the free radical at the nanoscale with subcellular resolution. We further compared the results from our new technique with a conventional fluorescence assay for the detection of reactive oxygen species. This provides a new method to investigate the relationship between drug release and the response by the cell via radical formation or inhibition.

A novel method for the selective catalytic N-dealkylation of drug molecules on a nanoporous gold (NPG) catalyst producing valuable N-dealkylated metabolites and intermediates is described. Drug metabolites are important chemical entities at every stage of drug discovery and development, from exploratory discovery to clinical development, providing the safety profiles and the ADME (adsorption, distribution, metabolism, and elimination) of new drug candidates. Synthesis was carried out in aqueous solution at 80 °C using air (oxygen source) as oxidant, in single step with good isolated yields. Different examples examined in this study showed that aerobic catalytic N-dealkylation of drug molecules on NPG has a broad scope supporting N-deethylation, N-deisopropylation and N-demethylation, converting either 3° amines to 2° amines, or 2° amines to 1° amines.

A practical, efficient, and selective electrochemicalN-demethylation method of tropane alkaloids to their nortropane derivatives is described. Nortropanes, such as noratropine and norscopolamine, are important intermediates for the semi-synthesis of the medicines ipratropium or oxitropium bromide, respectively. Synthesis was performed in a simple home-made electrochemical batch cell using a porous glassy carbon electrode. The reaction proceeds at room temperature in one step in a mixture of ethanol or methanol and water. The method avoids hazardous oxidizing agents such as H(2)O(2)orm-chloroperbenzoic acid (m-CPBA), toxic solvents such as chloroform, as well as metal-based catalysts. Various key parameters were investigated in electrochemical batch or flow cells, and the optimized conditions were used in batch and flow-cells at gram scale to synthesize noratropine in high yield and purity using a convenient liquid-liquid extraction method without any need for chromatographic purification. Mechanistic studies showed that the electrochemicalN-demethylation proceeds by the formation of an iminium intermediate which is converted by water as the nucleophile. The optimized method was further applied to scopolamine, cocaine, benzatropine, homatropine and tropacocaine, showing that this is a generic way ofN-demethylating tropane alkaloids to synthesize valuable precursors for pharmaceutical products.

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.

Targeted epitope-based mass spectrometry imaging (MSI) utilizes laser cleavable mass-tags bound to targeting moieties for detecting proteins in tissue sections. Our work constitutes the first proof-of-concept of a novel laser desorption ionization (LDI)-MSI strategy using photocleavable Ru(ii) polypyridine complexes as mass-tags for imaging of integrins avß3 in human cancer tissues.

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.

Methylphenidate is one of the most widely used oral treatments for attention-deficit/hyperactivity disorder (ADHD). The drug is mainly absorbed in the small intestine and has low bioavailability. Accordingly, a high interindividual variability in terms of response to the treatment is known among ADHD patients treated with methylphenidate. Nonetheless, very little is known about the factors that influence the drug’s absorption and bioavailability. Gut microbiota has been shown to reduce the bioavailability of a wide variety of orally administered drugs. Here, we tested the ability of small intestinal bacteria to metabolize methylphenidate. In silico analysis identified several small intestinal bacteria to harbor homologues of the human carboxylesterase 1 enzyme responsible for the hydrolysis of methylphenidate in the liver into the inactive form, ritalinic acid. Despite our initial results hinting towards possible bacterial hydrolysis of the drug, up to 60% of methylphenidate is spontaneously hydrolyzed in the absence of bacteria and this hydrolysis is pH-dependent. Overall, our results indicate that the stability of methylphenidate is compromised under certain pH conditions in the presence or absence of gut microbiota.

The recent development of mass spectrometry imaging (MSI) technology allowed to obtain extremely detailed images of the spatial distribution of proteins in tissue at high spatial resolution reaching cell dimensions, high target specificity and a large dynamic concentration range. This review focusses on the development of two main MSI principles, namely targeted and untargeted detection of protein distribution in tissue samples, with special emphasis on the improvements in analyzed mass range and spatial resolution over the last 10 years. Untargeted MSI of in situ digested proteins with matrix-assisted laser desorption ionization is the most widely used approach, but targeted protein MSI technologies using laser ablation inductively coupled plasma (LA-ICP) and photocleavable mass tag chemical labeling strategies are gaining momentum. Moreover, this review also provides an overview of the effect of sample preparation on image quality and the bioinformatic challenge to identify proteins and quantify their distribution in complex MSI data.

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.

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.

Methylphenidate is absorbed in the small intestine. The drug is known to have low bioavailability and a high interindividual variability in terms of response to the treatment. Gut microbiota has been shown to reduce the bioavailability of a wide variety of orally administered drugs. Here, we tested the ability of small intestinal bacteria to metabolize methylphenidate. In silico analysis identified several small intestinal bacteria to harbor homologues of the human carboxylesterase 1 enzyme responsible for the hydrolysis of methylphenidate in the liver. Despite our initial results hinting towards possible bacterial hydrolysis of the drug, up to 60% of methylphenidate was spontaneously hydrolyzed in the absence of bacteria and this hydrolysis was pH-dependent. Overall, the study shows that pH-dependent spontaneous hydrolysis rather than gut bacterial metabolism reduces levels of methylphenidate and suggest a role of the luminal pH in the bioavailability of the drug.

Cisplatin occupies a crucial role in the treatment of various malignant tumours. 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 αvβ3 integrins. Moreover, ex vivo studies in tissue slices indicated reduced toxicity towards healthy liver and kidney tissues for cage-encapsulated cisplatin. Analysis of metal content by ICP-MS demonstrated that encapsulated drug is less accumulated in these organs compared to the 'free' one.

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. Electronic supplementary material The online version of this article (10.1007/s00216-018-1200-x) contains supplementary material, which is available to authorized users.

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.

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.

Supramolecular Pd2L4 cages (L = ligand) hold promise as drug delivery systems. With the idea of achieving targeted delivery of the metallacages to tumor cells, the bioconjugation of exo-functionalized self-assembled Pd2L4 cages to peptides following two different approaches is reported for the first time. The obtained bioconjugates were analyzed and identified by high-resolution mass spectrometry.

Aromatic hydroxylation reactions, which are mainly catalyzed by cytochrome P450 (CYP) enzymes in vivo, are some of the most important reactions of Phase I metabolism, because insertion of a hydroxyl group into a lipophilic drug compound increases its hydrophilicity and prepares it for subsequent Phase II metabolic conjugation reactions as a prerequisite to excretion. Aromatic hydroxylation metabolites of pharmaceuticals may be obtained through various synthetic and enzymatic methods Electrochemical oxidation is an alternative with advantages in terms of mild reaction conditions and less hazardous chemicals. In the present study, we report that aromatic hydroxylation metabolites of lidocaine can be readily obtained electrochemically under aqueous acidic conditions at platinum electrodes. Our results show that the dominant N-deallcylation reaction can be suppressed by decreasing the solution pH below 0.5 resulting in selective 3-hydroxylidocaine, which is an in vivo metabolite of lidocaine. Experiments in O-18 labelled water indicated that water is the primary source of oxygen, while dissolved molecular oxygen contributes to a minor extent to the hydroxylation reaction. (C) 2016 Elsevier Ltd. All rights reserved.

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.

Several studies show effects of yolk androgens in avian eggs on the phenotype of the offspring. Yolk hormone concentrations decline strongly already in 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), while 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 five days of incubation the androgens are not taken up by the embryo. Yet their concentrations in the entire yolk-albumen homogenates decline strongly, even when corrected for dilution by albumen and water. Our results indicate metabolism of maternal androgens, very likely to 5β-androstane-3α,17β-diol, etiocholanolone, and their conjugated forms. The results imply that the effects of increased exposure of the embryo to maternal androgens either take place before this early conversion or are mediated by these metabolites with a so far unknown function, opening new avenues for understanding hormone mediated maternal effects in vertebrates.

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.

The feasibility of titanium dioxide (TiO2) photocatalysis, electrochemically assisted Fenton reaction (EC-Fenton) and direct electrochemical oxidation (EC) for simulation of phase I metabolism of drugs was studied by comparing the reaction products of buspirone, promazine, testosterone and 7-ethoxycoumarin with phase I metabolites of the same compounds produced in vitro by human liver microsomes (HLM). Reaction products were analysed by UHPLC–MS. TiO2 photocatalysis simulated the in vitro phase I metabolism in HLM more comprehensively than did EC-Fenton or EC. Even though TiO2 photocatalysis, EC-Fenton and EC do not allow comprehensive prediction of phase I metabolism, all three methods produce several important metabolites without the need for demanding purification steps to remove the biological matrix. Importantly, TiO2 photocatalysis produces aliphatic and aromatic hydroxylation products where direct EC fails. Furthermore, TiO2 photocatalysis is an extremely rapid, simple and inexpensive way to generate oxidation products in a clean matrix and the reaction can be simply initiated and quenched by switching the UV lamp on/off.

Protein heterogeneity may result from many factors often closely related to the regulation of biological mechanisms. This review addresses one source of protein heterogeneity, the translation of genetic variability and transcriptional modulation to the protein level. We provide an overview how customized protein sequence databases generated using genomic and transcriptomic sequence information in conjunction with approaches to increase protein sequence coverage can aid in gaining a deeper insight into variability at the protein level. Modem approaches of DNA/RNA sequencing open the possibility to obtain detailed sequence information from individual genomes and transcriptomes at single nucleotide resolution. Further studies tried to correlate genetic variability with important biological consequences such as the risk for developing a disease or defining a personalized approach towards therapy (also called "personalized or precision medicine"). Linking genomic and transcriptomic information to complex biological mechanisms has, however, remained elusive due to the fact that there is no direct cause and effect relationship between changes at the DNA/RNA level and downstream consequences. In this review we give an overview of the challenges of integrating genomics and transcriptomics data with proteomics data and link variability at the DNA/RNA level to protein variability and protein species.Biological significance: The manuscript focuses on a recent trend in proteomics, namely the integration of genomic and proteomic data. Genetic and transcriptomic variability accounts for a considerable part of protein variability and is at the basis of many protein species, many of which not yet described at the protein level but many also identified as proteins or peptides with unknown function. The review highlights the challenges of current proteomics methodology, notably incomplete sequence coverage, which make it difficult to appreciate the full complexity of any proteome and leads to the fact that much variability at the DNA/RNA level is not captured at the protein level. We outline a few strategies to ameliorate this situation. (C) 2015 Elsevier B.V. All rights reserved.

A generic approach for flavoenzyme immobilization was developed in which the flavin cofactor is used for anchoring enzymes onto the carrier. It exploits the tight binding of flavin cofactors to their target apo proteins. The method was tested for phenylacetone monooxygenase (PAMO) which is a well-studied and industrially interesting biocatalyst. Also a fusion protein was tested: PAMO fused to phosphite dehydrogenase (PTDH-PAMO). The employed flavin cofactor derivative, N6-(6-carboxyhexyl)-FAD succinimidylester (FAD*), was covalently anchored to agarose beads and served for apo enzyme immobilization by their reconstitution into holo enzymes. The thus immobilized enzymes retained their activity and remained active after several rounds of catalysis. For both tested enzymes, the generated agarose beads contained 3 U per g of dry resin. Notably, FAD-immobilized PAMO was found to be more thermostable (40% activity after 1 h at 60 degrees C) when compared to PAMO in solution (no activity detected after 1 h at 60 degrees C). The FAD-decorated agarose material could be easily recycled allowing multiple rounds of immobilization. This method allows an efficient and selective immobilization of flavoproteins via the FAD flavin cofactor onto a recyclable carrier. (C) 2015 Elsevier Inc. All rights reserved.

Electrochemical protein digestion prior to mass spectrometric analysis is a purely instrumental approach to protein identification, offering reduced consumption of chemicals and shorter analysis times compared to the use of enzymes and chemical cleavage agents. Here we demonstrate the possibilities of site-specific peptide bond cleavage and disulphide bond reduction in a microfluidic electrochemical cell. The use of microfluidics in this context is beneficial for increased electrochemical cleavage yields, small sample volumes and the possibility of rapid on-line analysis, thereby providing a versatile tool for routine proteomics studies.

Lipidomics is the most recent member in the family of the omic technologies. Its main objective is the identification and quantitation of the entire collection of chemically distinct lipid species in a cell, tissue, or biological fluid. Despite 15 years of development in the field, issues regarding the stability of the lipidome during the preanalytical phase have not been assessed in great detail. With this article, our aim is to focus attention on possible pitfalls of typical lipidomics strategies and to provide the reader with background information to approach this challenge in a systematic manner.

Identification of potentially toxic oxidative drug metabolites is a crucial step in the development of new drugs. Electrochemical methods are useful to study oxidative drug metabolism, but are not widely used to synthesize metabolites for follow-up studies. Careful optimization of reaction parameters is important for scaling up the electrochemical synthesis of metabolites. In the present study, lidocaine was used as a drug compound in order to optimize electrochemical reaction parameters employing a design of experiments approach to improve the yield of N-dealkylated lidocaine, a major in vivo metabolite. pH and electrode material were found to have a major effect on the final yield.

Recent advances in analytical chemistry have set the stage for metabolite profiling to help understand complex molecular processes in physiology. Despite ongoing efforts, there are concerns regarding metabolomics workflows, since it has been shown that internal (enzyme activity, blood contamination, and the dynamic nature of metabolite concentrations) as well as external factors (storage, handling, and analysis method) may affect the metabolome profile. Many metabolites are intrinsically instable, particularly some of those associated with central carbon metabolism. While enzymatic conversions have been studied in great detail, nonenzymatic, chemical conversions received comparatively little attention. This review aims to give an in-depth overview of nonenzymatic energy metabolite degradation/interconversion chemistry focusing on a selected range of metabolites. Special attention will be given to qualitative (degradation pathways) as well as quantitative aspects, that may affect the acquisition of accurate data in the context of metabolomics studies. Problems related to the use of isotopically labeled internal standards hindering the quantitative analysis of common metabolites will be presented with an experimental example. Finally, general conclusions and perspectives are given.

This review discusses different approaches that have been taken to mimic oxidative drug metabolism as executed by members of the Cytochrome P450 (CYP450) family of enzymes in humans. Non-modified electrodes can be used to produce some of the oxidative drug metabolites observed in vivo but their scope is rather limited. Modifying electrodes with simple cofactors in analogy to those observed in CYP450 but without the protein scaffold extends these possibilities and notably allows driving reactions following a CYP450-like mechanism. The review ends with approaches to immobilize CYP450s or analogs thereof on electrodes to mimic the in vivo drug metabolism fully. We discuss future perspectives with respect to the advantages and the disadvantages of each level of complexity and possible ways forward.

Identification of potentially toxic metabolites is important for drug discovery and development. Synthesis of drug metabolites is typically performed by organic synthesis or enzymatic methods, but is not always straightforward. Electrochemical (EC) methods are increasingly used to study drug oxidation and to identify potential metabolites of new drug candidates, but the absolute yield of metabolites of these methods is low. This review discusses the challenges and recent developments of EC synthesis in terms of instrumental aspects, EC-reaction parameters and reaction-monitoring approaches, in an effort to produce drug metabolites selectively from parent drugs on a preparative scale (1–10 mg).

Native enzyme and a mutant containing an extra disulphide bridge of recombinant Saccharomycopsis fibuligera R64 α-amylase, designated as Sfamy01 and Sfamy02, respectively, have successfully been overexpressed in the yeast Pichia pastoris KM71H . The purified α-amylase variants demonstrated starch hydrolysis resulting in a mixture of maltose, maltotriose, and glucose, similar to the wild type enzyme. Introduction of the disulphide bridge shifted the melting temperature ( T M ) from 54.5 to 56 °C and nearly tripled the enzyme half-life time at 65 °C. The two variants have similar k cat / K M values. Similarly, inhibition by acarbose was only slightly affected, with the IC50 of Sfamy02 for acarbose being 40 ± 3.4 μM, while that of Sfamy01 was 31 ± 3.9 μM. On the other hand, the IC50 of Sfamy02 for EDTA was 0.45 mM, nearly two times lower than that of Sfamy01 at 0.77 mM. These results show that the introduction of a disulphide bridge had little effect on the enzyme activity, but made the enzyme more susceptible to calcium ion extraction. Altogether, the new disulphide bridge improved the enzyme stability without affecting its activity, although minor changes in the active site environment cannot be excluded.

Introduction Boiling ethanol extraction is a frequently used method for metabolomics studies of biological samples. However, the stability of several central carbon metabolites, including nucleotide triphosphates, and the influence of the cellular matrix on their degradation have not been addressed. Objectives To study how a complex cellular matrix extracted from yeast (Saccharomyces cerevisiae) may affect the degradation profiles of nucleotide triphosphates extracted under boiling ethanol conditions. Methods We present a double-labelling LC–MS approach with a 13C-labeled yeast cellular extract as complex surrogate matrix, and 13C15N-labeled nucleotides as internal standards, to study the effect of the yeast matrix on the degradation of nucleotide triphosphates. Results While nucleotide triphosphates were degraded to the corresponding diphosphates in pure solutions, degradation was prevented in the presence of the yeast matrix under typical boiling ethanol extraction conditions. Conclusions Extraction of biological samples under boiling ethanol extraction conditions that rapidly inactivate enzyme activity are suitable for labile central energy metabolites such as nucleotide triphosphates due to the stabilizing effect of the yeast matrix. The basis of this phenomenon requires further study. Graphical abstract Electronic supplementary material The online version of this article (doi:10.1007/s11306-016-1140-4) contains supplementary material, which is available to authorized users.

We report the development of a 3D-printed cartridge for paper spray ionization (PSI) that can be used almost immediately after solvent introduction in a dedicated reservoir and allows prolonged spray generation from a paper tip. The fast wetting feature described in this work is based on capillary action through paper and movement of fluid between paper and the cartridge material (polylactic acid, PLA). The influence of solvent composition, PLA conditioning of the cartridge with isopropanol, and solvent volume introduced into the reservoir have been investigated with relation to wetting time and the amount of solvent consumed for wetting. Spray has been demonstrated with this cartridge for tens of minutes, without any external pumping. It is shown that fast wetting and spray generation can easily be achieved using a number of solvent mixtures commonly used for PSI. The PSI cartridge was applied to the analysis of lidocaine from a paper tip using different solvent mixtures, and to the analysis of lidocaine from a serum sample. Finally, a demonstration of online paper chromatography-mass spectrometry is given.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0327-5) contains supplementary material, which is available to authorized users.

Electrochemical oxidation of peptides and proteins is traditionally performed on carbon-based electrodes. Adsorption caused by the affinity of hydrophobic and aromatic amino acids toward these surfaces leads to electrode fouling. We compared the performance of boron-doped diamond (BDD) and glassy carbon (GC) electrodes for the electrochemical oxidation and cleavage of peptides. An optimal working potential of 2000 mV was chosen to ensure oxidation of peptides on BDD by electron transfer processes only. Oxidation by electrogenerated OH radicals took place above 2500 mV on BDD, which is undesirable if cleavage of a peptide is to be achieved. BDD showed improved cleavage yield and reduced adsorption for a set of small peptides, some of which had been previously shown to undergo electrochemical cleavage C-terminal to tyrosine (Tyr) and tryptophan (Trp) on porous carbon electrodes. Repeated oxidation with BDD electrodes resulted in progressively lower conversion yields due to a change in surface termination. Cathodic pretreatment of BDD at a negative potential in an acidic environment successfully regenerated the electrode surface and allowed for repeatable reactions over extended periods of time. BDD electrodes are a promising alternative to GC electrodes in terms of reduced adsorption and fouling and the possibility to regenerate them for consistent high-yield electrochemical cleavage of peptides. The fact that OH-radicals can be produced by anodic oxidation of water at elevated positive potentials is an additional advantage as they allow another set of oxidative reactions in analogy to the Fenton reaction, thus widening the scope of electrochemistry in protein and peptide chemistry and analytics.

The aim of this study was to investigate the effect of divalent metal ions (Ca, Mg 2+ , and Zn 2+ ) on the stability of oxytocin in aspartate buffer (pH 4.5) and to determine their interaction with the peptide in aqueous solution. Reversed-phase high-performance liq- uid chromatography and high-performance size-exclusion chromatography measurements indi- cated that after 4 weeks of storage at 55 ◦ C, all tested divalent metal ions improved the stability of oxytocin in aspartate-buffered solutions (pH 4.5). However, the stabilizing effects of Zn 2+ were by far superior compared with Ca 2+ and Mg 2+ . Liquid chromatography-tandem mass spectrom- etry showed that the combination of aspartate and Zn 2+ in particular suppressed the formation of peptide dimers. As shown by isothermal titration calorimetry, Zn 2+ interacted with oxytocin in the presence of aspartate buffer, whereas Ca 2+ or Mg 2+ did not. In conclusion, the stability of oxytocin in the aspartate-buffered solution is strongly improved in the presence of Zn 2+ ,a nd the stabilization effect is correlated with the ability of the divalent metal ions in aspartate buffer to interact with oxytocin. The reported results are discussed in relation to the possible mode of interactions among the peptide, Zn 2+ , and buffer components leading to the observed stabilization effects. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1734-1741, 2013

We investigated the effect of myriocin treatment, which extensively depletes sphingolipids from cells, on multidrug resistance-related protein 1 (MRP1) efflux activity in MRP1 expressing cells and isolated plasma membrane vesicles. Our data reveal that both short term (3 days) and long term (7 days) treatment effectively reduce the cellular sphingolipid content to the same level. Intriguingly, a two-fold increase in MRP1-mediated efflux activity was observed following long term treatment, while short term treatment had no impact. Very similar data were obtained with plasma membrane vesicles isolated from myriocin-treated cells. Exploiting the cell-free vesicle system, Michaelis-Menten analysis revealed that the intrinsic MRP1 activity remained unaltered; however, the fraction of active transporter molecules increased. We demonstrate that the latter effect is due to an enhanced recruitment of MRP1 into lipid raft fractions, thereby promoting MRP1 activity. (C) 2012 Elsevier Ltd. All rights reserved.

RATIONALE Cleavage of peptide bonds C-terminal to tyrosine and tryptophan after electrochemical oxidation may become a complementary approach to chemical and enzymatic cleavage. A chemical labeling approach specifically targeting reactive cleavage products is presented here and constitutes a promising first step towards the development of a new proteomics workflow. METHODS Hexylamine was used to react with the spirolactone moieties generated after electrochemical oxidation and cleavage of tripeptides. The influence of pH and reaction time on the yield was determined and the excess of tagging reagent was optimized. Selective detection of the tagged cleavage products was achieved by precursor ion scanning in a triple quadrupole mass spectrometer. RESULTS Optimal labeling was reached under aqueous conditions when working at pH 10 with a reaction time of 0.5?min. The excess of hexylamine over spirolactone groups can be significantly decreased by working under non-aqueous conditions in pure acetonitrile to prevent spirolactone hydrolysis. The specific formation of hexylamine-containing y1 reporter ions generated by collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) allows for selective detection by precursor ion scanning of the cleaved and labeled peptides. CONCLUSIONS This work presents a method for selective labeling and detection of electrochemically cleaved Tyr- and Trp-containing peptides for which reaction conditions have been optimized with hexylamine as labeling agent. This workflow offers new possibilities for electrochemical oxidation, cleavage and labeling of peptides and proteins. Copyright (c) 2013 John Wiley & Sons, Ltd.

Important biological consequences are related to the reaction of HO center dot radicals with methionine ( Met). Several fundamental aspects remain to be defined when Met is an amino acid residue incorporated in the interior of peptides and proteins. The present study focuses on Gly-Met-Gly, the simplest peptide where Met is not a terminal residue. The reactions of HO center dot with Gly-Met-Gly and its N-acetyl derivative were studied by pulse radiolysis technique. The transient absorption spectra were resolved into contributions from specific components of radical intermediates. Moreover, a detailed product analysis is provided for the first time for Met-containing peptides in radiolytic studies to support the mechanistic proposal. By parallel radiolytical and electrochemical reactions and consequent product identification, the formation of sulfoxide attributed to the direct HO center dot radical attack on the sulfide functionality of the Met residue could be excluded, with the in situ generated hydrogen peroxide responsible for this oxidation. LC-MS and high resolution MS/MS were powerful analytical tools to envisage the structures of five products, thus allowing to complete the mechanistic picture of the overall Met-containing peptide reactivity.

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.

We report on the suppression of localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering (SERS) of 60 nm thick gold nanostructures by titanium adhesion layers. Significant plasmon damping is observed for titanium layer thicknesses up to t(Ti) = 5 nm, which cannot be fully explained by simulation models that account for the overlap of the electric field in the bimetal layer with the titanium layer. Furthermore, the relationship between the SERS enhancement factor and the LSPR quality-factor G proportional to Q(4) breaks down as t(T1), is increased beyond 1 nm. We attribute the LSPR and SERS suppression to plasmon damping and spectral broadening of interband absorption due to the field overlap with the titanium layer and titanium impurities that interdiffuse through the grain boundaries of the polycrystalline gold layer during the deposition of the Au-Ti bimetal. These observations and analyses serve as a guide for improving the reliability of gold nanoplasmonic sensors.

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 beta-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.

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.

Quaternary ammonium salts are among the most widely used electrolytes in organic electrochemistry, but there is little known about their unwanted side oxidation reactions. We have, therefore, studied the constant potential oxidation products of quaternary ammonium electrolytes using mass spectrometry, and the proposed oxidation reaction mechanisms were verified by stable isotope labeling studies. Oxidation of tetrabutylammonium at +2.5 V vs Ag showed oxidation products at m/z 256, 258, and 299 which were shown corresponding to alcohol, ketone, and acetonitrile-derived amide products, respectively. We propose a mechanism based on the anodic oxidation of the side chain, to generate a carbocation, followed by nucleophilic attack by water or acetonitrile. Incorporation of a water molecule and/or an acetonitrile molecule in the structure was verified by mass analysis of the isotopically labeled oxidation products. Oxidation can be avoided by use of quaternary ammonium ions with shorter alkyl chains, where electrostatic repulsion presumably prohibits carbocation generation. Keywords: Electrolyte, Mass spectrometry, Oxidation, Quaternary ammonium

Electrochemistry combined with mass spectrometry (EC-MS) is an emerging analytical technique in the imitation of oxidative drug metabolism at the early stages of new drug development. Here, we present the benefits of electrochemical oxidation by square-wave potential pulses for the oxidation of lidocaine, a test drug compound, on a platinum electrode. Lidocaine was oxidized at constant potential and by square-wave potential pulses with different cycle times, and the reaction products were analyzed by liquid chromatography-mass spectrometry [LC-MS(/MS)]. Application of constant potentials of up to +5.0 V resulted in relatively low yields of N-dealkylation and 4-hydroxylation products, while oxidation by square-wave potential pulses generated up to 50 times more of the 4-hydroxylation product at cycle times between 0.2 and 12s (estimated yield of 10%). The highest yield of the N-dealkylation product was obtained at cycle times shorter than 0.2 s. Tuning of the cycle time is thus an important parameter to modulate the selectivity of electrochemical oxidation reactions. The N-oxidation product was only obtained by electrochemical oxidation under air atmosphere due to reaction with electrogenerated hydrogen peroxide. Square-wave potential pulses may also be applicable to modulate the selectivity of electrochemical reactions with other drug compounds in order to generate oxidation products with greater selectivity and higher yield based on the optimization of cycle times and potentials. This considerably widens the scope of direct electrochemistry-based oxidation reactions for the imitation of in vivo oxidative drug metabolism.

Prediction of oxidative drug metabolism at the early stages of drug discovery and development requires fast and accurate analytical techniques to mimic the in vivo oxidation reactions by cytochrome P450s (CYP). Direct electrochemical oxidation combined with mass spectrometry, although limited to the oxidation reactions initiated by charge transfer, has shown promise in the mimicry of certain CYP-mediated metabolic reactions. The electrochemical approach may further be utilized in an automated manner in microfluidics devices facilitating fast screening of oxidative drug metabolism. A wide range of in vivo oxidation reactions, particularly those initiated by hydrogen atom transfer, can be imitated through the electrochemically-assisted Fenton reaction. This reaction is based on O-O bond activation in hydrogen peroxide and oxidation by hydroxyl radicals, wherein electrochemistry is used for the reduction of molecular oxygen to hydrogen peroxide, as well as the reduction of Fe(3+) to Fe(2+). Metalloporphyrins, as surrogates for the prosthetic group in CYP, utilizing metallo-oxo reactive species, can also be used in combination with electrochemistry. Electrochemical reduction of metalloporphyrins in solution or immobilized on the electrode surface activates molecular oxygen in a manner analogous to the catalytical cycle of CYP and different metalloporphyrins can mimic selective oxidation reactions. Chemoselective, stereoselective, and regioselective oxidation reactions may be mimicked using electrodes that have been modified with immobilized enzymes, especially CYP itself. This review summarizes the recent attempts in utilizing electrochemistry as a versatile analytical and preparative technique in the mimicry of oxidative drug metabolism by CYP.