Effects of lysine deacetylase inhibitor treatment on LPS responses of alveolar-like macrophages.

Macrophages are key immune cells that can adapt their metabolic phenotype in response to different stimuli. Lysine deacetylases are important enzymes regulating inflammatory gene expression and lysine deacetylase inhibitors have been shown to exert anti-inflammatory effects in models of chronic obstructive pulmonary disease. We hypothesized that these anti-inflammatory effects may be associated with metabolic changes in macrophages. To validate this hypothesis, we used an unbiased and a targeted proteomic approach to investigate metabolic enzymes, as well as liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry, to quantify metabolites in combination with the measurement of functional parameters in primary murine alveolar-like macrophages after lipopolysaccharide-induced activation in the presence or absence of lysine deacetylase inhibition. We found that lysine deacetylase inhibition resulted in reduced production of inflammatory mediators such as tumor necrosis factor α and interleukin 1ß. However, only minor changes in macrophage metabolism were observed, as only one of the lysine deacetylase inhibitors slightly increased mitochondrial respiration while no changes in metabolite levels were seen. However, lysine deacetylase inhibition specifically enhanced expression of proteins involved in ubiquitination, which may be a driver of the anti-inflammatory effects of lysine deacetylase inhibitors. Our data illustrate that a multiomics approach provides novel insights into how macrophages interact with cues from their environment. More detailed studies investigating ubiquitination as a potential driver of lysine deacetylase inhibition will help developing novel anti-inflammatory drugs for difficult-to-treat diseases such as chronic obstructive pulmonary disease.

Nanoporous Gold Catalyst for the Oxidative N-Dealkylation of Drug Molecules: A Method for Synthesis of N-Dealkylated Metabolites.

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.

Change of charge variant composition of trastuzumab upon stressing at physiological conditions.

Cation-exchange chromatography is a widely used approach to study charge heterogeneity of monoclonal antibodies. Heterogeneity may arise both in vitro and in vivo because of the susceptibility of monoclonal antibodies to undergo chemical modifications. Modifications may adversely affect the potency of the drug, induce immunogenicity or affect pharmacokinetics. In this study, we evaluated the application of optimized pH gradient systems for the separation of charge variants of trastuzumab after forced degradation study. pH gradient-based elution resulted in high-resolution separation of some 20 charge variants after 3 weeks at 37°C under physiological conditions. The charge variants were further characterized by LC-MS-based peptide mapping. There was no significant difference in the binding properties to HER2 or a range of Fcγ receptors between non-stressed and stressed trastuzumab.

Automated Electrical Quantification of Vitamin†B1 in a Bodily Fluid using an Engineered Nanopore Sensor.

The ability to measure the concentration of metabolites in biological samples is important, both in the clinic and for home diagnostics. Here we present a nanopore-based biosensor and automated data analysis for quantification of thiamine in urine in less than a minute, without the need for recalibration. For this we use the Cytolysin A nanopore and equip it with an engineered periplasmic thiamine binding protein (TbpA). To allow fast measurements we tuned the affinity of TbpA for thiamine by redesigning the π-π stacking interactions between the thiazole group of thiamine and TbpA. This substitution resulted furthermore in a marked difference between unbound and bound state, allowing the reliable discrimination of thiamine from its two phosphorylated forms by residual current only. Using an array of nanopores, this will allow the quantification within seconds, paving the way for next-generation single-molecule metabolite detection systems.

Development of Lactococcus lactis Biosensors for Detection of Sulfur-Containing Amino Acids.

The sulfur-containing amino acids methionine and cysteine play an important role in food industry. These amino acids are used to confer a sulfur smell or meat-related aroma to food products. Besides their use as food additives, methionine and cysteine participate in flavor formation in dairy fermentations. For instance, the characteristic aroma of Cheddar cheeses is derived from methionine. Therefore, bacterial strains with the ability to overproduce and secrete these amino acids are relevant for the food industry. In addition, the quantification of these compounds in food matrices is a laborious task that involves sample preparation and specific analytical methods such as high-performance liquid chromatography. The ability of bacteria to naturally sense metabolites has successfully been exploited to develop biosensors. The presence of a specific metabolite is sensed by the biosensors, and it is subsequently translated into the expression of one or more reporter genes. In this study we aim to develop biosensors to detect methionine and cysteine, which are produced and secreted by wild-type Lactococcus lactis strains. We employed two strategies to create L. lactis biosensors, the first one is based on the methionine auxotrophy of this bacterium and the second strategy is based on a cysteine-responsive promoter. The characterization of the biosensors showed their specific response to the presence of these amino acids. Subsequently, we applied the methionine biosensor to quantify the presence of methionine in bacterial supernatants of wild-type L. lactis that naturally secretes methionine to benchmark the performance of our biosensors. The methionine biosensor responded linearly to the amounts of methionine present in the bacterial supernatants, i.e., the increases in the biosensor cell densities were proportional to the amounts of methionine present in the supernatants. The biosensors developed in this study tackle the limitations of amino acid quantification and the selection of strains with secretion of amino acids. These biosensors may eventually be used for screening of engineered strains to increase methionine and cysteine production, and may facilitate the detection of these amino acids in complex food matrices.

Enhancement of amino acid production and secretion by Lactococcus lactis using a droplet-based biosensing and selection system.

Amino acids are attractive metabolites for the pharmaceutical and food industry field. On one hand, the construction of microbial cell factories for large-scale production aims to satisfy the demand for amino acids as bulk biochemical. On the other hand, amino acids enhance flavor formation in fermented foods. Concerning the latter, flavor formation in dairy products, such as cheese is associated with the presence of lactic acid bacteria (LAB). In particular, Lactococcus lactis, one of the most important LAB, is used as a starter culture in fermented foods. The proteolytic activity of some L. lactis strains results in peptides and amino acids, which are flavor compounds or flavor precursors. However, it is still a challenge to isolate bacterial cells with enhanced amino acid production and secretion activity. In this work, we developed a growth-based sensor strain to detect the essential amino acids isoleucine, leucine, valine, histidine and methionine. Amino acids are metabolites that can be secreted by some bacteria. Therefore, our biosensor allowed us to identify wild-type L. lactis strains that naturally secrete amino acids, by using co-cultures of the biosensor strain with potential amino acid producing strains. Subsequently, we used this biosensor in combination with a droplet-based screening approach, and isolated three mutated L. lactis IPLA838 strains with 5-10 fold increased amino acid-secretion compared to the wild type. Genome re-sequencing revealed mutations in genes encoding proteins that participate in peptide uptake and peptide degradation. We argue that an unbalance in the regulation of amino acid levels as a result of these gene mutations may drive the accumulation and secretion of these amino acids. This biosensing system tackles the problem of selection for overproduction of secreted molecules, which requires the coupling of the product to the producing cell in the droplets.

Direct electrical quantification of glucose and asparagine from bodily fluids using nanopores.

Crucial steps in the miniaturisation of biosensors are the conversion of a biological signal into an electrical current as well as the direct sampling of bodily fluids. Here we show that protein sensors in combination with a nanopore, acting as an electrical transducer, can accurately quantify metabolites in real time directly from nanoliter amounts of blood and other bodily fluids. Incorporation of the nanopore into portable electronic devices will allow developing sensitive, continuous, and non-invasive sensors for metabolites for point-of-care and home diagnostics.

A fully validated liquid chromatography-mass spectrometry method for the quantification of the soluble receptor of advanced glycation end-products (sRAGE) in serum using immunopurification in a 96-well plate format.

The study of proteins is central to unraveling (patho)physiological processes and has contributed greatly to our understanding of biological systems. Corresponding studies often employ procedures to enrich proteins from their biological matrix using antibodies or other affinity binders coupled to beads with a large surface area and a correspondingly high binding capacity. Striving for maximal binding capacity may, however, not always be required or desirable, for example for proteins of low abundance. Here we describe a simplified immunoprecipitation in 96-well ELISA format (IPE) approach for fast and easy enrichment of proteins. The applicability of this approach for enriching low-abundant proteins was demonstrated by an IPE-based quantitative workflow using liquid chromatography-mass spectrometry (LC-MS) for the soluble Receptor of Advanced Glycation End-products (sRAGE), a promising biomarker in chronic obstructive pulmonary disease (COPD). The method was validated according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and enabled accurate quantitation of sRAGE between 0.1 and 10 ng/mL in 50 µL serum. The assay showed substantial correlation with the two most commonly-used sRAGE immunoassays (ELISAs) (R2-values between 0.7 and 0.8). However, the LC-MS method reported 2-4 times higher sRAGE levels compared to the ELISAs, which is largely due to a suboptimal amount of capturing antibody and/or calibration strategy used by the immunoassays. In conclusion, our simplified IPE approach proved to be an efficient strategy for enriching the low-abundant protein sRAGE from serum and may provide an easy to use platform for enriching other (low-abundant) proteins from complex, biological matrices.

Physicochemical Parameters Affecting the Electrospray Ionization Efficiency of Amino Acids after Acylation.

Electrospray ionization (ESI) is widely used in liquid chromatography coupled to mass spectrometry (LC-MS) for the analysis of biomolecules. However, the ESI process is still not completely understood, and it is often a matter of trial and error to enhance ESI efficiency and, hence, the response of a given set of compounds. In this work we performed a systematic study of the ESI response of 14 amino acids that were acylated with organic acid anhydrides of increasing chain length and with poly(ethylene glycol) (PEG) changing certain physicochemical properties in a predictable manner. By comparing the ESI response of 70 derivatives, we found that there was a strong correlation between the calculated molecular volume and the ESI response, while correlation with hydrophobicity (log P values), pKa, and the inverse calculated surface tension was significantly lower although still present, especially for individual derivatized amino acids with increasing acyl chain lengths. Acylation with PEG containing five ethylene glycol units led to the largest gain in ESI response. This response was maximal independent of the calculated physicochemical properties or the type of amino acid. Since no actual physicochemical data is available for most derivatized compounds, the responses were also used as input for a quantitative structure-property relationship (QSPR) model to find the best physicochemical descriptors relating to the ESI response from molecular structures using the amino acids and their derivatives as a reference set. A topological descriptor related to molecular size (SPAN) was isolated next to a descriptor related to the atomic composition and structural groups (BIC0). The validity of the model was checked with a test set of 43 additional compounds that were unrelated to amino acids. While prediction was generally good (R2 > 0.9), compounds containing halogen atoms or nitro groups gave a lower predicted ESI response.

The relevance of Ki calculation for bi-substrate enzymes illustrated by kinetic evaluation of a novel lysine (K) acetyltransferase 8 inhibitor.

Histone acetyltransferases (HATs) are important mediators of epigenetic post-translational modifications of histones that play important roles in health and disease. A disturbance of these modifications can result in disease states, such as cancer or inflammatory diseases. Inhibitors of HATs (HATi) such as lysine (K) acetyltransferase 8 (KAT8), could be used to study the epigenetic processes in diseases related to these enzymes or to investigate HATs as therapeutic targets. However, the development of HATi is challenged by the difficulties in kinetic characterization of HAT enzymes and their inhibitors to enable calculation of a reproducible inhibitory potency. In this study, a fragment screening approach was used, enabling identification of 4-amino-1-naphthol, which potently inhibited KAT8. The inhibitor was investigated for enzyme inhibition using kinetic and calorimetric binding studies. This allowed for calculation of the Ki values for both the free enzyme as well as the acetylated intermediate. Importantly, it revealed a striking difference in binding affinity between the acetylated enzyme and the free enzyme, which could not be revealed by the IC50 value. This shows that kinetic characterization of inhibitors and calculation of Ki values is crucial for determining the binding constants of HAT inhibitors. We anticipate that more comprehensive characterization of enzyme inhibition, as described here, is needed to advance the field of HAT inhibitors.

A 6-alkylsalicylate histone acetyltransferase inhibitor inhibits histone acetylation and pro-inflammatory gene expression in murine precision-cut lung slices.

Lysine acetylations are post-translational modifications of cellular proteins, that are crucial in the regulation of many cellular processes. Lysine acetylations on histone proteins are part of the epigenetic code regulating gene expression and are installed by histone acetyltransferases. Observations that inflammatory lung diseases, such as asthma and chronic obstructive pulmonary disease, are characterized by increased histone acetyltransferase activity indicate that development of small molecule inhibitors for these enzymes might be a valuable approach towards new therapies for these diseases. The 6-alkylsalicylate MG149 is a candidate to explore this hypothesis because it has been demonstrated to inhibit the MYST type histone acetyltransferases. In this study, we determined the Ki value for inhibition of the MYST type histone acetyltransferase KAT8 by MG149 to be 39 ± 7.7 µM. Upon investigating whether the inhibition of histone acetyltransferases by MG149 correlates with inhibition of histone acetylation in murine precision-cut lung slices, inhibition of acetylation was observed using an LC-MS/MS based assay on histone H4 res 4-17, which contains the target lysine of KAT8. Following up on this, upon treatment with MG149, reduced pro-inflammatory gene expression was observed in lipopolysaccharide and interferon gamma stimulated murine precision-cut lung slices. Based on this, we propose that 6-alkylsalicylates such as MG149 have potential for development towards applications in the treatment of inflammatory lung diseases.

Quantitative antibody-free LC-MS/MS analysis of sTRAIL in sputum and saliva at the sub-ng/mL level.

Soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) induces apoptosis via the extrinsic death receptor pathway and may be a biomarker in the pathogenesis of a broad range of diseases. To investigate the role of sTRAIL in asthma, we developed a quantitative LC-MS/MS method with a lower limit of quantitation (LLOQ) of ≈3pM in induced sputum (174pg/mL) and saliva (198pg/mL) without the use of antibodies. sTRAIL was enriched by immobilized metal affinity chromatography (IMAC) solid-phase extraction (SPE) followed by tryptic digestion and subsequent enrichment of a signature peptide by strong cation exchange (SCX) SPE. The method was validated with respect to stability, accuracy and precision using the standard addition approach and fully metabolically (15)N-labelled hrTRAIL as internal standard. Our results indicate that it is possible to quantify cytokines like sTRAIL at the pM level by LC-MS/MS without the use of antibodies, which has, to our knowledge, never been shown before.

Highly sensitive antibody-free µLC-MS/MS quantification of rhTRAIL in serum.

BACKGROUND: We describe an antibody-free approach to quantify rhTRAIL(WT) (wild-type) and its closely related death receptor 4 selective variant rhTRAIL(4C7) in human and murine serum by multiplex LC-MS/MS on a microfluidics interface. METHODOLOGY: Enrichment of rhTRAIL was performed by strong cation-exchange (SCX) followed by immobilized metal affinity (IMAC) solid-phase extraction. This was followed by trypsin digestion and using methionine-containing signature peptides after fully oxidizing the methionine residue with 0.25% (w/w) hydrogen peroxide. CONCLUSION: Absolute quantification was reaching down to 0.5 ng/ml for rhTRAIL(WT) (8.5 pM) and 2 ng/ml for rhTRAIL(4C7) (34 pM) in 100 µl human serum. To support preclinical studies in mice, the analysis was optimized further, for a sample volume of 20 µl murine serum.

Site-specific quantification of lysine acetylation in the N-terminal tail of histone H4 using a double-labelling, targeted UHPLC MS/MS approach.

We developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 by combining chemical derivatization at the protein and peptide levels with digestion using chymotrypsin and trypsin. Unmodified ε-amino groups were first modified with propionic acid anhydride and the derivatized protein digested with trypsin and chymotrypsin. The newly formed peptide N-termini were subjected to a second derivatization step with d6- (heavy) or d0- (light) acetic acid anhydride. Samples were mixed at different ratios and peptides monitored by multiple reaction monitoring (MRM) LC-MS/MS. The method was validated in terms of linearity (R(2) ≥ 0.94), precision (RSD ≤ 10%), and accuracy (≤27%) and used to assess the effect of the histone deacetylase (HDAC) inhibitors SAHA and MS-275 in the murine macrophage-like cell line RAW 264.7. SAHA and MS-275 showed site-specific effects on the acetylation levels of K5 and K8 with the K5(Ac)-K8 and K5-K8(Ac) peptides increasing 2.5-fold and 5-fold upon treatment with SAHA and MS-275, respectively. Assessing lysine acetylation in a site-specific manner is important for gaining a better understanding of the effects of HDAC inhibitors and for clarifying disease mechanisms where lysine acetylation plays a role.

Free Urinary Desmosine and Isodesmosine as COPD Biomarkers: The Relevance of Confounding Factors.

Background: Desmosine (DES) and isodesmosine (IDES) have been widely discussed as potential biomarkers of COPD. However, their clinical utility and validity remains unproven. Aim: This study aims to progress DES/IDES evaluation as a chronic obstructive pulmonary disease (COPD) biomarker by investigating its urinary excretion in a large sample cohort with respect to a) which factors influence DES/IDES levels in a population of healthy control individuals and COPD individuals; b) whether DES/IDES levels enable the differentiation between COPD individuals and healthy control individuals; c) whether DES/IDES can be used to differentiate between fast and slow decliners in lung function. Methods: Urinary DES and IDES were quantified in 365 individuals (147 healthy control individuals and 218 COPD individuals) from the Evaluation of COPD Longitudinally to Indentify Predictive Surrogate Endpoints (ECLIPSE) study (NCT00292552) by employing a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Results: Age, gender, body mass index (BMI) and smoking have a significant (p<0.05) influence on DES/IDES urinary excretion and need to be corrected for when investigating DES/IDES as a disease biomarker. Urinary DES/IDES allowed a statistically relevant differentiation (p<0.05) between stable COPD individuals and healthy control individuals, however, assay sensitivity and specificity were low (62% and 73%, respectively). Furthermore, urinary DES/IDES does not allow the differentiation of fast and slow decliners in lung function. Conclusions: The present results suggest that while urinary DES/IDES excretion is related to COPD, it is not a sensitive or specific biomarker for COPD diagnosis or prognosis.

Quantification of free and total desmosine and isodesmosine in human urine by liquid chromatography tandem mass spectrometry: a comparison of the surrogate-analyte and the surrogate-matrix approach for quantitation.

In spite of the data suggesting the potential of urinary desmosine (DES) and isodesmosine (IDS) as biomarkers for elevated lung elastic fiber turnover, further validation in large-scale studies of COPD populations, as well as the analysis of longitudinal samples is required. Validated analytical methods that allow the accurate and precise quantification of DES and IDS in human urine are mandatory in order to properly evaluate the outcome of such clinical studies. In this work, we present the development and full validation of two methods that allow DES and IDS measurement in human urine, one for the free and one for the total (free+peptide-bound) forms. To this end we compared the two principle approaches that are used for the absolute quantification of endogenous compounds in biological samples, analysis against calibrators containing authentic analyte in surrogate matrix or containing surrogate analyte in authentic matrix. The validated methods were employed for the analysis of a small set of samples including healthy never-smokers, healthy current-smokers and COPD patients. This is the first time that the analysis of urinary free DES, free IDS, total DES, and total IDS has been fully validated and that the surrogate analyte approach has been evaluated for their quantification in biological samples. Results indicate that the presented methods have the necessary quality and level of validation to assess the potential of urinary DES and IDS levels as biomarkers for the progression of COPD and the effect of therapeutic interventions.

Antibody-free LC-MS/MS quantification of rhTRAIL in human and mouse serum.

The major challenge in targeted protein quantification by LC-MS/MS in serum lies in the complexity of the biological matrix with regard to the wide diversity of proteins and their extremely large dynamic concentration range. In this study, an LC-MS/MS method was developed for the simultaneous quantification of the 60-kDa biopharmaceutical proteins recombinant human tumor necrosis factor-related apoptosis-inducing ligand wild type (rhTRAIL(WT)) and its death receptor 4 (DR4)-specific variant rhTRAIL(4C7) in human and mouse serum. Selective enrichment of TRAIL was accomplished by immobilized metal affinity chromatography (IMAC), which was followed by tryptic digestion of the enriched sample and quantification of a suitable signature peptide. For absolute quantification, (15)N-metabolically labeled internal standards of rhTRAIL(WT) and rhTRAIL(4C7) were used. Since the signature peptides that provided the highest sensitivity and allowed discrimination between rhTRAIL(WT) and rhTRAIL(4C7) contained methionine residues, we oxidized these quantitatively to their sulfoxides by the addition of 0.25% (w/w) hydrogen peroxide. The final method has a lower limit of quantification of 20 ng/mL (ca. 350 pM) and was fully validated according to current international guidelines for bioanalysis. To show the applicability of the LC-MS/MS method for pharmacokinetic studies, we quantified rhTRAIL(WT) and rhTRAIL(4C7) simultaneously in serum from mice injected intraperitoneally at a dose of 5 mg/kg for each protein. This is the first time that two variants of rhTRAIL differing by only a few amino acids have been analyzed simultaneously in serum, an approach that is not possible by conventional enzyme-linked immuno-sorbent assay (ELISA) analysis.

Electron transfer and collision induced dissociation of non-derivatized and derivatized desmosine and isodesmosine.

Electron transfer dissociation (ETD) has attracted increasing interest due to its complementarity to collision-induced dissociation (CID). ETD allows the direct localization of labile post-translational modifications, which is of main interest in proteomics where differences and similarities between ETD and CID have been widely studied. However, due to the fact that ETD requires precursor ions to carry at least two charges, little is known about differences in ETD and CID of small molecules such as metabolites. In this work, ETD and CID of desmosine (DES) and isodesmosine (IDS), two isomers that due to the presence of a pyridinium group can carry two charges after protonation, are studied and compared. In addition, the influence of DES/IDS derivatization with propionic anhydride and polyethyleneglycol (PEG) reagents on ETD and CID was studied, since this is a common strategy to increase sensitivity and to facilitate the analysis by reversed-phase chromatography. Clear differences between ETD and CID of non-derivatized and derivatized-DES/IDS were observed. While CID is mainly attributable to charge-directed fragmentation, ETD is initiated by the generation of a hydrogen atom at the initial protonation site and its subsequent transfer to the pyridinium ring of DES/IDS. These differences are reflected in the generation of complex CID spectra dominated by the loss of small, noninformative molecules (NH(3), CO, H(2)O), while ETD spectra are simpler and dominated by characteristic side-chain losses. This constitutes a potential advantage of ETD in comparison to CID when employed for the targeted analysis of DES/IDS in biological samples.

Quantification of matrix metalloprotease-9 in bronchoalveolar lavage fluid by selected reaction monitoring with microfluidics nano-liquid-chromatography-mass spectrometry.

Quantitative protein analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the selected reaction monitoring (SRM) mode was used to quantify matrix metalloprotease-9 (MMP-9; ∼90 kDa) in bronchoalveolar lavage fluid (BALF) from patients having undergone lung transplantation. We developed an SRM assay for microfluidics-based nanoLC-MS/MS on a triple quadrupole mass spectrometer based on two signature peptides. Samples were prepared by chloroform-methanol precipitation followed by trypsin digestion in the presence of stable-isotope-labeled internal peptide standards. The method allows accurate quantification of MMP-9 in BALF with an LLOQ of 2.9 ng/mL and an LLOD of 0.25 ng/mL without the use of extensive fractionation or antibodies.