Three-Minute Enantioselective Amino Acid Analysis by Ultra-High-Performance Liquid Chromatography Drift Tube Ion Mobility-Mass Spectrometry Using a Chiral Core-Shell Tandem Column Approach.

Fast liquid chromatography (LC) amino acid enantiomer separation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives using a chiral core-shell particle tandem column with weak anion exchange and zwitterionic-type quinine carbamate selectors in less than 3 min was achieved. Enantiomers of all AQC-derivatized proteinogenic amino acids and some isomeric ones (24 in total plus achiral glycine) were baseline separated (Rs > 1.5 except for glutamic acid with Rs = 1.3), while peaks of distinct amino acids and structural isomers (constitutional isomers and diastereomers of leucine and threonine) of the same configuration overlapped to various degrees. For this reason, drift tube ion mobility-mass spectrometry was added (i.e., LC-IM-MS) as an additional selectivity filter without extending run time. The IM separation dimension in combination with high-resolution demultiplexing enabled confirmation of threonine isomers (threonine, allo-threonine, homoserine), while leucine, isoleucine, and allo-isoleucine have almost identical collisional cross-section (DTCCSN2) values and added no selectivity to the partial LC separation. Density functional theory (DFT) calculations show that IM separation of threonine isomers was possible due to conformational stabilization by hydrogen bond formation between the hydroxyl side chain and the urea group. Generally, the CCSN2 of protonated ions increased uniformly with addition of the AQC label, while outliers could be explained by consideration of intramolecular interactions and additional structural analysis. Preliminary validation of the enantioselective LC-IM-MS method for quantitative analysis showed compliance of accuracy and precision with common limits in bioanalytical methods, and applicability to a natural lipopeptide and a therapeutic synthetic peptide could be demonstrated.

Non-enantioselective, enantioselective, and two-dimensional liquid chromatography coupled with tandem mass spectrometry for the study of stereochemical disposition of oxylipins in cGMP-regulated hemin-treated platelets.

Oxylipins are important low abundant signaling molecules in living organisms. In platelets they play a primary role in platelet activation and aggregation in the course of thrombotic events. In vivo, they are enzymatically synthesized by cyclooxygenases, lipoxygenases, or cytochrome P450 isoenzmes, resulting in diverse polyunsaturated fatty acid (FA) metabolites including hydroxy-, epoxy-, oxo-FAs, and endoperoxides with pro-thrombotic or anti-thrombotic effects. In a recent study, it was reported that hemin induces platelet death which was accompanied by enhanced reactive oxygen species (ROS) production (measured by flow cytometry) and lipid peroxidation (as determined by proxy using flow cytometry with BODIPY-C11 as sensor). Lipidomic studies further indicated significant changes of the platelet lipidome upon ex vivo hemin treatment, amongst others oxylipins were increased. The effect could be (at least partly) reversed by riociguat/diethylamine NONOate diethylammonium salt (DEA/NO) which modulates the soluble guanylate cyclase(sGC)-cGMP-cGMP-dependent protein kinase I(cGKI) signaling axis. In the original work, oxylipins were measured by a non-enantioselective UHPLC-tandem-MS assay which may not give the full picture whether oxylipin elevation is due to ROS or by enzymatic processes. We present here the study of the stereochemical disposition of hemin-induced platelet lipidome alterations using Chiralpak IA-U column with amylose tris(3,5-dimethylphenylcarbamate) chiral selector immobilized on 1.6 µm silica particles. It was found that the major platelet oxylipins 12-HETE, 12-HEPE and 14-HDoHE (from 12-LOX) and 12-HHT (from COX-1) were present in S-configuration indicating their enzymatic formation. On the other hand, both R and S enantiomers of 9- and 13-HODE, 11- and 15-HETE were detected, possibly due to enzyme promiscuity rather than non-specific oxidation (by ROS or autoxidation), as confirmed by multi-loop based two-dimensional LC-MS using selective comprehensive mode with achiral RPLC in the 1st dimension and chiral LC in the 2nd using a multiple heart-cutting interface. For 12-HETrE, a peak at the retention time of the R-enantiomer was ruled out as isobaric interference by 2D-LC-MS. In particular, arachidonic acid derivates 12(S)-HHT, 11(R)-HETE and 15(S)-HETE were found to be sensitive to hemin and cGMP modulation.

Solving the retention time repeatability problem of hydrophilic interaction liquid chromatography.

Hydrophilic interaction (liquid) chromatography (HILIC) has become the first choice LC mode for the separation of hydrophilic analytes. Numerous studies reported the poor retention time repeatability of HILIC. The problem was often ascribed to slow equilibration and insufficient re-equilibration time to establish the sensitive semi-immobilized water layer at the interface of the polar stationary phase and the bulk mobile phase. In this study, we compare retention time repeatability in HILIC for borosilicate glass and PFA (co-polymer of tetrafluoroethylene and perfluoroalkoxyethylene) solvent bottles. During this study, we observed peak patterns shifting towards higher retention times (for metabolites and peptides) and lower retention times (oligonucleotide sample) with ongoing analysis time when standard borosilicate glass bottles were used as solvent reservoirs. It was hypothesized that release of ions (sodium, potassium, borate, etc.) from the borosilicate glass bottles leads to alterations (thickness and electrostatic screening effects) in the semi-immobilized water layer which is adsorbed to the polar stationary phase surface under acetonitrile-rich eluents in HILIC with concomitant shifts in retention. When PFA solvent bottles were employed instead of borosilicate glass, retention time repeatability was greatly improved and changed from average 8.4 % RSD for the tested metabolites with borosilicate glass bottles to 0.14 % RSD for the PFA solvent bottles (30 injections over 12 h). Similar improvements were observed for peptides and oligonucleotides. This simple solution to the retention time repeatability problem in HILIC might contribute to a better acceptance of HILIC, especially in fields like targeted and untargeted metabolomics, peptide and oligonucleotide analysis.

Two-dimensional sequential selective comprehensive chiral×reversed-phase liquid chromatography of synthetic phosphorothioate oligonucleotide diastereomers.

In recent years, many nucleic acid-based pharmaceuticals have been approved and entered the market, and even a larger number are in late stage clinical trials. Conventional oligonucleotides are facing issues in vivo like fast renal clearance and nuclease degradation. Therefore, to increase their stability, phosphorothioation is a frequent modification of therapeutic oligonucleotides (ONs) which also leads to improved binding affinity facilitating cell internalization and intracellular distribution. At the same time, by replacing a phosphodiester linkage with a phosphorothioate group, a phosphorous stereogenic center is generated which causes the formation of Rp- and Sp-diastereomers. It increases the structural diversity. For example, with 15 of those phosphorothioate (PS) linkages, 32,768 different diastereomers are expected. Since the phosphorothioate is introduced non-stereoselectively, the molecular complexity of the resultant phosphorothioate ON products is tremendously increased impeding the chromatographic separation in the course of quality control. Since distinct phosphorothioate diastereomers have different bioactivities and pharmacological properties, there is increasing interest in implications of stereoisomerism of phosphorothiate oligonucleotides. From a quality and regulatory viewpoint, batch-to-batch reproducibility of the diastereomer profile may be of significant concern. In order to address this issue, this study investigates the stereoselectivity of LC methods for two phosphorothioate oligonucleotide (PSO) compounds differing in their molecular size and numbers of PS linkages. Diastereoselectivity of ion-pairing reversed-phase liquid chromatography (IP-RPLC), RPLC without ion-pairing agents and LC with chiral polysaccharide-based column were evaluated for model PSOs and an active pharmaceutical ingredient (API) of PSO with trivalent N-acetylgalactosamine (GalNAc) conjugate. Due to the structural complexity of PSOs, the separation power for the diastereomer mixture was increased by using sequential selective comprehensive two-dimensional chromatography with an amylose tris(α-methylbenzylcarbamate)-immobilized chiral stationary phase (CSP) in the first dimension and ion-pair RPLC with ethylammonium acetate in the second dimension. Improved diastereomer selectivity was obtained and a larger number of peaks could be separated.

Evaluation of a Covalent Library of Diverse Warheads (CovLib) Binding to JNK3, USP7, or p53.

Purpose: Over the last few years, covalent fragment-based drug discovery has gained significant importance. Thus, striving for more warhead diversity, we conceived a library consisting of 20 covalently reacting compounds. Our covalent fragment library (CovLib) contains four different warhead classes, including five α-cyanoacacrylamides/acrylates (CA), three epoxides (EO), four vinyl sulfones (VS), and eight electron-deficient heteroarenes with a leaving group (SNAr/SN). Methods: After predicting the theoretical solubility of the fragments by LogP and LogS during the selection process, we determined their experimental solubility using a turbidimetric solubility assay. The reactivities of the different compounds were measured in a high-throughput 5,5'-dithiobis-(2-nitrobenzoic acid) DTNB assay, followed by a (glutathione) GSH stability assay. We employed the CovLib in a (differential scanning fluorimetry) DSF-based screening against different targets: c-Jun N-terminal kinase 3 (JNK3), ubiquitin-specific protease 7 (USP7), and the tumor suppressor p53. Finally, the covalent binding was confirmed by intact protein mass spectrometry (MS). Results: In general, the purchased fragments turned out to be sufficiently soluble. Additionally, they covered a broad spectrum of reactivity. All investigated α-cyanoacrylamides/acrylates and all structurally confirmed epoxides turned out to be less reactive compounds, possibly due to steric hindrance and reversibility (for α-cyanoacrylamides/acrylates). The SNAr and vinyl sulfone fragments are either highly reactive or stable. DSF measurements with the different targets JNK3, USP7, and p53 identified reactive fragment hits causing a shift in the melting temperatures of the proteins. MS confirmed the covalent binding mode of all these fragments to USP7 and p53, while additionally identifying the SNAr-type electrophile SN002 as a mildly reactive covalent hit for p53. Conclusion: The screening and target evaluation of the CovLib revealed first interesting hits. The highly cysteine-reactive fragments VS004, SN001, SN006, and SN007 covalently modify several target proteins and showed distinct shifts in the melting temperatures up to +5.1 °C and -9.1 °C.

Machine learning insights into thrombo-ischemic risks and bleeding events through platelet lysophospholipids and acylcarnitine species.

Coronary artery disease (CAD) often leads to adverse events resulting in significant disease burdens. Underlying risk factors often remain inapparent prior to disease incidence and the cardiovascular (CV) risk is not exclusively explained by traditional risk factors. Platelets inherently promote atheroprogression and enhanced platelet functions and distinct platelet lipid species are associated with disease severity in patients with CAD. Lipidomics data were acquired using mass spectrometry and processed alongside clinical data applying machine learning to model estimates of an increased CV risk in a consecutive CAD cohort (n = 595). By training machine learning models on CV risk measurements, stratification of CAD patients resulted in a phenotyping of risk groups. We found that distinct platelet lipids are associated with an increased CV or bleeding risk and independently predict adverse events. Notably, the addition of platelet lipids to conventional risk factors resulted in an increased diagnostic accuracy of patients with adverse CV events. Thus, patients with aberrant platelet lipid signatures and platelet functions are at elevated risk to develop adverse CV events. Machine learning combining platelet lipidome data and common clinical parameters demonstrated an increased diagnostic value in patients with CAD and might improve early risk discrimination and classification for CV events.

Lipase-mediated detoxification of host-derived antimicrobial fatty acids by Staphylococcus aureus.

Long-chain fatty acids with antimicrobial properties are abundant on the skin and mucosal surfaces, where they are essential to restrict the proliferation of opportunistic pathogens such as Staphylococcus aureus. These antimicrobial fatty acids (AFAs) elicit bacterial adaptation strategies, which have yet to be fully elucidated. Characterizing the pervasive mechanisms used by S. aureus to resist AFAs could open new avenues to prevent pathogen colonization. Here, we identify the S. aureus lipase Lip2 as a novel resistance factor against AFAs. Lip2 detoxifies AFAs via esterification with cholesterol. This is reminiscent of the activity of the fatty acid-modifying enzyme (FAME), whose identity has remained elusive for over three decades. In vitro, Lip2-dependent AFA-detoxification was apparent during planktonic growth and biofilm formation. Our genomic analysis revealed that prophage-mediated inactivation of Lip2 was rare in blood, nose, and skin strains, suggesting a particularly important role of Lip2 for host - microbe interactions. In a mouse model of S. aureus skin colonization, bacteria were protected from sapienic acid (a human-specific AFA) in a cholesterol- and lipase-dependent manner. These results suggest Lip2 is the long-sought FAME that exquisitely manipulates environmental lipids to promote bacterial growth in otherwise inhospitable niches.

cGMP modulates hemin-mediated platelet death.

BACKGROUND AND AIMS: Hemolysis is a known risk factor for thrombosis resulting in critical limb ischemia and microcirculatory disturbance and organ failure. Intravasal hemolysis may lead to life-threatening complications due to uncontrolled thrombo-inflammation. Until now, conventional antithrombotic therapies failed to control development and progression of these thrombotic events. Thus, the pathophysiology of these thrombotic events needs to be investigated to unravel underlying pathways and thereby identify targets for novel treatment strategies. METHODS: Here we used classical experimental set-ups as well as high-end flow cytometry, metabolomics and lipidomic analysis to in-depth analyze the effects of hemin on platelet physiology and morphology. RESULTS: Hemin does strongly and swiftly induce platelet activation and this process is modulated by the sGC-cGMP-cGKI signaling axis. cGMP modulation also reduced the pro-aggregatory potential of plasma derived from patients with hemolysis. Furthermore, hemin-induced platelet death evokes distinct platelet subpopulations. Typical cell death markers, such as ROS, were induced by hemin-stimulation and the platelet lipidome was specifically altered by high hemin concentration. Specifically, arachidonic acid derivates, such as PGE2, TXB2 or 12-HHT, were significantly increased. Balancing the cGMP levels by modulation of the sGC-cGMP-cGKI axis diminished the ferroptotic effect of hemin. CONCLUSION: We found that cGMP modulates hemin-induced platelet activation and thrombus formation in vitro and cGMP effects hemin-mediated platelet death and changes in the platelet lipidome. Thus, it is tempting to speculate that modulating platelet cGMP levels may be a novel strategy to control thrombosis and critical limb ischemia in patients with hemolytic crisis.

mitoBKCa is functionally expressed in murine and human breast cancer cells and potentially contributes to metabolic reprogramming.

Alterations in the function of K+ channels such as the voltage- and Ca2+-activated K+ channel of large conductance (BKCa) reportedly promote breast cancer (BC) development and progression. Underlying molecular mechanisms remain, however, elusive. Here, we provide electrophysiological evidence for a BKCa splice variant localized to the inner mitochondrial membrane of murine and human BC cells (mitoBKCa). Through a combination of genetic knockdown and knockout along with a cell permeable BKCa channel blocker, we show that mitoBKCa modulates overall cellular and mitochondrial energy production, and mediates the metabolic rewiring referred to as the 'Warburg effect', thereby promoting BC cell proliferation in the presence and absence of oxygen. Additionally, we detect mitoBKCa and BKCa transcripts in low or high abundance, respectively, in clinical BC specimens. Together, our results emphasize, that targeting mitoBKCa could represent a treatment strategy for selected BC patients in future.

Commensal production of a broad-spectrum and short-lived antimicrobial peptide polyene eliminates nasal Staphylococcus aureus.

Antagonistic bacterial interactions often rely on antimicrobial bacteriocins, which attack only a narrow range of target bacteria. However, antimicrobials with broader activity may be advantageous. Here we identify an antimicrobial called epifadin, which is produced by nasal Staphylococcus epidermidis IVK83. It has an unprecedented architecture consisting of a non-ribosomally synthesized peptide, a polyketide component and a terminal modified amino acid moiety. Epifadin combines a wide antimicrobial target spectrum with a short life span of only a few hours. It is highly unstable under in vivo-like conditions, potentially as a means to limit collateral damage of bacterial mutualists. However, Staphylococcus aureus is eliminated by epifadin-producing S. epidermidis during co-cultivation in vitro and in vivo, indicating that epifadin-producing commensals could help prevent nasal S. aureus carriage. These insights into a microbiome-derived, previously unknown antimicrobial compound class suggest that limiting the half-life of an antimicrobial may help to balance its beneficial and detrimental activities.