Liquid Chromatography-mass Spectrometry Approach Research Articles

A Targeted, Bioinert LC-MS/MS Method for Sensitive, Comprehensive Analysis of Signaling Lipids.

Signaling lipids are key players in cellular processes. Despite their importance, no method currently allows their comprehensive monitoring in one analytical run. Challenges include a wide dynamic range, isomeric and isobaric species, and unwanted interaction along the separation path. Herein, we present a sensitive and robust targeted liquid chromatography-mass spectrometry (LC-MS/MS) approach to overcome these challenges, covering a broad panel of 17 different signaling lipid classes. It involves a simple one-phase sample extraction and lipid analysis using bioinert reversed-phase liquid chromatography coupled to targeted mass spectrometry. The workflow shows excellent sensitivity and repeatability in different biological matrices, enabling the sensitive and robust monitoring of 388 lipids in a single run of only 20 min. To benchmark our workflow, we characterized the human plasma signaling lipidome, quantifying 307 endogenous molecular lipid species. Furthermore, we investigated the signaling lipidome during platelet activation, identifying numerous regulations along important lipid signaling pathways. This highlights the potential of the presented method to investigate signaling lipids in complex biological systems, enabling unprecedentedly comprehensive analysis and direct insight into signaling pathways.

Phenolic Compounds of Six Unexplored Asteraceae Species from Asia: Comparison of Wild and Cultivated Plants

The Asteraceae family in Siberian Asia exhibits remarkable biodiversity and has long served as a valuable resource for domesticating various beneficial plants with medicinal, therapeutic, and industrial significance to humanity. In this work, we studied for the first time the chemical composition of six understudied or previously unexplored plant species, Artemisia jacutica (AJ), Carduus nutans subsp. leiophyllus (CL), Cirsium heterophyllum (CH), Echinops davuricus (ED), Ixeris chinensis subsp. versicolor (IV), and Lactuca sibirica (LS), which were successfully cultivated under open-field conditions as biennial or perennial crops. We profiled these species, employing a liquid chromatography–mass spectrometry approach, identifying over 100 phenolic compounds. Among these compounds were hydroxybenzoic acid glucosides, hydroxybenzoyl/p-coumaroyl/feruloyl quinic acids, hydroxycoumarin O-glucosides, caffeoyl/p-coumaroyl/feruloyl glucaric/tartaric acids, O- and C-glucosides of apigenin, acacetin, luteolin, chrysoeriol, 6-hydroxyluteolin, pectolinarigenin, kaempferol, quercetin, isorhamnetin, and tri-/tetra-O-p-coumaroyl spermines and spermidines. All examined species exhibited a significant accumulation of phenolic compounds throughout the experimental period, reaching levels comparable to or exceeding those found in wild samples (WSs), with the best total phenolic content for AJ at 26.68 mg/g (vs. 26.68 mg/g in WS; second year), CL at 50.23 mg/g (vs. 38.32 mg/g in WS; second year), CH at 51.14 mg/g (vs. 40.86 mg/g in WS; sixth year), ED at 86.12 mg/g (vs. 78.08 mg/g in WS; seventh year), IV at 102.49 mg/g (vs. 88.58 mg/g in WS; fourth year), and LS at 127.34 mg/g (vs. 110.64 mg/g in WS; fifth year). Notably, in the first year of cultivation, approximately 40–60% of the wild-level target compounds accumulated in the plants, with even higher levels detected in subsequent years, particularly in the second and third years. This study highlights the potential of cultivation to produce new Asteraceae plants rich in bioactive phenolics.

Transcriptomic and metabolomic dissection of skeletal muscle of crossbred Chongming white goats with different meat production performance

BackgroundThe transcriptome and metabolome dissection of the skeletal muscle of high- and low- growing individuals from a crossbred population of the indigenous Chongming white goat and the Boer goat were performed to discover the potential functional differentially expressed genes (DEGs) and differential expression metabolites (DEMs).ResultsA total of 2812 DEGs were detected in 6 groups at three time stages (3,6,12 Month) in skeletal muscle using the RNA-seq method. A DEGs set containing seven muscle function related genes (TNNT1, TNNC1, TNNI1, MYBPC2, MYL2, MHY7, and CSRP3) was discovered, and their expression tended to increase as goat muscle development progressed. Seven DEGs (TNNT1, FABP3, TPM3, DES, PPP1R27, RCAN1, LMOD2) in the skeletal muscle of goats in the fast-growing and slow-growing groups was verified their expression difference by reverse transcription-quantitative polymerase chain reaction. Further, through the Liquid chromatography-mass spectrometry (LC-MS) approach, a total of 183 DEMs in various groups of the muscle samples and these DEMs such as Queuine and Keto-PGF1α, which demonstrated different abundance between the goat fast-growing group and slow-growing group. Through weighted correlation network analysis (WGCNA), the study correlated the DEGs with the DEMs and identified 4 DEGs modules associated with 18 metabolites.ConclusionThis study benefits to dissection candidate genes and regulatory networks related to goat meat production performance, and the joint analysis of transcriptomic and metabolomic data provided insights into the study of goat muscle development.

Lipid Mapping of Lipid Signaling Mediators in Skeletal Muscle

Recent advancements in lipidomic have provided profound insight that goes beyond considering lipids as simple organic compounds that constitute the fundamental components of cell membranes. Beyond their pivotal roles in processes such as energy storage and serving as the fundamental building blocks of life, lipids have emerged as potent signaling molecules of significance. The importance of lipid signaling stems from its ability to orchestrate cellular responses to many environmental stresses, including salinity and pathogen attacks. Lipid signaling plays a significant role by allowing cells to respond to their local environment through reactions such as cell differentiation, inflammation and even immunity. In context with this knowledge, our objective is to comprehensively identify key signaling lipid mediators documented from published scientific literature, followed by a systemic visualization of the resulting data through the process of lipid mediator mapping to identify common signaling mediators pathways/networks that are essential for musculoskeletal (MSK) health and disease. These bioinformatics studies are complemented by our experimental studies in muscle cells. To this end, we are using a customized liquid chromatography-mass spectrometry (LC-MS) approach to profile and quantify lipid mediators (LMs). We were able to identify and profile up to 214 LMs and to precisely quantify 60 of them from various samples including body fluid, plasma, serum, bone marrow, muscle tissue, and other cell tissues. Preliminary screening indicated that 11,12-EET, 12-HETE, and PGE2 are three LMs that regulate physiologic aspects in muscles. In addition, 11,12-EET contributes to the regulation of vasodilation, exhibits anti-inflammatory properties under some conditions and in some specific tissues, and maintains endothelial function, and vascular health. However, our data indicates that 11, 12-EET levels increase with aging, decrease with exercise, and is causal for osteopenia in a zebrafish model, supporting our hypothesis that the effects of LMs are tissue specific and related to environmental factors. PGE2, which also causes inflammation, seems to be a putative LM implicated in tissue regeneration in multiple organs. In skeletal muscles, we found that PGE2 enhances myogenic differentiation and proliferation of muscle cells up to nM concentration. PGE2 also possesses beneficial bronchoprotective and anti-inflammatory effects on the airways. In conclusion, this combination of bioinformatics and experimental studies to deepen the mapping of signaling pathways and networks of LMs is crucially needed to understand the full function of LMs in MSK health and disease. This work was directly supported by NIH-National Institutes of Aging P01AG039355 (MB) and the George W. and Hazel M. Jay and Evanston Research Endowments (MB). Authors were supported by NIH Grants: National Institutes of Aging (NINDS) 2-R01NS105621; NIA-R01AG056504, NIA-2R01AG060341, National Institutes of Diabetes, Digestive, and Kidney Diseases Kidney (NIDDK)-1R01DK119066 to MB, and National Institutes of Neurological Disorders and Stroke (NINDS) 2-R01NS105621 to MB. Also, NIH/NIDC 1R01DE031872-01 (VV). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Formulation and Evaluation of Herbal Ethosomal Cream of Passiflora foetida Linn. for the Treatment of Psoriasis

Psoriasis is an autoimmune condition with the most significant and far-reaching consequences for humans. In order to cure psoriasis, an herbal ethosomal cream was developed, and one of the plants that went into its production was the Passiflora foetida Linn. plant. A large number of distinct phytochemical identification tests were carried out with the use of a broad range of reagents. The ability of an antioxidant to reduce free radicals, as measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH), is one way that its effectiveness may be shown. Coconut oil, olive oil, and vitamin E oil are the three essential oils combined to make the final product. All of these oils bring its own unique set of medicinal benefits to the table. The liquid chromatography-mass spectrometry (LC-MS) analysis carried out on the herbal extract revealed the presence of phytoconstituents inside the sample. The overall quality of the cream that was produced was evaluated. The LC-MS approach was used to dissect the anti-inflammatory and psoriasis-fighting components included in herbal remedies. An attempt to treat psoriasis using herbal medicine was made using the ethosomes of the P. foetida Linn. plant.

Multi-omics analysis of the gut microbiome and metabolites associated with the psychoneurological symptom cluster in children with cancer receiving chemotherapy

BackgroundChildren with cancer receiving chemotherapy commonly report a cluster of psychoneurological symptoms (PNS), including pain, fatigue, anxiety, depression, and cognitive dysfunction. The role of the gut microbiome and its functional metabolites in PNS is rarely studied among children with cancer. This study investigated the associations between the gut microbiome–metabolome pathways and PNS in children with cancer across chemotherapy as compared to healthy children.MethodsA case–control study was conducted. Cancer cases were recruited from Children’s Healthcare of Atlanta and healthy controls were recruited via flyers. Participants reported PNS using the Pediatric Patient-Reported Outcomes Measurement Information System. Data for cases were collected pre-cycle two chemotherapy (T0) and post-chemotherapy (T1), whereas data for healthy controls were collected once. Gut microbiome and its metabolites were measured using fecal specimens. Gut microbiome profiling was performed using 16S rRNA V4 sequencing, and metabolome was performed using an untargeted liquid chromatography–mass spectrometry approach. A multi-omics network integration program analyzed microbiome–metabolome pathways of PNS.ResultsCases (n = 21) and controls (n = 14) had mean ages of 13.2 and 13.1 years. For cases at T0, PNS were significantly associated with microbial genera (e.g., Ruminococcus, Megasphaera, and Prevotella), which were linked with carnitine shuttle (p = 0.0003), fatty acid metabolism (p = 0.001) and activation (p = 0.001), and tryptophan metabolism (p = 0.008). Megasphaera, clustered with aspartate and asparagine metabolism (p = 0.034), carnitine shuttle (p = 0.002), and tryptophan (p = 0.019), was associated with PNS for cases at T1. Gut bacteria with potential probiotic functions, along with fatty acid metabolism, tryptophan, and carnitine shuttle, were more clustered in cancer cases than the control network and this linkage with PNS needs further studies.ConclusionsUsing multi-omics approaches, this study indicated specific microbiome–metabolome pathways linked with PNS in children with cancer across chemotherapy. Due to limitations such as antibiotic use in cancer cases, these findings need to be further confirmed in a larger cohort.

First comprehensive untargeted metabolomics study of suramin-treated Trypanosoma brucei: an integrated data analysis workflow from multifactor data modelling to functional analysis.

Human African trypanosomiasis, commonly known as sleeping sickness, is a vector-borne parasitic disease prevalent in sub-Saharan Africa and transmitted by the tsetse fly. Suramin, a medication with a long history of clinical use, has demonstrated varied modes of action against Trypanosoma brucei. This study employs a comprehensive workflow to investigate the metabolic effects of suramin on T. brucei, utilizing a multimodal metabolomics approach. The primary aim of this study is to comprehensively analyze the metabolic impact of suramin on T. brucei using a combined liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) approach. Statistical analyses, encompassing multivariate analysis and pathway enrichment analysis, are applied to elucidate significant variations and metabolic changes resulting from suramin treatment. A detailed methodology involving the integration of high-resolution data from LC-MS and NMR techniques is presented. The study conducts a thorough analysis of metabolite profiles in both suramin-treated and control T. brucei brucei samples. Statistical techniques, including ANOVA-simultaneous component analysis (ASCA), principal component analysis (PCA), ANOVA 2 analysis, and bootstrap tests, are employed to discern the effects of suramin treatment on the metabolomics outcomes. Our investigation reveals substantial differences in metabolic profiles between the control and suramin-treated groups. ASCA and PCA analysis confirm distinct separation between these groups in both MS-negative and NMR analyses. Furthermore, ANOVA 2 analysis and bootstrap tests confirmed the significance of treatment, time, and interaction effects on the metabolomics outcomes. Functional analysis of the data from LC-MS highlighted the impact of treatment on amino-acid, and amino-sugar and nucleotide-sugar metabolism, while time effects were observed on carbon intermediary metabolism (notably glycolysis and di- and tricarboxylic acids of the succinate production pathway and tricarboxylic acid (TCA) cycle). Through the integration of LC-MS and NMR techniques coupled with advanced statistical analyses, this study identifies distinctive metabolic signatures and pathways associated with suramin treatment in T. brucei. These findings contribute to a deeper understanding of the pharmacological impact of suramin and have the potential to inform the development of more efficacious therapeutic strategies against African trypanosomiasis.

Viral Infection Induces Changes to the Metabolome, Immune Response and Development of a Generalist Insect Herbivore.

Host plant consumption and pathogen infection commonly influence insect traits related to development and immunity, which are ultimately reflected in the behavior and physiology of the insect. Herein, we explored changes in the metabolome of a generalist insect herbivore, Vanessa cardui (Lepidoptera: Nymphalidae), in response to both dietary variation and pathogen infection in order to gain insight into tritrophic interactions for insect metabolism and immunity. Caterpillars were reared on two different host plants, Plantago lanceolata (Plantaginaceae) and Taraxacum officinale (Asteraceae) and subjected to a viral infection by Junonia coenia densovirus (JcDV), along with assays to determine the insect immune response and development. Richness and diversity of plant and caterpillar metabolites were evaluated using a liquid chromatography-mass spectrometry approach and showed that viral infection induced changes to the chemical content of V. cardui hemolymph and frass dependent upon host plant consumption. Overall, the immune response as measured by phenoloxidase (PO) enzymatic activity was higher in individuals feeding on P. lanceolata compared with those feeding on T. officinale. Additionally, infection with JcDV caused suppression of PO activity, which was not host plant dependent. We conclude that viral infection combined with host plant consumption creates a unique chemical environment, particularly within the insect hemolymph. Whether and how these metabolites contribute to defense against viral infection is an open question in chemical ecology.

Mass Spectrometry-Based Profiling of Histone Post-Translational Modifications in Uveal Melanoma Tissues, Human Melanocytes, and Uveal Melanoma Cell Lines - A Pilot Study.

Epigenetic alterations in uveal melanoma (UM) are still neither well characterized, nor understood. In this pilot study, we sought to provide a deeper insight into the possible role of epigenetic alterations in the pathogenesis of UM and their potential prognostic relevance. To this aim, we comprehensively profiled histone post-translational modifications (PTMs), which represent epigenetic features regulating chromatin accessibility and gene transcription, in UM formalin-fixed paraffin-embedded (FFPE) tissues, control tissues, UM cell lines, and healthy melanocytes. FFPE tissues of UM (n = 24), normal choroid (n = 4), human UM cell lines (n = 7), skin melanocytes (n = 6), and uveal melanocytes (n = 2) were analyzed through a quantitative liquid chromatography-mass spectrometry (LC-MS) approach. Hierarchical clustering showed a clear separation with several histone PTMs that changed significantly in a tumor compared to normal samples, in both tissues and cell lines. In addition, several acetylations and H4K20me1 showed lower levels in BAP1 mutant tumors. Some of these changes were also observed when we compared GNA11 mutant tumors with GNAQ tumors. The epigenetic profiling of cell lines revealed that the UM cell lines MP65 and UPMM1 have a histone PTM pattern closer to the primary tissues than the other cell lines analyzed. Our results suggest the existence of different histone PTM patterns that may be important for diagnosis and prognosis in UM. However, further analyses are needed to confirm these findings in a larger cohort. The epigenetic characterization of a panel of UM cell lines suggested which cellular models are more suitable for epigenetic investigations.

Quantification of mRNA in Lipid Nanoparticles Using Mass Spectrometry.

Lipid nanoparticle-encapsulated mRNA (LNP-mRNA) holds great promise as a novel modality for treating a broad range of diseases. The ability to quantify mRNA accurately in therapeutic products helps to ensure consistency and safety. Here, we consider a central aspect of accuracy, measurement traceability, which establishes trueness in quantity. In this study, LNP-mRNA is measured in situ using a novel liquid chromatography-mass spectrometry (LC-MS) approach with traceable quantification. Previous works established that oligonucleotide quantification is possible through the accounting of an oligomer's fundamental nucleobases, with traceability established through common nucleobase calibrators. This sample preparation does not require mRNA extraction, detergents, or enzymes and can be achieved through direct acid hydrolysis of an LNP-mRNA product prior to an isotope dilution strategy. This results in an accurate quantitative analysis of mRNA, independent of time or place. Acid hydrolysis LC-MS is demonstrated to be amenable to measuring mRNA as both an active substance or a formulated mRNA drug product.

Absolute Quantification of Human Serum Albumin Isoforms by Internal Calibration Based on a Top-Down LC-MS Approach.

Well-characterized biomarkers using reliable quantitative methods are essential for the management of various pathologies such as diabetes, kidney, and liver diseases. Human serum albumin (HSA) isoforms are gaining interest as biomarkers of advanced liver pathologies. In view of the structural alterations observed for HSA, insights into its isoforms are required to establish them as reliable biomarkers. Therefore, a robust absolute quantification method seems necessary. In this study, we developed and validated a far more advanced top-down liquid chromatography-mass spectrometry (LC-MS) method for the absolute quantification of HSA isoforms, using myoglobin (Mb) as an internal standard for quantification and for mass recalibration. Two different quantification approaches were investigated based on peak integration from the deconvoluted spectrum and extracted ion chromatogram (XIC). The protein mixture human serum albumin/myoglobin eluted in well-shaped separated peaks. Mb allowed a systematic mass recalibration for every sample, resulting in extremely low mass deviations compared to conventional deconvolution-based methods. In total, eight HSA isoforms of interest were quantified. Specific-isoform calibration curves showing good linearity were obtained by using the deconvoluted peaks. Noticeably, the HSA ionization behavior appeared to be isoform-dependent, suggesting that the use of an enriched isoform solution as a calibration standard for absolute quantification studies of HSA isoforms is necessary. Good repeatability, reproducibility, and accuracy were observed, with better sensitivity for samples with low albumin concentrations compared to routine biochemical assays. With a relatively simple workflow, the application of this method for absolute quantification shows great potential, especially for HSA isoform studies in a clinical context, where a high-throughput method and sensitivity are needed.

Discovery, verification, and validation of walnut protein marker peptides using LC-MS approaches.

A key requirement of liquid chromatography-mass spectrometry (LC-MS)-based allergenic food protein analysis methods is to use protein marker peptides with good analytical performances in LC-MS analysis of commercial processed foods. In this study, we developed a multi-stage walnut protein marker peptide selection strategy involving marker peptide discovery and verification and LC-MS validation of chemically equivalent stable isotope-labeled peptides. This strategy proposed three walnut protein marker peptides, including two new marker peptides. Our LC-MS-based walnut protein analysis method using the three stable isotope-labeled peptides showed acceptable linearity (R2>0.99), matrix effects (coefficient of variation <±15%), sensitivity (limit of detection>0.3pg/μL, limit of quantification>0.8pg/μL), recovery (85.1-103.4%), accuracy, and precision (coefficient of variation<10%). In conclusion, our multi-stage marker peptide selection strategy effectively selects specific protein marker peptides for sensitive detection and absolute quantification of walnut proteins in LC-MS analysis of commercial processed foods.

LC-MS Approaches for Oxysterols in Various Biosamples.

Oxysterols are involved in a plethora of biological processes, including a wide variety of diseases. Therefore, monitoring oxysterols is important for obtaining a deeper understanding of their biological roles and utilizing them as, for example, biomarkers. However, oxysterols can be challenging compounds to study, as they can be very similar in chemical structure but still have distinct biological roles. In addition, oxysterols may be difficult to detect, even with advanced analytical instrumentation. We here focus on the use of liquid chromatography-mass spectrometry (LC-MS) for the analysis of oxysterols, with an additional focus on the steps needed to prepare oxysterols for LC-MS. Steps can include chemical modification of the oxysterols for improving LC-MS sensitivity and adding chemicals that can reveal if the oxysterol levels have been perturbed during preparation. We then round off with descriptions and applications of various sample preparations for different biological matrices, from blood to cells, and biosamples with emerging attention, for example, exosomes and organoids. Taken together, oxysterol analysis is highly compatible with a wide variety of biosamples, allowing for a deeper understanding of these challenging analytes.

Leukaemia Exposure Alters the Transcriptional Profile and Function of Macrophages in the Bone Marrow Niche

These authors contributed equally to this work: Martha M. Zarou &amp; Amy Dawson Macrophages are fundamental cells of the innate immune system. Bone marrow (BM) resident macrophages are involved in antigen presentation, phagocytosis, efferocytosis (clearing of apoptotic cells) and play a critical role in regulating haematopoietic stem cell (HSC) function. In chronic myeloid leukaemia (CML), the disease-initiating leukaemic stem cells (LSCs) modulate the BM microenvironment to support leukaemia maintenance and progression. However, whether the CML BM niche alters the function of macrophages remains unknown. Using single-cell RNA sequencing (scRNA-seq) of human BM mononuclear cells, we demonstrate heterogeneity of tissue resident macrophages, isolated from CML patients at diagnosis, which cluster separately from BM macrophages deriving from heathy individuals. As most BM macrophages at diagnosis express BCR::ABL1 and are sensitive to TKI treatment, we further investigated if Philadelphia chromosome negative (Ph -) macrophages play a major role in CML development. We applied a chimeric CML mouse model (SCLtTA/BCR::ABL1) and reveal that temporal depletion of macrophages prior to leukaemia induction (using CSF1R antagonist antibody), increases leukaemia burden and decreases survival of chimeric mice following BCR::ABL1 induction (p&amp;lt;0.05). Comparison of CML exposed Ph - BM macrophages to control counterparts by scRNA-seq demonstrates that the CML niche drives two unique subpopulations of immature and anti-inflammatory macrophages. Functionally, we discover that Ph - BM macrophages exposed to a CML microenvironment display reduced phagocytic and efferocytic capacity, which correlates with reduction in mitochondrial respiration (p&amp;lt;0.05). Mechanistically, we show that diminished efferocytosis is mediated by reduced expression of the plasma membrane fatty acid translocase CD36. Additionally, treatment of primary mouse and human BM derived macrophages (BMDM) with the irreversible CD36 inhibitor sulfosuccinimidyl oleate (SSO), or genetic deletion of CD36 in THP-1 derived macrophages, results in reduced clearance of apoptotic cells in vitro. Furthermore, CD36 deficient macrophages display decreased mitochondrial respiration, similarly to CML exposed Ph - BMDM. Through untargeted liquid chromatography-mass spectrometry approach we identified the immunomodulatory protein lactotransferrin (LTF) as differentially secreted protein by leukaemic mouse stem/progenitor cells, when compared with secretome of normal counterparts (p&amp;lt;0.05). Treatment with LTF replicates the effect of CML exposure on BMDM, including decreased phagocytic function of mouse BMDM and THP-1 derived macrophages (p&amp;lt;0.05). Furthermore, mouse/human BMDM display reduced clearance of CML apoptotic cells following LTF exposure. Notably, LTF treatment reduces CD36 transcript and protein levels on BMDM and/or THP-1 derived macrophages, which correlates with reduced oxygen consumption rate and a 40% decrease in expression of the monocytic marker CD11b during macrophage differentiation, suggesting that secreted LTF is a major contributor to the immature phenotype of CML exposed macrophages. Overall, we present novel findings that dysregulated CML microenvironment modifies BM macrophage function, including their phagocytic and efferocytic capacity. This is at least partially contributed by secretion of LTF from CML stem/progenitor cells and modulation of CD36 expression on altered BM macrophages.

Analysis of total thiol concentrations in environmental samples using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS)

In this study, we developed an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) approach to measure the concentration of total reactive thiol binding sites in a range of types of environmental samples by monitoring the decrease of monobromo-(trimethylammonio)-bimane (qBBr) caused by thiol-qBBr binding reactions. We first examined the stability of qBBr and potential side reactions that could reduce the accuracy of the UHPLC-MS approach. We found that significant loss of qBBr occurs in the absence of thiols in aqueous solutions after 2–4 h due to qBBr degradation and other side reactions, such as a chloride-for-bromide substitution reaction. To address these issues, in our approach we conduct the thiol-qBBr binding reaction in the presence of excessive qBBr for only 1 h, and we compare the MS signal of unbound qBBr in a thiol-free qBBr standard with that in a thiol-bearing qBBr standard in order to calculate the decrease in the concentration of free qBBr in solution that is caused by thiol-qBBr binding. Using 100–600 nM of N-Acetyl-L-cysteine (ACYS) as a model thiol in a 2 μM qBBr standard, we found that the measured decrease in the MS signal of unbound qBBr linearly correlates to the concentration of added ACYS, with a R2 of 0.988, demonstrating that monitoring the decrease in unbound qBBr can be used to calculate the concentration of total thiol sites in a sample. We demonstrate that the new UHPLC-MS approach yields accurate results when measuring a series of single and mixed thiol standards in the 200–500 nM range, with measured values ranging from 86% to 109% of the known concentration of thiols. In addition to the above control tests, we also used the UHPLC-MS approach to measure dissolved thiol concentrations in several lake water samples. These measurements revealed that each lake contained low μM levels of total thiols, not only demonstrating the ability of the analytical approach to determine thiol site concentrations in natural waters, but also suggesting the widespread importance of these sites in controlling chalcophile element behavior in these systems. The UHPLC-MS approach not only can be used to determine the concentration of dissolved thiol sites at nM levels, but can also be adapted to measure the concentration of thiol binding sites on other environmental samples such as soil, sediment, and solid-phase organic matter surfaces.

LC-MS Approach to Decipher a Light Chain Chromatographic Peak Splitting of a Monoclonal Antibody.

Monoclonal antibodies (mAbs), like other protein therapeutics, are prone to various forms of degradation, some of which are difficult to distinguish from the native form yet may alter potency. A generalizable LC-MS approach was developed to enable quantitative analysis of isoAsp. In-depth understanding of product quality attributes (PQAs) enables optimization of the manufacturing process, better formulation selection, and decreases risk associated with product handling in the clinic or during shipment. Reversed-phase chromatographic peak splitting was observed when a mAb was exposed to elevated temperatures. Multiple LC-MS based methods were applied to identify the reason for peak splitting. The approach involved the use of complementary HPLC columns, multiple enzymatic digestions and different MS/MS ion dissociation methods. In addition, mAb potency was measured by enzyme-linked immunosorbent assay (ELISA). The split peaks had identical masses, and the root cause of the peak splitting was identified as isomerization of an aspartic acid located in the complementarity-determining region (CDR) of the light chain. And the early eluting and late eluting peaks were collected and performed enzymatic digestion to confirm the isoAsp enrichment in the early eluting peak. In addition, decreased potency was observed in the same heat-stressed sample, and the increased isoAsp levels in the CDR correlate well with a decrease of potency. Liquid chromatography-mass spectrometry (LC-MS) has been utilized extensively to assess PQAs of biological therapeutics. In this study, a generalizable LC-MS-based approach was developed to enable identification and quantitation of the isoAsp-containing peptides.

Characterization of Gla proteoforms and non-Gla peptides of gamma carboxylated proteins: Application to quantification of prothrombin proteoforms in human plasma

Gamma (γ) carboxylation is an essential post-translational modification in vitamin K-dependent proteins (VKDPs), involved in maintaining critical biological homeostasis. Alterations in the abundance or activity of these proteins have pharmacological and pathological consequences. Importantly, low levels of fully γ-carboxylated clotting factors increase plasma des-γ-carboxy precursors resulting in little or no biological activity. Therefore, it is important to characterize the levels of γ-carboxylation that reflect the active state of these proteins. The conventional enzyme-linked immunosorbent assay for protein induced by vitamin K absence or antagonist II (PIVKA-II) quantification uses an antibody that is not applicable to distinguish different γ-carboxylation states. Liquid chromatography-mass spectrometry (LC-MS) approaches have been utilized to distinguish different γ-carboxylated proteoforms, however, these attempts were impeded by poor sensitivity due to spontaneous neutral loss of CO2 and simultaneous cleavage of the backbone bond in the collision cell. In this study, we utilized an alkaline mobile phase in combination with polarity switching (positive and negative ionization modes) to simultaneously identify and quantify γ-carboxylated VKDPs. The method was applied to compare Gla proteomics of prothrombin (FII) in 10 μL plasma samples of healthy control and warfarin-treated adults. We also identified surrogate non-Gla peptides for seven other VKDPs to quantify total (active plus inactive) protein levels. The total protein approach (TPA) was used to quantify absolute levels of the VKDPs in human plasma.

Identifying Serum Metabolomic Markers Associated with Skin Disease Activity in Patients with Psoriatic Arthritis.

Psoriatic arthritis (PsA) is a chronic, systemic, immune-mediated inflammatory disease causing cutaneous and musculoskeletal inflammation that affects 25% of patients with psoriasis. Current methods for evaluating PsA disease activity are not accurate enough for precision medicine. A metabolomics-based approach can elucidate psoriatic disease pathogenesis, providing potential objective biomarkers. With the hypothesis that serum metabolites are associated with skin disease activity, we aimed to identify serum metabolites associated with skin activity in PsA patients. We obtained serum samples from patients with PsA (n = 150) who were classified into mild, moderate and high disease activity groups based on the Psoriasis Area Severity Index. We used solid-phase microextraction (SPME) for sample preparation, followed by data acquisition via an untargeted liquid chromatography-mass spectrometry (LC-MS) approach. Disease activity levels were predicted using identified metabolites and machine learning algorithms. Some metabolites tentatively identified include eicosanoids with anti- or pro-inflammatory properties, like 12-Hydroxyeicosatetraenoic acid, which was previously implicated in joint disease activity in PsA. Other metabolites of interest were associated with dysregulation of fatty acid metabolism and belonged to classes such as bile acids, oxidized phospholipids, and long-chain fatty acids. We have identified potential metabolites associated with skin disease activity in PsA patients.

Revolutions in Lipid Isomer Resolution: Application of Ultrahigh-Resolution Ion Mobility to Reveal Lipid Diversity.

Many families of lipid isomers remain unresolved by contemporary liquid chromatography-mass spectrometry approaches, leading to a significant underestimation of the structural diversity within the lipidome. While ion mobility coupled to mass spectrometry has provided an additional dimension of lipid isomer resolution, some isomers require a resolving power beyond the capabilities of conventional platforms. Here, we present the application of high-resolution traveling-wave ion mobility for the separation of lipid isomers that differ in (i) the location of a single carbon-carbon double bond, (ii) the stereochemistry of the double bond (cis or trans), or, for glycerolipids, (iii) the relative substitution of acyl chains on the glycerol backbone (sn-position). Collisional activation following mobility separation allowed identification of the carbon-carbon double-bond position and sn-position, enabling confident interpretation of variations in mobility peak abundance. To demonstrate the applicability of this method, double-bond and sn-position isomers of an abundant phosphatidylcholine composition were resolved in extracts from a prostate cancer cell line and identified by comparison to pure isomer reference standards, revealing the presence of up to six isomers. These findings suggest that ultrahigh-resolution ion mobility has broad potential for isomer-resolved lipidomics and is attractive to consider for future integration with other modes of ion activation, thereby bringing together advanced orthogonal separations and structure elucidation to provide a more complete picture of the lipidome.

Quantitative Analysis of Acetyl-CoA, Malonyl-CoA, and Succinyl-CoA in Myocytes.

Several analytical challenges make it difficult to accurately measure coenzyme A (CoA) metaboforms, including insufficient stability and a lack of available metabolite standards. Consequently, our understanding of CoA biology and the modulation of human diseases may be nascent. CoA's serve as lipid precursors, energy intermediates, and mediators of post-translational modifications of proteins. Here, we present a liquid chromatography-mass spectrometry (LC-MS) approach to measure malonyl-CoA, acetyl-CoA, and succinyl-CoA in complex biological samples. Additionally, we evaluated workflows to increase sample stability. We used reference standards to optimize CoA assay sensitivity and test CoA metabolite stability as a function of the reconstitution solvent. We show that using glass instead of plastic sample vials decreases CoA signal loss and improves the sample stability. We identify additives that improve CoA stability and facilitate accurate analysis of CoA species across large sample sets. We apply our optimized workflow to biological samples of skeletal muscle cells cultured under hypoxic and normoxia conditions. Together, our workflow improves the detection and identification of CoA species through targeted analysis in complex biological samples.