Quantitative Liquid Chromatography-mass Spectrometry Research Articles

Circulating amino acid signature features urea cycle alterations associated with coronary artery disease

Coronary artery disease (CAD) remains a leading cause of death worldwide and imposes a substantial socioeconomic burden on healthcare. Improving risk stratification in clinical practice could help to combat this burden. As amino acids are biologically active metabolites whose involvement in CAD remains largely unknown, this study investigated associations between circulating amino acid levels and CAD phenotypes. A high-coverage quantitative liquid chromatography-mass spectrometry approach was applied to acquire the serum amino acids profile of age- and sex-coarsened-matched patients with CAD (n = 46, 66.9 years, 74.7% male) and healthy individuals (n = 120, 67.4 years, 74.7% male) from the COmPLETE study. Multiple linear regressions were performed to investigate associations between amino acid levels and (a) the health status (CAD vs. healthy), (b) the number of affected coronary arteries, or (c) the left ventricular ejection fraction. Regressions were adjusted for age, sex, daily physical activity, sampling, and fasting time. Urea cycle amino acids (ornithine, citrulline, homocitrulline, aspartate, and arginine) were significantly and negatively associated with CAD, the number of affected coronary arteries, and the left ventricular ejection fraction. Lysine, histidine, and the glutamine/glutamate ratio were also significantly and negatively associated with the CAD phenotypes. Overall, patients with CAD displayed lower levels of urea cycle amino acids, highlighting a potential role for urea cycle amino acid profiling in cardiovascular risk stratification.Trial registrationThe study was registered on https://www.clinicaltrials.gov (NCT03986892) on June 5, 2019.

Regioselective analysis of heat-induced conformational changes of β-lactoglobulin by quantitative liquid chromatography–mass spectrometry analysis of chemical labeling kinetics

β-Lactoglobulin is a main allergen in cow's milk; its allergenicity is strongly impacted by processing. To understand heat-induced epitope-specific effects, the present study analyzed regiospecific conformational changes of heated native β-lactoglobulin variant A (BLG-A). Complementary fluorescence spectroscopy methods indicated two denaturation phases comprising minor sequential conformational changes (25–75 °C) and complete transitions (80–90 °C). Regioselective conformational changes of BLG-A in the native state (25 °C), sequential (70 °C) and complete transition (90 °C) were determined by quantitative liquid chromatography–mass spectrometry analysis of chemical labeling kinetics covering 14 lysine residues and the N-terminus. Conformational changes in two phases were observed for N-terminus, K8 (both N-terminal chain), K60 (β-sheet C), K75 (β-sheet D), K77 (DE loop), K83 (β-sheet E), K100 and K101 (FG loop). The residues K14 (β-sheet A1), K47 (β-sheet B), K69, K70 (both β-sheet D), and K91 (β-sheet F) were not involved in conformational changes.

SCALiR: A Web Application for Automating Absolute Quantification of Mass Spectrometry-Based Metabolomics Data.

Quantitative liquid chromatography-mass spectrometry (LC-MS)-based metabolomics is becoming an important approach for studying complex biological systems but presents several technical challenges that limit its widespread use. Computing metabolite concentrations using standard curves generated from standard mixtures of known concentrations is a labor-intensive process that is often performed manually. Currently, there are few options for open-source software tools that can automatically calculate metabolite concentrations. Herein, we introduce SCALiR (standard curve application for determining linear ranges), a new web-based software tool specifically built for this task, which allows users to automatically transform LC-MS signals into absolute quantitative data (https://www.lewisresearchgroup.org/software). SCALiR uses an algorithm that automatically finds the equation of the line of best fit for each standard curve and uses this equation to calculate compound concentrations from the LC-MS signal. Using a standard mix containing 77 metabolites, we show a close correlation between the concentrations calculated by SCALiR and the expected concentrations of each compound (R2 = 0.99 for a y = x curve fitting). Moreover, we demonstrate that SCALiR reproducibly calculates concentrations of midrange standards across ten analytical batches (average coefficient of variation 0.091). SCALiR can be used to calculate metabolite concentrations either using external calibration curves or by using internal standards to correct for matrix effects. This open-source and vendor agnostic software offers users several advantages in that (1) it requires only 10 s of analysis time to compute concentrations of >75 compounds, (2) it facilitates automation of quantitative workflows, and (3) it performs deterministic evaluations of compound quantification limits. SCALiR therefore provides the metabolomics community with a simple and rapid tool that enables rigorous and reproducible quantitative metabolomics studies.

Mass Spectrometry-Based Method to Measure Aflatoxin B1 DNA Adducts in Formalin-Fixed Paraffin-Embedded Tissues.

Aflatoxin B1 (AFB1) is a potent human liver carcinogen produced by certain molds, particularly Aspergillus flavus and Aspergillus parasiticus, which contaminate peanuts, corn, rice, cottonseed, and ground and tree nuts, principally in warm and humid climates. AFB1 undergoes bioactivation in the liver to produce AFB1-exo-8,9-epoxide, which forms the covalently bound cationic AFB1-N7-guanine (AFB1-N7-Gua) DNA adduct. This adduct is unstable and undergoes base-catalyzed opening of the guanine imidazolium ring to form two ring-opened diastereomeric 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy-aflatoxin B1 (AFB1-FapyGua) adducts. The AFB1 formamidopyrimidine (Fapy) adducts induce G → T transversion mutations and are likely responsible for the carcinogenic effects of AFB1. Quantitative liquid chromatography-mass spectrometry (LC-MS) methods have shown that AFB1-N7-Gua is eliminated in rodent and human urine, whereas ring-opened AFB1-FapyGua adducts persist in rodent liver. However, fresh frozen biopsy tissues are seldom available for biomonitoring AFB1 DNA adducts in humans, impeding research advances in this potent liver carcinogen. In contrast, formalin-fixed paraffin-embedded (FFPE) specimens used for histopathological analysis are often accessible for molecular studies. However, ensuring nucleic acid quality presents a challenge due to incomplete reversal of formalin-mediated DNA cross-links, which can preclude accurate quantitative measurements of DNA adducts. In this study, employing ion trap or high-resolution accurate Orbitrap mass spectrometry, we demonstrate that ring-opened AFB1-FapyGua adducts formed in AFB1-exposed newborn mice are stable to the formalin fixation and DNA de-cross-linking retrieval processes. The AFB1-FapyGua adducts can be detected at levels comparable to those in a match of fresh frozen liver. Orbitrap MS2 measurements can detect AFB1-FapyGua at a quantification limit of 4.0 adducts per 108 bases when only 0.8 μg of DNA is assayed on the column. Thus, our breakthrough DNA retrieval technology can be adapted to screen for AFB1 DNA adducts in FFPE human liver specimens from cohorts at risk of this potent liver carcinogen.

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.

Biomarkers for predicting the severity of spinal cord injury by proteomic analysis.

Currently, there is a shortage of the protein biomarkers for classifying spinal cord injury (SCI) severity. We attempted to explore the candidate biomarkers for predicting SCI severity. SCI rat models with mild, moderate, and severe injury were constructed with an electro-mechanic impactor. The behavior assessment and pathological examinations were conducted before and after SCI. Then, quantitative liquid chromatography-mass spectrometry (LC-MS/MS) was performed in spinal cord tissues with different extents of injury. The differentially expressed proteins (DEPs) in SCI relative to controls were identified, followed by Mfuzz clustering, function enrichment analysis, and protein-protein interaction (PPI) network construction. The differential changes of candidate proteins were validated by using a parallel reaction monitoring (PRM) assay. After SCI modeling, the motor function and mechanical pain sensitivity of SCI rats were impaired, dependent on the severity of the injury. A total of 154 DEPs overlapped in the mild, moderate, and severe SCI groups, among which 82 proteins were classified in clusters 1, 2, 3, 5, and 6 with similar expression patterns at different extents of injury. DEPs were closely related to inflammatory response and significantly enriched in the IL-17 signaling pathway. PPI network showed that Fgg (Fibrinogen gamma chain), Fga (Fibrinogen alpha chain), Serpinc1 (Antithrombin-III), and Fgb (Fibrinogen beta chain) in cluster 1 were significant nodes with the largest degrees. The upregulation of the significant nodes in SCI samples was validated by PRM. Fgg, Fga, and Fgb may be the putative biomarkers for assessing the extent of SCI.

LC‐MS/MS‐based proteome profiling of CSF‐derived extracellular vesicles in Alzheimer’s disease

AbstractBackgroundExtracellular vesicles (EVs) participate in the spread of pathological proteins between cells, contributing to the progression of neurodegenerative disorders, including Alzheimer’s disease (AD). As cerebrospinal fluid (CSF) reflects the biochemical environment of the brain, EVs from CSF constitutes a promising source to investigate novel biomarkers and therapeutic targets. We aimed to explore the unbiased proteome profile of CSF‐derived EVs from individuals with AD by label‐free quantitative liquid chromatography mass spectrometry (LC‐MS/MS).MethodEVs were obtained from 500 µL of CSF from sex and age‐matched cognitively healthy donors (HC, n = 10, mean age = 73±1.4) and patients with AD dementia (AD, n = 10, mean age = 76.2±0.9) by a high‐throughput size exclusion chromatography method (SmartSEC). Resultant EV fractions were characterized by nanoparticle tracking analysis (NTA). EVs were lysed and proteins were trypsinized overnight via S‐Trap filters (Protifi). The peptides were separated on an Evosep nano LC system and analyzed by the TimsTOF Pro (Bruker) mass spectrometer. Peptides were identified using PEAKS Online software (1% FDR). DAVID Bioinformatics Resources 6.8 was used to determine the gene ontology (GO) enrichment. We normalized log2 peptide intensities (quantiles) and determined the differential protein expression across groups by a linear mixed effects (fixed effects = diagnosis, random effects = peptide sequence) model using the MsqRob package in R. P‐values were adjusted using Benjamini‐Hochberg.ResultNTA showed homogeneous EV size profiles (mean size = 113.4±1.41nm). We identified 2096 proteins with at least 1 proteotypic peptide. GO enrichment analyses showed “extracellular exosome” as the most statistically significant enriched term (p = 1.8×10−212), confirming the enrichment of EVs. Statistical inference analysis revealed 18 differentially expressed proteins between both diagnostic groups (adj.p<0.05), all of them increased in the AD group. (Fig 1). Some of the greatest changes include CLUS (log2FC = 0.61, adj.p = 8.38×10−4, Fig.2), SPB1 (log2FC = 1.15, adj.p = 1.23×10−2, Fig.3), LRP1 (log2FC = 3.43, adj.p = 1.23×10−2), KLK7 (log2FC = 2.59, adj.p = 1.23×10−2), SAP3 (log2FC = 2.09, adj.p = 3.1×10−2) and SHPS1 (log2FC = 1.03, adj.p = 4.25×10−3), which are proteins that have been previously associated with inflammation.ConclusionOur results reveal novel protein changes in CSF‐derived EVs from individuals with AD, which could be explored further to their applicability in distinguishing the different clinical stages across the AD continuum.

Urinary phenotyping of SARS-CoV-2 infection connects clinical diagnostics with metabolomics and uncovers impaired NAD+ pathway and SIRT1 activation.

The stratification of individuals suffering from acute and post-acute SARS-CoV-2 infection remains a critical challenge. Notably, biomarkers able to specifically monitor viral progression, providing details about patient clinical status, are still not available. Herein, quantitative metabolomics is progressively recognized as a useful tool to describe the consequences of virus-host interactions considering also clinical metadata. The present study characterized the urinary metabolic profile of 243 infected individuals by quantitative nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography mass spectrometry (LC-MS). Results were compared with a historical cohort of noninfected subjects. Moreover, we assessed the concentration of recently identified antiviral nucleosides and their association with other metabolites and clinical data. Urinary metabolomics can stratify patients into classes of disease severity, with a discrimination ability comparable to that of clinical biomarkers. Kynurenines showed the highest fold change in clinically-deteriorated patients and higher-risk subjects. Unique metabolite clusters were also generated based on age, sex, and body mass index (BMI). Changes in the concentration of antiviral nucleosides were associated with either other metabolites or clinical variables. Increased kynurenines and reduced trigonelline excretion indicated a disrupted nicotinamide adenine nucleotide (NAD+) and sirtuin 1 (SIRT1) pathway. Our results confirm the potential of urinary metabolomics for noninvasive diagnostic/prognostic screening and show that the antiviral nucleosides could represent novel biomarkers linking viral load, immune response, and metabolism. Moreover, we established for the first time a casual link between kynurenine accumulation and deranged NAD+/SIRT1, offering a novel mechanism through which SARS-CoV-2 manipulates host physiology.

Analysis of insecticides in long-lasting insecticidal nets using X-ray fluorescence spectroscopy and correlation with bioefficacy

BackgroundLong-lasting insecticidal nets (LLINs) are a key vector control tool used for the prevention of malaria. Active ingredient (AI) measurements in LLINs are essential for evaluating their quality and efficacy. The main aim of the present study was to determine the utility of X-ray fluorescence (XRF) spectroscopy as a suitable field-deployable tool for total AI quantification in LLINs.MethodsNew and unused LLIN samples containing deltamethrin (PermaNet® 2.0, n = 35) and alpha-cypermethrin (SafeNet®, n = 43) were obtained from batches delivered to Papua New Guinea (PNG) for mass distribution. Insecticides were extracted from the LLINs using a simple extraction technique and quantified using liquid chromatography mass spectrometry (LC-MS). The LC-MS results were correlated with XRF spectroscopy measurements on the same nets. Operators were blinded regarding the type of net. Bioefficacy of the LLIN samples was tested using WHO cone bioassays and test results were correlated with total AI content.ResultsThe results indicate correlation between quantitative XRF and LC-MS. Interestingly, the total AI content was negatively correlated with bioefficacy in PermaNet® 2.0 (especially in recently manufactured nets). In contrast, AI content was positively correlated with bioefficacy in SafeNet®. These results indicate that the chemical content analysis in predelivery inspections does not always predict bioefficacy.ConclusionXRF is a promising field-deployable tool for quantification of both deltamethrin- and alpha-cypermethrin-coated LLINs. Because total AI content is not always a predictor of the efficacy of LLINs to kill mosquitoes, bioefficacy measurements should be included in predelivery inspections.

Meta-Analysis of COVID-19 Metabolomics Identifies Variations in Robustness of Biomarkers.

The global COVID-19 pandemic resulted in widespread harms but also rapid advances in vaccine development, diagnostic testing, and treatment. As the disease moves to endemic status, the need to identify characteristic biomarkers of the disease for diagnostics or therapeutics has lessened, but lessons can still be learned to inform biomarker research in dealing with future pathogens. In this work, we test five sets of research-derived biomarkers against an independent targeted and quantitative Liquid Chromatography-Mass Spectrometry metabolomics dataset to evaluate how robustly these proposed panels would distinguish between COVID-19-positive and negative patients in a hospital setting. We further evaluate a crowdsourced panel comprising the COVID-19 metabolomics biomarkers most commonly mentioned in the literature between 2020 and 2023. The best-performing panel in the independent dataset-measured by F1 score (0.76) and AUROC (0.77)-included nine biomarkers: lactic acid, glutamate, aspartate, phenylalanine, β-alanine, ornithine, arachidonic acid, choline, and hypoxanthine. Panels comprising fewer metabolites performed less well, showing weaker statistical significance in the independent cohort than originally reported in their respective discovery studies. Whilst the studies reviewed here were small and may be subject to confounders, it is desirable that biomarker panels be resilient across cohorts if they are to find use in the clinic, highlighting the importance of assessing the robustness and reproducibility of metabolomics analyses in independent populations.

Hibernation and plasma lipids in free-ranging brown bears-implications for diabetes.

Brown bears (Ursus arctos) prepare for winter by overeating and increasing adipose stores, before hibernating for up to six months without eating, drinking, and with minimal movement. In spring, the bears exit the den without any damage to organs or physiology. Recent clinical research has shown that specific lipids and lipid profiles are of special interest for diseases such as diabetes type 1 and 2. Furthermore, rodent experiments show that lipids such as sulfatide protects rodents against diabetes. As free-ranging bears experience fat accumulation and month-long physical inactivity without developing diabetes, they could possibly be affected by similar protective measures. In this study, we investigated whether lipid profiles of brown bears are related to protection against hibernation-induced damage. We sampled plasma from 10 free-ranging Scandinavian brown bears during winter hibernation and repeated sampling during active state in the summer period. With quantitative shotgun lipidomics and liquid chromatography-mass spectrometry, we profiled 314 lipid species from 26 lipid classes. A principal component analysis revealed that active and hibernation samples could be distinguished from each other based on their lipid profiles. Six lipid classes were significantly altered when comparing plasma from active state and hibernation: Hexosylceramide, phosphatidylglycerol, and lysophosphatidylglycerol were higher during hibernation, while phosphatidylcholine ether, phosphatidylethanolamine ether, and phosphatidylinositol were lower. Additionally, sulfatide species with shorter chain lengths were lower, while longer chain length sulfatides were higher during hibernation. Lipids that are altered in bears are described by others as relevant for and associated with diabetes, which strengthens their position as potential effectors during hibernation. From this analysis, a range of lipids are suggested as potential protectors of bear physiology, and of potential importance in diabetes.

Dissection of schistosome tissues under LC-MS compatible preservative conditions for quantitative proteomics.

Schistosomes are blood flukes with specialised tissues and organs, each one playing a pivotal role in perpetuating the parasite life cycle. Herein, we describe a detailed methodology for preserving the proteome of adult Schistosoma mansoni worms during manual dissection for enrichment of tissues associated with the parasite's alimentary tract. We provide step-by-step directions for specimen storage and dissection while in preservative solution, tissue homogenisation, protein extraction and digestion using a methodology fully compatible with downstream quantitative liquid chromatography-mass spectrometry analysis. Our methodology uses label-free and QconCAT-based absolute quantification for detection of S. mansoni oesophageal gland products proposed as vaccine candidates. Through stabilisation of the proteome and minimising sample degradation during dissection our approach has allowed us to access the hidden proteome of target tissues not readily available from total lysates because of their small volume. This protocol can be replicated or adapted to other Schistosoma species lacking quantitative proteomics characterisation of specialised tissues for discovery of proteins with potential diagnostic and therapeutic utility.

The Ubiquitin-Proteasome System Participates in Sperm Surface Subproteome Remodeling during Boar Sperm Capacitation.

Sperm capacitation is a complex process endowing biological and biochemical changes to a spermatozoon for a successful encounter with an oocyte. The present study focused on the role of the ubiquitin-proteasome system (UPS) in the remodeling of the sperm surface subproteome. The sperm surface subproteome from non-capacitated and in vitro capacitated (IVC) porcine spermatozoa, with and without proteasomal inhibition, was selectively isolated. The purified sperm surface subproteome was analyzed using high-resolution, quantitative liquid chromatography-mass spectrometry (LC-MS) in four replicates. We identified 1680 HUGO annotated proteins, out of which we found 91 to be at least 1.5× less abundant (p < 0.05) and 141 to be at least 1.5× more abundant (p < 0.05) on the surface of IVC spermatozoa. These proteins were associated with sperm capacitation, hyperactivation, metabolism, acrosomal exocytosis, and fertilization. Abundances of 14 proteins were found to be significantly different (p < 0.05), exceeding a 1.5-fold abundance between the proteasomally inhibited (100 µM MG132) and vehicle control (0.2% ethanol) groups. The proteins NIF3L1, CSE1L, NDUFB7, PGLS, PPP4C, STK39, and TPRG1L were found to be more abundant; while BPHL, GSN, GSPT1, PFDN4, STYXL1, TIMM10, and UBXN4 were found to be less abundant in proteasomally inhibited IVC spermatozoa. Despite the UPS having a narrow range of targets, it modulated sperm metabolism and binding by regulating susceptible surface proteins. Changes in CSE1L, PFDN4, and STK39 during in vitro capacitation were confirmed using immunocytochemistry, image-based flow cytometry, and Western blotting. The results confirmed the active participation of the UPS in the extensive sperm surface proteome remodeling that occurs during boar sperm capacitation. This work will help us to identify new pharmacological mechanisms to positively or negatively modulate sperm fertilizing ability in food animals and humans.

Distinguishing Natural Infections of the Bovine Mammary Gland by Staphylococcus from Streptococcus spp. Using Quantitative Milk Proteomics.

Bovine mastitis is the most frequent disease on dairy farms, which leads to a decrease in the health welfare of the animals and great economic losses. This study was aimed at determining the quantitative variations in the milk proteome caused by natural infection by Staphylococcus and Streptococcus species in order to gain further understanding of any discrepancies in pathophysiology and host immune responses, independent of the mastitis level. After identification of Staphylococcus (N = 51) and Streptococcus (N = 67) spp., tandem mass tag (TMT)-labeled quantitative proteomic and liquid chromatography-mass spectrometry (LC-MS/MS) techniques on a modular Ultimate 3000 RSLCnano system coupled to a Q Exactive Plus was applied on aseptically sampled milk from Holstein cows. Proteome Discoverer was used for protein identification and quantitation through the SEQUEST algorithm. Statistical analysis employing R was used to identify differentially abundant proteins between the groups. Protein classes, functions and functional-association networks were determined using the PANTHER and STRING tools and pathway over-representation using the REACTOME. In total, 156 master bovine proteins were identified (two unique peptides, p < 0.05 and FDR < 0.001), and 20 proteins showed significantly discrepant abundance between the genera (p < 0.05 and FDR < 0.5). The most discriminatory proteins per group were odorant-binding protein (higher in staphylococci) and fibrinogen beta chain protein (higher in streptococci). The receiver operating characteristic (ROC) curve showed that protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I have diagnostic potential for differentiating staphylococci and streptococci intramammary infection and inflammation. Improved understanding of the host response mechanisms and recognition of potential biomarkers of specific-pathogen mastitis, which may aid prompt diagnosis for control implementation, are potential benefits of this study.

Comparative study of phenolic compounds reveals a positive relationship between astringency and the phenolic composition in table grape varieties

Phenolic compounds and their derivatives play a major role in the intensity and characteristics of grape (Vitis vinifera) astringency. The present study investigated the relationship between phenolic composition and astringency of six commercial table grape varieties (two of each white-, red-, and black-skinned). Qualitative and quantitative liquid chromatography-mass spectrometry analysis was used to identify the variety-specific phenolic profiles in the skins and total astringency intensity was assessed and described by a trained sensory panel. Thirty phenolic compounds were identified among the six varieties. Principal component analysis of the phenolic profiles revealed that the intensity of astringency of grape skin was positively correlated with catechin, epicatechin, epicatechin-3-O-gallate, and proanthocyanidin dimers B1, B2, and B3. A further orthogonal partial least-squares discrimination analysis of these compounds showed that catechin was the substance most strongly and positively correlated (R=0.904) with grape skin astringency. PRACTICAL APPLICATION: This study provided a better understanding of the relationships between phenolic composition and table grape astringency and highlighted a potential metabolic marker that could be used as a predictor for the complex astringency sensory attributes of table grape berries.

Use of Affinity Purification-Mass Spectrometry to Identify Phosphorylated Tau Interactors in Alzheimer's Disease.

Phosphorylated tau is the main protein present in neurofibrillary tangles, the presence of which is a key neuropathological hallmark of Alzheimer's disease (AD). The toxic effects of phosphorylated tau are likely mediated by interacting proteins; however, methods to identify these interacting proteins comprehensively in human brain tissue are limited. Here, we describe a method that enables the efficient identification of hundreds of proteins that interact with phosphorylated tau (pTau), using affinity purification-mass spectrometry (AP-MS) on human, fresh-frozen brain tissue from donors with AD. Tissue is homogenized using a gentle technique that preserves protein-protein interactions, and co-immunoprecipitation of pTau and its interacting proteins is performed using the PHF1 antibody. The resulting protein interactors are then identified using label-free quantitative liquid chromatography-mass spectrometry (LC-MS)/MS. The Significance Analysis of INTeractome (SAINT) algorithm is used to determine which proteins significantly interact with pTau. This approach enables the detection of an abundance of all 6 isoforms of tau, 23 phosphorylated residues on tau, and 125 significant pTau protein interactors, in human AD brain tissue.

Discovery of novel ascorbic acid derivatives and other metabolites in fruit of Rosa roxburghii Tratt through untargeted metabolomics and feature-based molecular networking

Fruit of Rosa roxburghii Tratt (FRR) is widely used in functional food industry while short of metabolites research. Herein, we firstly identified 251 metabolites in FRR based on untargeted liquid chromatography-mass spectrometry (LC-MS) approach. Then, 42 differential compounds were sought out to avoid the confusing use of FRR and fruit of R. sterilis S. D. Shi (FRS), and FRR was evaluated exhibiting higher biofunction potential. Moreover, a quantitative LC-MS approach was established to determine the contents of 3 ascorbyl hexosides, and FRR with higher contents should be better source than FRS. Additionally, 17 ascorbic acid (AA) derivatives formed by conjugation of ascorbyl unit with organic acids, flavonoids, or glucuronic acid were also discovered in FRR through characteristic ions of AA and feature-based molecular networking (FBMN), enlightening that AA derivatives were not limited to ascorbyl glycosides. This study provided abundant metabolites information of FRR, laying the basis for exploitation of FRR.

Deeper Understanding of Solvent-Based Ambient Ionization Mass Spectrometry: Are Molecular Profiles Primarily Dictated by Extraction Mechanisms?

Solvent-based ambient ionization mass spectrometry (MS) techniques provide a powerful approach for direct chemical analysis and molecular profiling of biological tissues. While molecular profiling of tissues has been widely used for disease diagnosis, little is understood about how the interplay among solvent properties, matrix effects, and ion suppression can influence the detection of biological molecules. Here, we perform a systematic investigation of the extraction processes of lipids using an ambient ionization droplet microsampling platform to investigate how the physicochemical properties of the solvent systems and extraction time influence molecular extraction and detection. Direct molecular profiling and quantitative liquid chromatography-mass spectrometry (LC-MS) of discrete solvent droplets after surface sampling were investigated to provide insights into extraction and ionization mechanisms. The results of this study suggest that intermolecular interactions such as hydrogen bonding play a major role in extraction and detection of lipids using solvent-based ambient ionization techniques. In addition, extraction time was observed to impact the molecular profiles obtained, suggesting optimization of this parameter can be performed to favor detection of specific analytes.

Bio-analytical Method Development and Validation for Simultaneous Determination of Bictegravir, Emtricitabine, and Tenofovir Alafenamide Fumarate in Human Plasma by LC-MS/MS

Abstract: Aim: The current method was developed as a novel and reliable quantitative liquid chromatography-mass (tandem) spectrometry (LC-MS/MS) method for the estimation of analytes like Bictegravir (BIC) Tenofovir Alafenamide Fumarate (TNF), and Emtricitabine (EMT) in plasma of human simultaneously. Materials: Naproxen (NPX) is used as the internal standard for the current study. The ‘Precipitation Extraction technique’ is used for the present study. The Zorbax XDB C18 analytical column (2.1 X 50 and particle size of 5μm) is used for the chromatographic separation with isocratic natured mobile phase, which consists of Acetonitrile: Formic acid (0.1%) in water (70:30, v/v), at a flow rate of 0.15 mL/minute. Methods: The parent→production conversions were observed at m/z 450.1 → 289.1 (BIC), m/z 248.3 → 130.03 (EMT), m/z 477.3 → 270.04 (TNF), and m/z 231.12 → 184.82 (NPX) on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) positive ion mode. The three compounds were found to possess primary groups hence positive manner was selected for the LC-MS/MS study. Results: The subject method validation was performed for the concentration of range 2-500 ng for Bictegravir (BIC), Emtricitabine (EMT), and Tenofovir Alafenamide Fumarate (TNF). The obtained mean recoveries for the three-drug moieties from samples of spiked plasma were found reproducible. Conclusion: Hence, based on the above, the method was proved to be rugged and rapid, with a least total run time of 3.0 min. The current method was successfully validated according to the FDA, EMA, and ICH guidelines, and the stability studies were evaluated accordingly. Keywords: Bictegravir, Emtricitabine, Tenofovir Alafenamide Fumarate, Liquid Chromatography-Mass Spectrometry, Extraction, Stability.

Quantitation of plasma angiotensin II in healthy Chinese subjects by a validated liquid chromatography tandem mass spectrometry method.

Quantitation of plasma angiotensin (Ang) II, the active mediator of the renin-angiotensin system, is challenging owing to its low physiological concentration. We report a validated liquid chromatography-mass spectrometry (LCMS) method to overcome this challenge. Ang II was extracted from EDTA plasma by an offline solid-phase extraction procedure with a Waters MAX μElution plate. LCMS quantitation was performed on the Waters TQS system, monitoring the 3+ ions of the peptide. The analytical performance of the LCMS method was validated. The stability of Ang II was studied with or without the presence of a protease inhibitor. Local reference intervals were established from 143 healthy normotensive subjects (57% female, 21-60 years old). The Ang II LCMS method had a measurable range of 3.3-700 pmol/L. The between-batch precision coefficient of variation was <7% over Ang II concentrations of 8.6-110 pmol/L. No significant matrix interference and carryover were observed. There was no significant difference in Ang II concentration in EDTA blood and plasma for at least 2h and 1h at room temperature, respectively. Ang II was stable for at least 1 year when stored at -80°C, with or without the protease inhibitor. Age-dependent Ang II reference intervals were established: 4.4-17.7pmol/L (21-30 years) and 3.9-12.8pmol/L (31-60 years). The present LCMS method is suitable for quantitation of plasma Ang II to study the renin-angiotensin system.