Mass Spectrometry Assay Research Articles

A highly efficient liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for etomidate and etomidate acid in urine, liver and kidney

Etomidate (ETO) is a highly-efficient drug that can induce anesthesia with increasing doses, thus subject to strict regulation. However, an accurate and efficient method for ETO intake detection is currently lacking. Therefore, this study developed a straightforward sample preparation method using LC-MS/MS to analyze ETO and its primary metabolite, etomidate acid (ETA), in urine, liver, and kidney samples. Snap frozen pig liver and kidney samples were ground into a fine powder. Then, all the biological samples, including human urine, pig liver and kidney tissues, were deproteinized using acetonitrile and filtered for analysis. The separation was achieved in 9.01 min with gradient elution. The calibration curves ranged from 0.5 to 50 ng/mL for ETO in urine and 0.5 to 50 ng/g in liver and kidney, while the curves ranged from 1 to 100 ng/mL for ETA in urine and 1 to 100 ng/g in liver and kidney. The correlation coefficients (R2) were greater than 0.9957. The Limit of detection (LOD) and limit of quantitation (LOQ) for ETO were 0.2 and 0.5 ng/mL in urine samples and 0.2 and 0.5 ng/g in liver and kidney samples, respectively. For ETA, the LOD and LOQ were 0.5 and 1 ng/mL in urine samples and 0.5 and 1 ng/g in liver and kidney samples. This method was assessed by validation parameters, including selectivity, intra- and inter-day precision and accuracy, recovery, matrix effect, dilution integrity and stability. It was successfully applied to a practical case, revealing ETO and ETA concentrations in urine of 1.01 and 5.58 μg/mL, in liver samples of 12.30 and 1.13 μg/g, and in kidney samples of 6.95 and 4.23 μg/g. This suggests that the method is suitable for routine forensic detection of illicit ETO abuse.

Dual-Specificity Inhibitor Targets Enzymes of the Trehalose Biosynthesis Pathway.

To reduce the risk of resistance development, a novel fungicide with dual specificity is demanded. Trehalose is absent in animals, and its synthases, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), are safe fungicide targets. Here, we report the discovery of a dual-specificity inhibitor of MoTps1 (Magnaporthe oryzae Tps1, TPS) and MoTps2 (M. oryzae Tps2, TPP). The inhibitor, named A1-4, was obtained from a virtual screening and subsequent surface plasmon resonance screening. In in vitro assays, A1-4 interacts with MoTps1 and MoTps2-TPP (MoTps2 TPP domain) and inhibits their enzyme activities. In biological activity assays, A1-4 not only inhibits the virulence of M. oryzae on host but also causes aggregation of conidia cytosol, which is a characteristic phenotype of MoTps2. Furthermore, hydrogen/deuterium exchange mass spectrometry assays support the notion that A1-4 binds to the substrate pockets of TPS and TPP. Collectively, A1-4 is a promising hit compound for the development of safe fungicide with dual-target specificity.

IDENTIFYING SENOLYTIC TARGETS OF DIETARY FLAVONOIDS IN RENAL CELLS BY THERMAL PROTEOME PROFILING

Abstract Senescent cell accumulation is a known driver of aging and age-related pathologies. Clearance of senescent cells is a promising approach to increase longevity and reduce multiple age-related diseases in humans. Flavonoid compounds are present in fruits and vegetables, of which certain flavonoids (e.g., quercetin and fisetin) have demonstrated senolytic, or ‘senescent-cell-killing’, activity in culture and in mice. However, the cellular mechanism of senolysis and the efficacy of these compounds are not known in renal epithelial cells. Here, we combine flavonoid drug screening, targeted metabolomics, and thermal proteome profiling (TPP) with analysis by mass spectrometry (MS) to explore protein targets of senolytic drugs in senescent human renal cortical epithelial cells and human renal proximal tubular epithelial cells. Senescence was induced by exposure to ionizing radiation and the senescence phenotype was validated through a rigorous panel of senescence and viability markers. Proliferating and senescent renal epithelial cells were screened with a panel of 8 flavonoids to identify senolytic drugs and their concentrations for selectively killing senescent cells. Targeted MS assays were developed to assess intracellular drug uptake, a potential mechanism of senescent-specific killing. To identify protein targets of the senolytic flavonoids in senescent cells, we performed a variation of TPP in combination with liquid chromatography (LC)-MS/MS proteomics analysis, carefully controlling for non-senolytic interactions by excluding proteins bound by a non-senolytic flavonoid. The results of this study pave the way for the development of a more specific generation of senolytic compounds and identify novel candidate senolytic pathways engaged by dietary flavonoids.

Targeting lncRNA16 by GalNAc-siRNA conjugates facilitates chemotherapeutic sensibilization via the HBB/NDUFAF5/ROS pathway.

Chemoresistance is a significant barrier to effective cancer treatment. Potential mechanisms for chemoresistance include reactive oxygen species (ROS) accumulation and expression of chemoresistance-promoting genes. Here, we report a novel function of lncRNA16 in the inhibition of ROS generation and the progression of chemoresistance. By analyzing the serum levels of lncRNA16 in a cohort of 35 patients with non-small cell lung cancer (NSCLC) and paired serum samples pre- and post-treatment from 10 NSCLC patients receiving neoadjuvant platinum-based chemotherapy, performing immunohistochemistry (IHC) assays on 188 NSCLC tumor samples, using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) assays, as well as RNA immunoprecipitation (RIP) and RNA pull-down analyses, we discovered that patients with increased serum levels of lncRNA16 exhibited a poor response to platinum-based chemotherapy. The expression of hemoglobin subunit beta (HBB) and NDUFAF5 significantly increases with the development of chemoresistance. LncRNA16 binds to HBB and promotes HBB accumulation by inhibiting autophagy. LncRNA16 can also inhibit ROS generation via the HBB/NDUFAF5 axis and function as a scaffold to facilitate the colocalization of HBB and NDUFAF5 in the mitochondria. Importantly, preclinical studies in mouse models of chemo-resistant NSCLC have suggested that lncRNA16 targeting by trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNA restores chemosensitivity and results in tumor growth inhibition with no detectable toxicity in vivo. Overall, lncRNA16 is a promising therapeutic target for overcoming chemoresistance, and the combination of first-line platinum-based chemotherapy with lncRNA16 intervention can substantially enhance anti-tumor efficacy.

Solid-Phase Extraction at High pH as a Promising Tool for Targeted Isolation of Biologically Active Fractions of Humic Acids.

A search for novel sources of biologically active compounds is at the top of the agenda for biomedical technologies. Natural humic substances (HSs) contain a large variety of different chemotypes, such as condensed tannins, hydrolyzable tannins, terpenoids, lignins, etc. The goal of this work was to develop an efficient separation technique based on solid-phase extraction (SPE) for the isolation of narrow fractions of HS with higher biological activity compared to the initial material. We used lignite humic acid as the parent humic material, which showed moderate inhibition activity toward beta-lactamase TEM 1 and antioxidant activity. We applied two different SPE techniques: the first one was based on a gradient elution with water/methanol mixtures of the humic material sorbed at pH 2, and the second one implied separation by a difference in the pKa value by the use of sequential sorption of HS at pH from 8 to 3. SPE cartridges Bond Elute PPL (Agilent) were used in the fractionation experiments. The first and second techniques yielded 9 and 7 fractions, respectively. All fractions were characterized using high-resolution mass spectrometry and biological assays, including the determination of beta-lactamase (TEM 1) inhibition activity and antioxidant activity. The acidity-based separation technique demonstrated substantial advantages: it enabled the isolation of components, outcompeting the initial material at the first step of separation (sorption at pH 8). It showed moderate orthogonality in separation with regard to the polarity-based technique. Good perspectives are shown for developing a 2D separation scheme using a combination of polarity and acidity-based approaches to reduce structural heterogeneity of the narrow fractions of HS.

Stable isotope-assisted mass spectrometry reveals in vivo distribution, metabolism, and excretion of tire rubber-derived 6PPD-quinone in mice

6PPD-quinone (6PPD-Q) has been identified as a ubiquitous contaminant in the surrounding locality, including air particles, roadside soils, dust, and water. Recently, the prevalence of 6PPD-Q in human urine has accentuated the urgency for investigating its biological fate. To address this, we conducted a stable isotope-assisted high-resolution mass spectrometry (HRMS) assay to unveil the distribution, metabolism, excretion, and toxicokinetic properties of this contaminant in a mouse model. Mice were fed with a single dose of deuterated 6PPD-Q-d5 at human-relevant exposure levels. Results indicated that 6PPD-Q was quickly assimilated and distributed into bloodstream and main organs of mice, with the concentrations reaching peaks under 1 h following administration. Notably, 6PPD-Q was primarily distributed in the adipose tissue, marked by a significant Cmax (p < 0.05), followed by the kidney, lung, testis, liver, spleen, heart, and muscle. In addition, our measurement demonstrated that 6PPD-Q can penetrate the blood-brain barrier of mice within 0.5 h after exposure. The half-lives (t1/2) of 6PPD-Q in serum, lung, kidney, and spleen of mice were measured at 12.7 ± 0.3 h, 20.7 ± 1.4 h, 21.6 ± 5.3 h, and 20.6 ± 2.8 h, respectively. Using HRMS combined with isotope tracing techniques, two novel hydroxylated metabolites of 6PPD-Q in the mice liver were identified for the first time, which provides new insights into its rapid elimination in-vivo. Meanwhile, fecal excretion was identified as the main excretory pathway for 6PPD-Q and its hydroxylated metabolites. Collectively, our findings extend the current knowledge on the biological fate and exposure status of 6PPD-Q in a mouse model, which has the potential to be extrapolated to humans.

Simultaneous determination of five compounds of fried Radix Paeoniae Alba extract in beagle dogs plasma by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry and its application in a pharmacokinetic study.

In this present study, we developed a reliable and simple ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for the simultaneous quantification of paeoniflorin, albiflorin, oxypaeoniflorin, benzoylpaeoniflorin and isomaltopaeoniflorin in beagle dog plasma. We also analyzed the pharmacokinetics of those components after oral administration of fried Radix Paeoniae Alba (FRPA) in beagle dogs. Plasma samples were processed by protein precipitation with methanol. Chromatographic separation was performed with a Waters HSS-T3 C18 column (100 × 2.1mm, 1.8μm, kept at 40°C) using multiple reaction monitoring mode. A gradient elution procedure was used with solvent A (0.02% formic acid-water) and solvent B (0.02% formic acid-acetonitrile) as mobile phases. Method validation was performed as US Food and Drug Administration guidelines, and the results met the acceptance criteria. The method we establish in this experiment was successfully applied to the pharmacokinetic study after oral administration of FRPA extract to beagle dogs.

Development and validation of a liquid chromatography tandem mass spectrometry assay for the analysis of bedaquiline and M2 in breast milk

ObjectiveTo develop and validate an assay for the analysis of bedaquiline and its M2 metabolite in human breast milk. MethodsThe analytes were extracted using solid phase extraction following protein precipitation. Quantification was performed with liquid chromatography coupled with tandem mass spectrometry. Chromatographic separation was achieved using gradient chromatography on a Poroshell 120 SB-C18 analytical column at 40 °C, with a flow rate of 350 µL/minute and a total run time of eight minutes. An AB Sciex 3000 mass spectrometer with electrospray ionization in the positive mode was used for detection, employing multiple reaction monitoring scan mode. Bedaquiline-d6 and M2-d3-13C were used as internal standards. ResultsCalibrations curves for bedaquiline and M2 exhibited quadratic (weighted 1/x concentration) regressions over the respective concentration ranges of 0.0780 to 5.00 µg/mL and 0.0312 to 2.00 µg/mL. Inter- and intra-day validation accuracies ranged between 96.7 % and 103.5 % for bedaquiline, and 104.2 % to 106.5 % for M2, with a coefficient of variation below 9.2 % for both compounds. ConclusionThe developed assay demonstrated selectivity and robustness, enabling differentiation between bedaquiline and M2 within the context of endogenous compounds from six separate lots of breast milk samples. Successful application was observed in the analysis of breast milk samples sourced from patients treated for multidrug-resistant tuberculosis within a clinical study setting.

Differential roles for the oxygen sensing enzymes PHD1 and PHD3 in the regulation of neutrophil metabolism and function

Background Neutrophils are essential in the early innate immune response to pathogens. Harnessing their antimicrobial powers, without driving excessive and damaging inflammatory responses, represents an attractive therapeutic possibility. The neutrophil population is increasingly recognised to be more diverse and malleable than was previously appreciated. Hypoxic signalling pathways are known to regulate important neutrophil behaviours and, as such, are potential therapeutic targets for regulating neutrophil antimicrobial and inflammatory responses. Methods We used a combination of in vivo and ex vivo models, utilising neutrophil and myeloid specific PHD1 or PHD3 deficient mouse lines to investigate the roles of oxygen sensing prolyl hydroxylase enzymes in the regulation of neutrophilic inflammation and immunity. Mass spectrometry and Seahorse metabolic flux assays were used to analyse the role of metabolic shifts in driving the downstream phenotypes. Results We found that PHD1 deficiency drives alterations in neutrophil metabolism and recruitment, in an oxygen dependent fashion. Despite this, PHD1 deficiency did not significantly alter ex vivo neutrophil phenotypes or in vivo outcomes in mouse models of inflammation. Conversely, PHD3 deficiency was found to enhance neutrophil antibacterial properties without excessive inflammatory responses. This was not linked to changes in the abundance of core metabolites but was associated with increased oxygen consumption and increased mitochondrial reactive oxygen species (mROS) production. Conclusions PHD3 deficiency drives a favourable neutrophil phenotype in infection and, as such, is an important potential therapeutic target.

CircGPRC5A enhances colorectal cancer progress by stabilizing PPP1CA and inducing YAP dephosphorylation

BackgroundWith the advancements in bioinformatic technology, an increasing number of circular RNAs (circRNAs) have been discovered and their crucial roles in the development and progression of various malignancies have been confirmed through multiple pathways. However, the specific mechanisms involving protein-binding circRNAs in colorectal cancer (CRC) remain largely unexplored.MethodsDifferential circRNA expression was assessed using a human circRNA microarray in five CRC tissue and paired normal samples. CircGPRC5A expression was then confirmed in the CRC tissues and paired normal samples using qRT-PCR. The biological function of circGPRC5A in CRC were studied in vitro and in vivo. Western blotting, fluorescence in situ hybridization, immunofluorescence, RNA pulldown, mass spectrometry, immunoprecipitation, quantitative phosphoproteomics, and RNA-binding protein immunoprecipitation assays were used to study circGPRC5A.ResultsOur analysis revealed that circGPRC5A expression was higher in CRC tissues compared to normal tissues and was associated with tumor size, tumor stage and lymph node status. CircGPRC5A promoted CRC cell proliferation, migration, and metastasis in vitro and in vivo. CircGPRC5A could stabilize PPP1CA protein by inhibiting the binding between UBA1 and PPP1CA, and increasing YAP dephosphorylation.ConclusionsOur study revealed that circGPRC5A plays an essential function in CRC progression by stabilizing PPP1CA protein and enhancing YAP dephosphorylation. CircGPRC5A could act as a novel and potential target for CRC.

Use of volumetric absorptive microsampling and parallel reaction monitoring mass spectrometry for tacrolimus blood trough measurements at home in pediatric heart transplant patients

BackgroundMeasurement of trough levels for calcineurin inhibitors by venipuncture sampling is a mainstay of patient management in solid organ transplant recipients but challenging in pediatric patients. Volumetric Absorptive Microsampling (VAMS) is a patient-friendly, minimally invasive sampling technique to accurately collect blood. An assay for measurement of tacrolimus in blood using VAMS, coupled with parallel reaction monitoring (PRM) mass spectrometry, was validated in pediatric heart transplant patients. MethodsTacrolimus was measured by a newly developed high-resolution PRM assay and compared with low-resolution tandem mass spectrometry (MRM). Dried blood samples were collected from pediatric heart transplant patients (n = 35) using VAMS devices and a satisfaction survey was completed by patients/guardians. Tacrolimus concentrations were compared across whole liquid blood, dried blood spots, and capillary blood, and shipping stability determined. ResultsThe PRM assay was linear over a range 1–50 ng/mL, similar to MRM but had greater specificity due to reduced background noise. No significant differences in tacrolimus concentrations were observed between VAMS and venous blood. Tacrolimus dried on VAM tips was stable for 14 days and concentrations were unaffected by postal shipping. The variability in two simultaneously collected at-home patient samples was minimal – average concentration difference was 0.12 ± 0.94 ng/mL (p = 0.6) between paired samples. ConclusionA high resolution PRM mass spectrometry assay was developed for home-based dried blood collections for therapeutic monitoring of tacrolimus. The advantage of PRM was enhanced specificity and the VAMS devices provided a simple and convenient approach to blood sampling at home in pediatric heart transplant patients.

USP7 promotes non-small-cell lung cancer cell glycolysis and survival by stabilizing and activating c-Abl.

Abelson tyrosine kinase (c-Abl) is frequently mutated and highly expressed, and promotes non-small-cell lung cancer (NSCLC) survival, metastasis and tumorigenesis. c-Abl could also be modified through ubiquitination, but the underlying mechanism is not well understood. Mass spectrometry assays were performed to search c-Abl deubiquitination enzymes. The molecular mechanism was determined using Co-IP assays, pull-down assays, Western blotting upon gene knockdown or overexpression. Cell lines and animal models were used to investigate the role of c-Abl and USP7 in NSCLC. EdU staining assay and Transwell assay were performed to evaluate the proliferation and migration ability of NSCLC cells, respectively. Ubiquitin-specific protease 7 (USP7) is found to upregulate c-Abl via the deubiquitinase screen. USP7 interacts with c-Abl and decreases its K48-linked polyubiquitination, thereby increasing the stability of c-Abl. In addition to the wild-type one, c-Abl mutants can also be deubiquitinated and stabilized by USP7. Moreover, USP7 promotes c-Abl accumulation in cytoplasm by increasing its binding to 14-3-3α/β and activates the oncogenic c-Abl signalling pathway. Furthermore, the USP7/c-Abl axis promotes NSCLC cell glycolysis by direct phosphorylating and stabilizing hexokinase-2 (HK2). Knockdown of USP7 or c-Abl suppresses NSCLC cell glycolysis and reduces lactate production. Further studies revealed that overexpression of USP7 facilitates NSCLC cell growth and metastasis as well as xenograft growth in nude mice, while these activities are suppressed with USP7 or c-Abl being knocked down. USP7 is a deubiquitinase of c-Abl and upregulates its oncogenic activity. USP7 promotes NSCLC cell metabolism by activating c-Abl and HK2. Targeting the USP7/c-Abl/HK2 axis might be a potential strategy to the precision therapy of NSCLC.

Multiomics Blood Test for Increasing Asymptomatic AD Patients Proportion in Preventive Clinical Trials

AbstractBackgroundWith the first positive phase III trials for anti‐amyloid approaches in MCI participants, the drug evaluation during the asymptomatic Alzheimer’s disease (AD) stage is now more relevant than ever. Currently, cognitively unimpaired participants are included in preventive trials based on risk factors such as amyloid positivity. The lifetime risks for an individual without cognitive impairment does not exceed 30% for a 65‐year‐old with amyloidosis alone (Brookmeyer et al., 2018). Based on this risk factor, most participants included will never develop AD and therefore will not respond to anti‐AD therapies. There is a need to develop new biomarkers with high specificity predicting which individuals in the cognitively unimpaired population will exhibit AD symptoms and which will not to conduct effective preventive clinical trials.MethodTargeted mass spectrometry assays were developed for 81 blood biomarkers (45 proteins and 36 metabolites) pre‐identified in AAV‐AD rats (Audrain et al. 2018). 287 participants were collected in plasma at baseline and followed clinically from 1 to 18 years. Participants’ diagnosis (AD, or Healthy Controls) was established at the last clinical visit based on reference diagnosis standard. 73.9% of participants had available amyloid status, 42.9% were determined as amyloid positive. Predictive machine learning models for asymptomatic AD participants (n = 48, followed for an average of 6.1 years) among individuals without cognitive decline (HC, n = 239, followed for an average of 4.6 years) was developed. The training dataset (70%) aimed to select the biomarkers subset, train the algorithm, and define the cut‐off. External test dataset (30%) aimed to validate in blind the locked predictive model.ResultWith 20 blood biomarkers and 2 covariates (age and gender), the model predicted asymptomatic AD (n = 16) from HC participants (n = 72) with 81.9% specificity and 56.3% sensitivity during external validation (AUROC = 69.1%, p = 0.02). When current amyloid tests (CSF or PET) and this predictive ML model were applied in series, and participants positive for both were predicted as asymptomatic AD, 90.9% specificity and 54.5% sensitivity were achieved.ConclusionMultiomics blood peripheral biomarkers predict asymptomatic AD patients among cognitively unimpaired individuals with high specificity. This could constitute an additional tool for the inclusion of participants in preventive clinical trials.

Development of a targeted proteomic assay for the quantitation of neurofilament light in cerebrospinal fluid: Correlation with Simoa‐based quantitation across neurodegenerative diseases and centres

AbstractBackgroundCerebrospinal fluid (CSF) neurofilament light (NfL) is a dynamic biomarker of neurodegeneration that is used for supporting clinical diagnosis and monitoring disease‐modifying therapies. Development of new analytical tests has been limited, with quantitation of NfL in biomarker studies largely restricted to immunoassay methods. Targeted mass spectrometry is a highly specific technique that can be easily translated into existing proteomic and clinical pathology laboratories. In this multi‐centre study, we have developed two targeted mass spectrometry assays which were compared to Simoa, the gold standard method for NfL quantitation. The developed methods included immunocapture of intact NfL protein or specific peptides (immune‐peptide capture) prior to targeted proteomic analyses. We discuss the development, validation and merits of each technology to quantitate NfL in CSF from healthy controls and several neurodegenerative diseases.MethodCSF was collected from patients with a range of neurological diseases at the National Hospital for Neurology and Neurosurgery (London, UK). Clinical diagnosis was determined by their treating clinician according to consensus clinical criteria. NfL concentrations were measured by Simoa (Quanterix, USA) and two immunoprecipitation‐targeted mass spectrometry (IP‐MS) assays developed by a collaboration between UCL and Washington University in St Louis. To evaluate replication of results across centres and mass spectrometry approaches, results were compared using Spearman’s correlation analysis and Bland‐Altman testing.ResultsA high correlation was observed for CSF NfL levels between Simoa and both IP‐MS assays (immune‐peptide capture, r = 0.90, P&lt;0.0001 and immune‐protein capture, r = 0.88, P&lt;0.0001). Relative differences in mean NfL concentration between clinical groups remained the same across the analytical methods, with the highest and lowest NfL concentrations observed in the Huntington’s disease and the healthy control groups respectively. Interestingly, differences in the extent of correlation between Simoa and IP‐MS were observed between the different clinical groups (r = 0.37 ‐ 0.94).ConclusionImmunoprecipitation‐targeted mass spectrometry quantitation of NfL correlates highly with NfL Simoa measures across a range of neurodegenerative and neuroinflammatory diseases, and is replicable between laboratories using different immunoprecipitation‐targeted mass spectrometry approaches.

Blood‐based biomarkers of Alzheimer disease as measured by a mass spectrometry assay are as accurate as CSF tests

AbstractBackgroundEspecially because disease‐modifying treatments (DMTs) for Alzheimer disease (AD) are likely to become more available soon, highly accurate AD blood tests for use in clinical diagnosis are critically needed. Although amyloid PET scans and cerebrospinal fluid (CSF) tests are highly accurate in the identification of individuals with brain amyloidosis, blood tests are likely the only test modality that will enable an adequate scale of AD biomarker testing. Mass spectrometry‐based AD blood tests have consistently demonstrated very high performance.MethodsResearch participants enrolled in studies at the Knight Alzheimer Disease Research Center who underwent amyloid PET and measurement of established CSF biomarkers of AD with Lumipulse automated assays were included. The presence and severity of dementia was evaluated with the Clinical Dementia Rating® (CDR®) and Clinical Dementia Rating Sum of Boxes (CDR‐SB). Plasma samples collected at the same session as CSF underwent analysis for specific phosphorylated and non‐phosphorylated tau species with an immunoprecipitation‐mass spectrometry assay. Plasma T217 occupancy was calculated as the percent of phosphorylated to non‐phosphorylated tau at threonine 217.ResultsThe study cohort included 337 individuals with an average age of 69.8 ± 8.3 years (mean ± standard deviation); 175 (52%) were female, 128 (38%) were APOE ε4 carriers, 112 (33%) were amyloid PET positive, and 50 (15%) were cognitively impaired. Classification accuracy of amyloid PET status was equivalent for plasma T217 occupancy (AUC 0.973) and CSF Aβ42/Aβ40 (AUC 0.970), (p = 0.72 for the difference). Interestingly, plasma T217 occupancy more accurately classified individuals as cognitively impaired (CDR&gt;0) or cognitively unimpaired (CDR 0), with an AUC of 0.876 versus 0.814 for CSF Aβ42/Aβ40 (p = 0.006 for the difference). Further, plasma T217 occupancy was better correlated with dementia severity as measured by the CDR‐SB (Spearman ρ = 0.455 versus ‐0.338 for CSF Aβ42/Aβ40, p = 0.02).ConclusionPlasma T217 occupancy as measured by mass spectrometry had equivalent classification accuracy of amyloid PET status compared to Lumipulse CSF Aβ42/Aβ40. Notably, plasma T217 occupancy was more strongly associated with cognitive impairment and dementia severity than CSF Aβ42/Aβ40. Overall, these data suggest that some AD blood tests have equivalent or better performance characteristics than clinically used CSF tests.

The synaptic biomarker contactin5 differentiates between neurodegenerative dementias

AbstractBackgroundSynapses are crucial for memory formation, and their disruption is among the earliest events in Alzheimer’s disease (AD) pathogenesis. Less is known about synaptic alterations in other types of dementia. Contactin5 (CNTN‐5) is a synaptic protein and one of the neural adhesion molecules. It contributes to synaptic formation, plasticity and is associated with dendritic morphogenesis. A GWAS identified a risk variant (rs 141684 G) in CNTN‐5 gene, which is associated with increased risk of AD development. We aimed to assess whether CNTN‐5 could be a differential diagnostic cerebrospinal fluid (CSF) biomarker for AD from other types of dementia.MethodCNTN‐5 levels in CSF samples were measured with two methods: an in‐house developed immunoassay on the automated ELLA platform, and a multiple reaction monitoring mass spectrometry (MRM) assay. The clinical cohort included controls (n = 27), patients with AD (n = 19), patients with behavioral variant frontotemporal dementia (bvFTD) (n = 18), and patients with dementia with Lewy bodies and Parkinson’s disease dementia (PDD/DLB) (n = 18; table 1). CNTN‐5 results obtained with both methods were correlated using spearman’s correlation. Differences in CNTN‐5 levels between the diagnostic groups were assessed with Kruskal‐Wallis test and corrected with Dunn´s factor for multiple comparison.ResultsAs shown in Figure 1, CNTN‐5 measurements between methods were strongly and positively correlated (rho = 0.58, p&lt;0.001). In AD patients, CNTN‐5 levels were significantly elevated in comparison to bvFTD patients with both assays (both: p&lt;0.05), as well as, compared to patients with DLB/PDD (MRM: p&lt;0.05 and ELLA: p&lt;0.01). There was no significant difference in CNTN‐5 levels between AD and controls, or between bvFTD and PDD/DLB, as shown in figure 2.ConclusionOur results showed that elevated CNTN‐5 is a promising synaptic biomarker, with specificity for AD. As a next step, we would look further into a larger clinical cohort, where associations between CNTN‐5 levels and cognitive stage could be thoroughly studied.

Immunophilin FKB20-2 participates in oligomerization of Photosystem I in Chlamydomonas.

PSI is a sophisticated photosynthesis protein complex that fuels the light reaction of photosynthesis in algae and vascular plants. While the structure and function of PSI have been studied extensively, the dynamic regulation on PSI oligomerization and high light response is less understood. In this work, we characterized a high light-responsive immunophilin gene FKB20-2 (FK506-binding protein 20-2) required for PSI oligomerization and high light tolerance in Chlamydomonas (Chlamydomonas reinhardtii). Biochemical assays and 77-K fluorescence measurement showed that loss of FKB20-2 led to the reduced accumulation of PSI core subunits and abnormal oligomerization of PSI complexes and, particularly, reduced PSI intermediate complexes in fkb20-2. It is noteworthy that the abnormal PSI oligomerization was observed in fkb20-2 even under dark and dim light growth conditions. Coimmunoprecipitation, MS, and yeast 2-hybrid assay revealed that FKB20-2 directly interacted with the low molecular weight PSI subunit PsaG, which might be involved in the dynamic regulation of PSI-light-harvesting complex I supercomplexes. Moreover, abnormal PSI oligomerization caused accelerated photodamage to PSII in fkb20-2 under high light stress. Together, we demonstrated that immunophilin FKB20-2 affects PSI oligomerization probably by interacting with PsaG and plays pivotal roles during Chlamydomonas tolerance to high light.

A Liquid Chromatography-Tandem Mass Spectrometry Assay for Quantification of Ethyl Glucuronide and Ethyl Sulfate in Urine.

Ethyl glucuronide and ethyl sulfate emerged as the biomarkers of choice for detection of ethanol use as the required sample is urine, enabling easy and noninvasive collection. Further, these biomarkers have a longer detection window in urine than blood ethanol. A liquid chromatography-tandem mass spectrometry method was developed and clinically validated using electrospray ionization in negative mode and selected reaction monitoring. A simple dilution was used for sample preparation on 100 microliters of urine. Gradient elution had a run time of 7min. The reportable range was established to be 180-100,000ng/mL for ethyl glucuronide and 50-46,600ng/mL for ethyl sulfate and between-run imprecision was <7% for both analytes.

Mass Spectrometry in Clinical Laboratory: Applications in Therapeutic Drug Monitoring and Toxicology.

Mass spectrometry (MS) coupled with liquid chromatography (LC) or gas chromatography (GC) has been proven to be a powerful platform in research and specialized clinical laboratories for decades. In clinical laboratories, it is used for compound identification and quantification. Due to the ability to provide specific identification, high sensitivity, and simultaneous analysis of multiple analytes (>100) in recent years, application of MS in routine clinical laboratories has increased significantly. Although MS is used in many laboratory areas, therapeutic drug monitoring, drugs of abuse, and clinical toxicology remain the primary focuses of the field. Due to rapid increase in the number of prescription drugs and drugs of abuse (e.g., novel psychoactive substances), clinical laboratories are challenged with developing new MS assays to meet the clinical needs of the patients. We are here to present "off-the-shelf" and "ready-to-use" protocols of recent developments in new assays to help the clinical laboratory community adopt the technology and analysis for the betterment of patient care.

Tandem mass spectrometric assay of N-acetylglucosamine-6-sulfatase for multiplex analysis of mucopolysaccharidosis-IIID in dried blood spots

Previously we developed a multiplex liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay using dried blood spots for all subtypes of mucopolysaccharidoses (MPS) except MPS-IIID. Here we show that the MPS-IIID enzyme N-acetylglucosamine-6-sulfatase (GNS) is inhibited in dried blood spot (DBS) extracts, but activity can be recovered if the extract is diluted to reduce the concentrations of endogenous inhibitors. The new GNS assay displays acceptable characteristics including linearity in product formation with incubation time and amount of enzyme, low variability, and ability to distinguish MPS-IIID-affected from healthy patients using DBS. The assay can be added to the LC-MS/MS multiplex panel for all MPS subtypes requiring ∼2 min per newborn for the LC-MS/MS run.