Monitoring Mass Spectrometry Research Articles

Mapping Reaction Pathways by In Situ Step Sweep Voltammetry Flow Electrochemical Mass Spectrometry.

A step sweep voltammetry (SSV) flow electrochemical (EC) mass spectrometry (MS) platform was developed for real-time and in situ mapping of EC reaction pathways. By integrating a flow EC cell into the pneumatic spray nozzle followed by atmospheric chemical ionization, this setup was capable of in situ MS monitoring of short-lived EC intermediates with enhanced sensitivity. This setup also realized precise measurement and control of the electrode potential during in situ EC-MS analysis, which can provide detailed information on the interplay of reaction pathways under different electrode potentials. Taking the EC reductive cross coupling of nitroarenes with arylboronic acids as an example, SSV-MS had identified 13 compounds among four reaction pathways. Among these, the electrode potential of active nitrene and cross coupling intermediates were measured for the first time and the structure of the nitroso coupling complex was also confirmed by MS. With the systematic measurement of electrode potential of the intermediates and products, SSV-MS had clearly mapped out the synergies and competitions between different reaction pathways, offering key insights for optimizing reaction conditions and investigating reaction mechanisms for EC research.

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<0.001). In AD patients, CNTN‐5 levels were significantly elevated in comparison to bvFTD patients with both assays (both: p<0.05), as well as, compared to patients with DLB/PDD (MRM: p<0.05 and ELLA: p<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.

Targeted Analysis of HSP70 Isoforms in Human Spermatozoa in the Context of Capacitation and Motility.

HSP70s constitute a family of chaperones, some isoforms of which appear to play a role in sperm function. Notably, global proteomic studies analyzing proteins deregulated in asthenozoospermia, a main cause of male infertility characterized by low sperm motility, showed the dysregulation of some HSP70 isoforms. However, to date, no clear trend has been established since the variations in the abundance of HSP70 isoforms differed between studies. The HSPA2 isoform has been reported to play a key role in fertilization, but its dysregulation and possible relocation during capacitation, a maturation process making the spermatozoon capable of fertilizing an oocyte, is debated in the literature. The aim of the present study was to investigate the fate of all sperm HSP70 isoforms during capacitation and in relation to sperm motility. Using Multiple-Reaction Monitoring (MRM) mass spectrometry, we showed that the relative abundance of all detected isoforms was stable between non-capacitated and capacitated spermatozoa. Immunofluorescence using two different antibodies also demonstrated the stability of HSP70 isoform localization during capacitation. We also investigated spermatozoa purified from 20 sperm samples displaying various levels of total and progressive sperm motility. We showed that the abundance of HSP70 isoforms is not correlated to sperm total or progressive motility.

An Optimized MRM-Based Workflow of the l-Arginine/Nitric Oxide Pathway Metabolites Revealed Disease- and Sex-Related Differences in the Cardiovascular Field.

Clinical data indicate that low circulating l-homoarginine (HArg) concentrations are associated with cardiovascular (CV) disease, CV mortality, and all-cause mortality. A high number of LC-based analytical methods for the quantification of HArg, in combination with the l-arginine (Arg)-related pathway metabolites, have been reported. However, these methods usually consider a limited panel of analytes. Thus, in order to achieve a comprehensive picture of the Arg metabolism, we described an improved targeted metabolomic approach based on a multiple reaction monitoring (MRM) mass spectrometry method for the simultaneous quantification of the Arg/nitric oxide (NO) pathway metabolites. This methodology was then employed to quantify the plasma concentrations of these analytes in a cohort of individuals with different grades/types of coronary artery disease (CAD) in order to increase knowledge about the role of HArg and its associated metabolites in the CV field. Our results showed that the MRM method here implemented is suitable for the simultaneous assessment of a wide panel of amino acids involved in the Arg/NO metabolic pathway in plasma samples from patients with CV disease. Further, our findings highlighted an impairment of the Arg/NO metabolic pathway, and suggest a sex-dependent regulation of this metabolic route.

Proton-transfer-reaction mass spectrometry (PTR-MS) for online monitoring of glucose depletion and cell concentrations in HEK 293 gene therapy processes

ObjectivesThe applicability of proton-transfer-reaction mass spectrometry (PTR-MS) as a versatile online monitoring tool to increase consistency and robustness for recombinant adeno-associated virus (rAAV) producing HEK 293 bioprocesses was evaluated. We present a structured workflow to extract process relevant information from PTR-MS data.ResultsReproducibility of volatile organic compound (VOC) measurements was demonstrated with spiking experiments and the process data sets used for applicability evaluation consisted of HEK 293 cell culture triplicates with and without transfection. The developed data workflow enabled the identification of six VOCs, of which two were used to develop a soft sensor providing better real-time estimates than the conventional capacitance sensor. Acetaldehyde, another VOC, provides online process information about glucose depletion that can directly be used for process control purposes.ConclusionsThe potential of PTR-MS for HEK 293 cell culture monitoring has been shown. VOC data derived information can be used to develop soft sensors and to directly set up new process control strategies.

Abstract 1891: Targeted mass spectrometry provides direct evidence of a human endogenous retrovirus type E (HERV-E) antigen presented on the surface of clear cell renal cell carcinoma cells

Abstract Background: Previously, our group identified donor-derived T cells that mediated regression of metastatic clear cell renal cell carcinoma (ccRCC) in a patient who underwent an allogeneic hematopoietic stem cell transplant. A T cell clone isolated from this patient had direct cytotoxicity against ccRCC cells. Its target antigen was discovered to be encoded by a human endogenous retrovirus type E (named CT-RCC HERV-E) that had selective expression in ccRCC. Subsequent experiments showed this T cell clone recognized a cryptic 10-mer peptide called CT-RCC1 (ATFLGSLTWK), derived from a noncanonical open reading frame of the CT-RCC HERV-E and presented in the context of HLA-A11. This study aimed to establish a proteomic workflow using targeted parallel reaction monitoring (PRM) mass spectrometry (MS) for the direct identification of CT-RCC1 peptide in ccRCC HLA ligandomes and to use that platform to identify other CT-RCC HERV-E derived peptides presented in the context of more common HLA alleles such as HLA-A02. Methods: In this study, four ccRCC cell lines were used: HLA-A11 positive/CT-RCC HERV-E positive, HLA-A11 negative/CT-RCC HERV-E positive, HLA-A11 positive/CT-RCC HERV-E negative and HLA-A11 negative/CT-RCC HERV-E negative. Additionally, we used the HLA-A11 positive/CT-RCC HERV-E positive cell line to create both beta 2-microglobulin and CT-RCC HERV-E CRISPR knockout lines. HLA-peptide complexes were immunopurified from cell lysates with anti-HLA Class I antibodies cross-linked to Protein A Sepharose beads. Peptides were separated from HLA molecules using Sep-Pak tC18 reverse phase separation columns and subjected to targeted parallel reaction monitoring (PRM) mass spectrometry (MS) analysis. Synthetic CT-RCC1 peptide was used to generate optimum PRM transitions, determine the best collision energy, and obtain the full fragment ion spectrum. Data analysis was done using the Skyline package. Results: CT-RCC1 peptide was identified by targeted MS to be presented on the surface of HLA-A11 positive/CT-RCC HERV-E positive ccRCC cells but not in those same cells that had CT-RCC HERV-E or beta 2-microglobulin knocked out using CRISPR. Additionally, the CT-RCC1 peptide was not detected in cells which were naturally HLA-A11 negative, CT-RCC HERV-E negative, or both. Conclusions: Targeted MS identified the HERV-E derived HLA-A11 restricted peptide CT-RCC1 to be expressed on the surface of ccRCC cells. This study defines a reproducible immunopeptidomic workflow using targeted MS for identifying other antigens derived from this HERV-E that may be presented in the context of common HLA alleles. Acknowledgments: We acknowledge the NIH Intramural Research Program and Rancic O'Neill Renal Cell Cancer Research Fellowship Fund for supporting this project. Citation Format: Stefan Barisic, Elena Cherkasova, Yong Chen, Stephanie Pierre, Richard W. Childs. Targeted mass spectrometry provides direct evidence of a human endogenous retrovirus type E (HERV-E) antigen presented on the surface of clear cell renal cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1891.

Targeted analysis of eicosanoids derived from cytochrome P450 pathway by high-resolution multiple-reaction monitoring mass spectrometry.

Cytochrome P450 (CYP450) pathway is one of the critical enzymatic via eicosanoid biosynthesis. Nevertheless, their metabolites are far less explored. This pathway plays a crucial role in converting arachidonic acid to hydroxyeicosatetraenoic (HETEs), epoxyeicosatrienoic (EETs), dihydroxyeicosatetraenoic acids (DiHETEs), and dihydroxyeicosatrienoic acids (DiHETrEs), which mediate several physiological and pathological functions. However, CYP450-derived eicosanoids are structurally complex, making those analyses a challenge in lipidomics studies. Herein, a high-resolution multiple-reaction monitoring (MRMHR ) method has been proposed as a powerful tool for the simultaneous analysis of CYP450-eicosanoids on different biological samples. The developed liquid chromatography (LC)-MRMHR method was partially validated according to the Food and Drug Administration (FDA) criteria, demonstrating adequate specificity, linearity, precision, and accuracy. Besides, several biological samples were analyzed to guarantee the feasibility of the method. The proposed strategy may improve the understanding of CYP450-derived eicosanoids in biological systems, which could be fundamental to reveal new aspects of those in physiologic and pathologic conditions.

Microdroplet Mass Spectrometry Enables Extremely Accelerated Pepsin Digestion of Proteins.

In microdroplets, rates of chemical or biomolecular reactions can exceed those in the bulk phase by more than a million times. As electrospray ionization-based mass spectrometry (MS) involves the formation of charged microdroplets, reaction acceleration and online MS monitoring of reaction products can readily be performed at the same time. We investigated accelerated enzymatic reactions in microdroplets and focused on the proteolytic enzyme pepsin. Electrosonic spray ionization (ESSI) was utilized for developing the ultrarapid pepsin in-spray digestion of two different proteins, cytochrome c and RocC, at low pH values. The optimization of the protein digestion aimed at achieving maximum sequence coverage for the analyzed proteins. Furthermore, carefully designed control experiments allowed us to unambiguously prove that enzymatic protein cleavage almost exclusively occurs within the spray at a millisecond time scale and not prior to microdroplet generation.

Detection and Structural Investigation of Elusive Palladium Hydride Intermediates Formed from Simple Metal Salts.

The Mizoroki-Heck reaction is one of the most known and best studied catalytic transformations and has provided an outstanding driving force for the development of catalysis and synthetic applications. Three out of four classical Mizoroki-Heck catalytic cycle intermediates contain Pd-C bonds and are well known and studied in detail. However, a simple palladium hydride (which is formed after the product-releasing β-H-elimination step) is a kind of elusive intermediate in the Mizoroki-Heck reaction. In the present study, we performed a combined theoretical and mass spectrometry (MS) study of palladium hydride complexes [PdX2H]- (X = Cl, Br, and I), which are reactive intermediates in the Mizoroki-Heck reaction. Static and molecular dynamic calculations revealed that these species have a T-shaped structure with a trans-arrangement of halogen atoms. Other isomers of [PdX2H]- are unstable and easily rearrange into the T-shaped form or decompose. These palladium hydride intermediates were detected by MS in precatalyst activation using NaBH4, Et3N, and a solvent molecule as reducing agents. Online MS monitoring allowed the detection of [PdX2H]- species in the course of the Mizoroki-Heck reaction.

Hydrophilic interaction liquid chromatography for removal of pesticide residues in ginseng extracts

亲水作用液相色谱法(HILIC)是一种用于改善强极性物质的保留和分离选择性的方法,广泛应用于药物分析、代谢组学、蛋白质组学等领域。该文利用农药分子与皂苷成分在HILIC上的保留行为差异,开发了一种农药残留脱除方法。以市售高纯人参提取物为例,该文评价了农药分子和人参皂苷在亲水色谱柱上的保留行为,并考察了上样量、淋洗体积、上样体积等因素对农残脱除效果的影响。实验结果证明:7种人参皂苷由于糖链上的羟基与亲水色谱固定相上的羧基形成氢键作用而具有较强保留,而农药分子由于亲水性较差且相对分子质量较小,保留很弱,从而一步实现了7种人参皂苷的富集与14种农残的脱除。在优化所得的最佳脱除工艺条件下,最终制备得到的人参总皂苷样品中,总皂苷的含量由59.87%提高到69.61%;总皂苷的回收率为94.4%;通过气相色谱-三重四极杆质谱(GC-MS/MS)对样品中的农残进行定量检测,发现原人参提取物中14种农残均得到了有效脱除,其中5种含量降至0.05 mg/kg以下,9种完全脱除。本研究是亲水色谱在中药提取物中农残脱除领域的应用,为天然产物的精制提供了一种新的技术手段,该技术对人参提取物中的农残脱除率高、人参总皂苷回收率高且安全、高效、无污染,为高品质人参提取物的研制提供了新的思路。

Development of a Gas Chromatography Tandem Mass Spectrometry-Based Analytical Method for the Quantitative Determination of Organochlorine Pesticide Residues in Potato Crops

A method validation study has been performed to analyze organochlorine pesticide residues in potato and sweet potato using gas chromatography-tandem mass spectrometry in a selective ion reaction monitoring (SRM) mode of GC-MS/MS scans. The method (QuEChERS) has been validated in compliance with the European SANTE. The validation parameters were assessed to verify the method’s fitness for the targeted analysis, and the obtained results were observed in the range of the criteria mentioned in the validation guidelines. The optimized method was utilized for the organochlorine pesticide analysis in potatoes and sweet potatoes. The analyzed samples were minimal in their pesticide content as per the standards; it has shown great effectiveness by offering excellent results in terms of accuracy (70-120 % recovery) and precision (<20 %), and retention time (13.5 minutes). As the analysis was well linearized with a regression coefficient of >0.99, it holds a much-reduced maximum relative standard deviation (RSD) of <6 %. The method can be utilized for the twentytwo organochlorine pesticides in potato and sweet potato.

Abstract LB-232: Deep integrated proteogenomic characterization of clear cell renal cell carcinoma

Abstract Clear cell renal cell carcinoma (ccRCC) is the predominant histology of renal cell carcinoma (RCC), representing 75% of all cases and accounting for the majority of associated deaths. Towards understanding the impact of genomic alterations on the functional modules that drive ccRCC tumorigenesis, the Clinical Proteomics Tumor Analysis Consortium (CPTAC) leveraged a proteogenomic approach to characterize 110 RCC tumors and 84 paired normal adjacent tissue (NAT) samples. Pairing state-of-the-art mass spectrometry based proteomic and phosphoproteomic strategies with comprehensive genomic and transcriptomic analyses, we integrated global and phosphoproteomic measurements with genomic and transcriptomic data to elucidate the dysregulated cellular mechanism resulting from genomic alterations. In addition to delineating chromosomal translocations involving chromosome 3p as a possible mechanism for 3p loss in ccRCC and the altered cellular pathways between tumors and NATs, we also identified several novel proteomic, phosphoproteomic, and immune ccRCC subtypes for personalized, precision-based care. Investigation of the phosphoproteome in our cohort revealed the activation of cell signaling pathways associated with ERK/MAPK, PI3K/AKT, metabolic reprogramming, and G2/M cell cycle regulation. Leveraging FDA-approved kinase inhibitors, prioritized by our phosphoproteomic results, allowed for the examination of the functional consequences of these therapies respective to mechanisms of action in ccRCC cells. Independently, the immunogenicity of ccRCC tumors prompted our exploration of the degree of immune infiltration in our cohort, wherein we identified four subtypes based on immune and stromal tumor microenvironment cell compositions: CD8+ Inflamed, CD8- Inflamed, VEGF Immune Desert, and Metabolic Immune Desert. These subtypes were further discriminated by proteomic and transcriptomic features, as well as displaying distinct predicted outcomes related to patient survival and response to targeted therapeutic intervention. We verified the protein features of these subtypes via orthogonal methodologies, including data-independent acquisition (DIA) and selective reaction monitoring (SRM) mass spectrometry, finding a high degree of correlation between the datasets. Further examination of these proteomic features in an independent cohort found the subtypes robustly identified; providing further evidence of ccRCC inter-tumor heterogeneity and rationale to stratify ccRCC patients for treatment selection. Overall, our study highlights the unique insight that can be gained when integrating multi-level “omics” analyses to broaden our understanding of ccRCC biology, as well as leading to the identification of several new hypothesis-driven investigations for therapeutic intervention in ccRCC. Citation Format: David J. Clark, Clinical Proteomic Tumor Analysis Consortium. Deep integrated proteogenomic characterization of clear cell renal cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-232.

Multiplexed MRM-based protein quantification of putative prognostic biomarkers for chronic kidney disease progression in plasma

Current diagnostic measures for Chronic Kidney Disease (CKD) include detection of reduced estimated glomerular filtration rate (eGFR) and albuminuria, which have suboptimal accuracies in predicting disease progression. The disease complexity and heterogeneity underscore the need for multiplex quantification of different markers. The goal of this study was to determine the association of six previously reported CKD-associated plasma proteins [B2M (Beta-2-microglobulin), SERPINF1 (Pigment epithelium-derived factor), AMBP (Protein AMBP), LYZ (Lysozyme C), HBB (Hemoglobin subunit beta) and IGHA1 (Immunoglobulin heavy constant alpha 1)], as measured in a multiplex format, with kidney function, and outcome. Antibody-free, multiple reaction monitoring mass spectrometry (MRM) assays were developed, characterized for their analytical performance, and used for the analysis of 72 plasma samples from a patient cohort with longitudinal follow-up. The MRM significantly correlated (Rho = 0.5–0.9) with results from respective ELISA. Five proteins [AMBP, B2M, LYZ, HBB and SERPINF1] were significantly associated with eGFR, with the three former also associated with unfavorable outcome. The combination of these markers provided stronger associations with outcome (p < 0.0001) compared to individual markers. Collectively, our study describes a multiplex assay for absolute quantification and verification analysis of previously described putative CKD prognostic markers, laying the groundwork for further use in prospective validation studies.

Changes in plasma bile acid profiles after partial internal biliary diversion in PFIC2 patients.

BackgroundWe ask if plasma bile acid profiles can be used to monitor the effectiveness of partial internal biliary diversion (PIBD) for treating uncontrolled cholestasis in progressive familial intrahepatic cholestasis type 2 (PFIC2) patients.MethodsPlasma bile acids were profiled in 3 cases of ATP-binding cassette, sub-family B member 11 (ABCB11)-mutated PFIC2 children before and after PIBD compared to healthy controls and 8 PFIC2 patients. The quantitation of bile acids was performed by reversed-phase ultrahigh-performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC/MRM-MS) with negative ion detection.ResultsBefore PIBD, all three patients presented with >50-fold higher levels of total plasma bile acids, 2–7 folds higher ratios of taurine: glycine conjugated primary bile acids, and unchanged secondary bile acids levels compared to healthy controls. After PIBD, only one of the three patients (P3) showed relief of cholestasis. The bile acid profiles of the two nonresponding patients showed little change while that of the responding patient showed a 5-fold reduction in total plasma primary bile acids, a reduced taurine: glycine conjugate ratio, and an unexpected 26- and 12-fold increase in secondary bile acids DCA and LCA respectively. One year later, the responder suffered a recurrence of cholestasis, and the bile acid profile shifted back to a more pre-PIBD-like profile.ConclusionsPlasma bile acid profiles may potentially be useful as sensitive biomarkers for monitoring the clinical course of PIBD patients. Relief of cholestasis after PIBD appears to be associated with significantly increased circulating toxic secondary bile acids and this may limit the utility of PIBD in PFIC2 patients in the long run.

Determination of coriatin and corianin in plasma and urine using ultra-performance liquid chromatography-triple quadrupole mass spectrometry

An ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS) method has been developed for the determination of coriatin and corianin in plasma and urine, which are the biomarkers of poisoning caused by Coriaria sinica Maxim. Plasma and urine samples were extracted and purified using a solid supported liquid/liquid extraction method. Chromatographic separation was performed on a Cortecs C18 column (100 mm×2.1 mm, 1.6 μm) using a gradient elution of methanol and water. Coriatin and corianin were detected using negative electrospray ionization tandem mass spectrometry in multiple reaction monitoring (MRM) mode and quantified via a matrix working standard curve internal standard method; florfenicol was used as the internal standard. The assay was linear in the calibration range of 0.03-5.0 μg/L for coriatin and 0.3-50 μg/L for corianin in plasma, and 0.1-10 μg/L and 1-100 μg/L for coriatin and corianin in urine, respectively. The average recoveries were 86.2%-110% for coriatin and corianin in plasma and urine with relative standard deviations of 5.1%-14.6% (n=6). The limits of detection (S/N=3) for coriatin and corianin were 0.01 μg/L and 0.1 μg/L in plasma, and 0.03 μg/L and 0.3 μg/L in urine, respectively. The method is simple, sensitive and accurate for the determination of coriatin and corianin in plasma and urine for toxicological purposes.

MALDI-TOF MS monitoring of PBMC activation status in sepsis

BackgroundMALDI-TOF mass spectrometry (MS) on whole cells enables the detection of different cell types and cell activation. Here, we wondered whether this approach would be useful to investigate the host response in sepsis.MethodsPeripheral blood mononuclear cells (PBMCs) from patients with severe sepsis and healthy donors were analyzed with MALDI-TOF MS. PBMCs from healthy donors were also stimulated with lipopolysaccharide, peptidoglycan, CpG oligonucleotides, polyinosinic polycytidylic acid, and with heat-inactivated bacteria. Averaged spectra of PBMCs stimulated in vitro by different agonists were generated from the database using the Biotyper software and matching scores between each spectrum from patients and averaged spectra from the database were calculated.ResultsWe show that the MALDI-TOF MS signature of PBMCs from septic patients was specific, compared with healthy controls. As the fingerprints observed in patients may be related to PBMC activation, PBMCs from healthy controls were stimulated with cytokines, soluble Pathogen-Associated Molecular Patterns (PAMPs) and heat-killed bacteria, and we created a database of reference spectra. The MALDI-TOF MS profiles of PBMCs from septic patients were then compared with the database. No differences were found between patients with documented infection (n = 6) and those without bacteriological documentation (n = 6). The spectra of PBMCs from septic patients matched with those of interferon-γ- and interleukin-10-stimulated PBMCs, confirming that sepsis is characterized by both inflammatory and immunoregulatory features. Interestingly, the spectra of PBMCs from septic patients without documented infection matched with the reference spectrum of PBMCs stimulated by CpG oligonucleotides, suggesting a bacterial etiology in these patients.ConclusionsDespite the limits of this preliminary study, these results indicate that the monitoring of functional status of PBMCs in peripheral blood by whole cell MALDI-TOF MS could provide unique opportunities to assess disease progression or resolution in clinical settings.

A Convenient and Sensitive LC-MS/MS Method for Simultaneous Determination of Carbadox- and Olaquindox-Related Residues in Swine Muscle and Liver Tissues

This paper presents a convenient and sensitive LC-MS/MS method for the simultaneous determination of carbadox and olaquindox residues, including desoxyolaquindox (DOLQ), desoxycarbadox (DCBX), quinoxaline-2-carboxylic acid (QCA), 3-methyl-quinoxaline-2-carboxylic acid (MQCA), and the glycine conjugates of QCA and MQCA (namely, QCA-glycine and MQCA-glycine, resp.) in swine muscle and liver tissues. Tissue samples were extracted with 2% metaphosphoric acid in 20% methanol and cleaned up by solid-phase extraction (SPE) on a mixed-mode anion-exchange column (Oasis MAX). Analysis was performed on a C18 column by detection with mass spectrometry in the multiple reaction monitoring (MRM) mode. The limits of detection (LODs) of the six analytes were determined to be 0.01 μg·kg−1 to 0.25 μg·kg−1, and the limits of quantification (LOQs) were 0.02 μg·kg−1 to 0.5 μg·kg−1. The total recoveries of the six analytes in all tissues were higher than 79.1% with the RSD% less than 9.2%. The developed method can determine the real residue level of QCA and MQCA, whether they are present in free form or as glycine conjugates in tissues, together with the carcinogenic desoxy metabolites DCBX and DOLQ with high recovery. Therefore, this method was suitable for routine analysis of residue control programmes and the residue depletion study of CBX and OLQ on swine.

Comprehensive bile acid profiling in hereditary intrahepatic cholestasis: Genetic and clinical correlations.

Genetic defects causing dysfunction in bile salt export pump (BSEP/ABCB11) lead to liver diseases. ABCB11 mutations alter the bile acid metabolome. We asked whether profiling plasma bile acids could reveal compensatory mechanisms and track genetic and clinical status. We compared plasma bile acids in 17 ABCB11-mutated patients, 35 healthy controls and 12 genetically undiagnosed cholestasis patients by ultra-high-performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC/MRM-MS). We developed an index to rank bile acid hydrophobicity, and thus toxicity, based on LC retention times. We recruited 42 genetically diagnosed hereditary cholestasis patients, of whom 12 were presumed to have impaired BSEP function but carried mutations in genes other than ABCB11, and 8 healthy controls, for further verification. The overall hydrophobicity indices of total bile acids in both the ABCB11-mutated group (11.89±1.07min) and the undiagnosed cholestasis group (11.46±1.07min) were lower than those of healthy controls (13.69±0.77min) (both p<0.005). This was owing to increased bile acid modifications. Secondary bile acids were detected in patients without BSEP expression, suggesting biliary bile acid secretion through alternative routes. A diagnostic panel comprising lithocholic acid (LCA), tauro-LCA, glyco-LCA and hyocholic acid was identified that could differentiate the ABCB11-mutated cohort from healthy controls and undiagnosed cholestasis patients (AUC=0.946, p<0.0001) and, in non-ABCB11-mutated cholestasis patients, could distinguish BSEP dysfunction from normal BSEP function (9/12 vs 0/38, p<0.0000001). Profiling of plasma bile acids has provided insights into cholestasis alleviation and may be useful for the clinical management of cholestatic diseases.

Choline and its metabolites are differently associated with cardiometabolic risk factors, history of cardiovascular disease, and MRI-documented cerebrovascular disease in older adults ,

Background: There is a potential role of choline in cardiovascular and cerebrovascular disease through its involvement in lipid and one-carbon metabolism.Objective: We evaluated the associations of plasma choline and choline-related compounds with cardiometabolic risk factors, history of cardiovascular disease, and cerebrovascular pathology.Design: A cross-sectional subset of the Nutrition, Aging, and Memory in Elders cohort who had undergone MRI of the brain (n = 296; mean ± SD age: 73 ± 8.1 y) was assessed. Plasma concentrations of free choline, betaine, and phosphatidylcholine were measured with the use of liquid-chromatography-stable-isotope dilution-multiple-reaction monitoring-mass spectrometry. A volumetric analysis of MRI was used to determine the cerebrovascular pathology (white-matter hyperintensities and small- and large-vessel infarcts). Multiple linear and logistic regression models were used to examine relations of plasma measures with cardiometabolic risk factors, history of cardiovascular disease, and radiologic evidence of cerebrovascular pathology.Results: Higher concentrations of plasma choline were associated with an unfavorable cardiometabolic risk-factor profile [lower high-density lipoprotein (HDL) cholesterol, higher total homocysteine, and higher body mass index (BMI)] and greater odds of large-vessel cerebral vascular disease or history of cardiovascular disease but lower odds of small-vessel cerebral vascular disease. Conversely, higher concentrations of plasma betaine were associated with a favorable cardiometabolic risk-factor profile [lower low-density lipoprotein (LDL) cholesterol and triglycerides] and lower odds of diabetes. Higher concentrations of plasma phosphatidylcholine were associated with characteristics of both a favorable cardiometabolic risk-factor profile (higher HDL cholesterol, lower BMI, lower C-reactive protein, lower waist circumference, and lower odds of hypertension and diabetes) and an unfavorable profile (higher LDL cholesterol and triglycerides).Conclusion: Choline and its metabolites have differential associations with cardiometabolic risk factors and subtypes of vascular disease, thereby suggesting differing roles in the pathogenesis of cardiovascular and cerebral large-vessel disease compared with that of small-vessel disease.

Rapid mass spectrometry analysis of a rectilinear ion trap by continuous secular frequency scanning.

Secular frequency scanning is a mass spectrometry (MS) analysis method in which the frequency of the auxiliary alternating current (AC) signal is scanned. It has low requirements for radio-frequency (RF) power, which is beneficial for the miniaturization of the mass spectrometer. In this study, the MS performance in the reverse secular frequency scanning (RSFS) mode is optimized for a rectilinear ion trap (RIT), and a method for rapid MS analysis using continuous secular frequency scanning (CSFS) is proposed. A RIT mass spectrometer with an auxiliary AC frequency scanning function was built. The resolution, tandem mass spectrometry (MS/MS) and quantitation capability in the RSFS mode were characterized and optimized. Operation in the CSFS mode was then performed by scanning the frequency of the auxiliary AC signal continuously and periodically while maintaining the RF signal and the front Z electrode in the ion injection state, so that the ion injection and cooling were performed at the same time as the mass analysis. With this system, the RSFS mode achieved unit mass resolution at 332 Th, and the MS/MS analysis was completed without changing the RF amplitude at q=0.4583 for reserpine. The limit of quantitation for imatinib was about 250ng/mL with the determination coefficient R2 =0.9981. In the CSFS mode, a single analysis cycle of less than 20ms could be achieved, which is 14 times faster than the traditional sweep modes. In addition, 100% ion utilization can theoretically be achieved in the CSFS mode. The CSFS mode is different from the traditional phased sequential operation mode of an ion trap mass spectrometer. By periodic scanning of the auxiliary AC frequency while maintaining ion injection, it is possible to improve the analysis efficiency of the mass spectrometer, which has the prospect of useful application in the field of rapid MS monitoring. Copyright © 2017 John Wiley & Sons, Ltd.