Monitoring of self-healing composites: a nonlinear ultrasound approach

NASA Astrophysics Data System (ADS)

Malfense Fierro, Gian-Piero; Pinto, Fulvio; Dello Iacono, Stefania; Martone, Alfonso; Amendola, Eugenio; Meo, Michele

2017-11-01

Self-healing composites using a thermally mendable polymer, based on Diels-Alder reaction were fabricated and subjected to various multiple damage loads. Unlike traditional destructive methods, this work presents a nonlinear ultrasound technique to evaluate the structural recovery of the proposed self-healing laminate structures. The results were compared to computer tomography and linear ultrasound methods. The laminates were subjected to multiple loading and healing cycles and the induced damage and recovery at each stage was evaluated. The results highlight the benefit and added advantage of using a nonlinear based methodology to monitor the structural recovery of reversibly cross-linked epoxy with efficient recycling and multiple self-healing capability.

Quantification of Peptides from Immunoglobulin Constant and Variable Regions by Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry for Assessment of Multiple Myeloma Patients

PubMed Central

Remily-Wood, Elizabeth R.; Benson, Kaaron; Baz, Rachid C.; Chen, Y. Ann; Hussein, Mohamad; Hartley-Brown, Monique A.; Sprung, Robert W.; Perez, Brianna; Liu, Richard Z.; Yoder, Sean; Teer, Jamie; Eschrich, Steven A.; Koomen, John M.

2014-01-01

Purpose Quantitative mass spectrometry assays for immunoglobulins (Igs) are compared with existing clinical methods in samples from patients with plasma cell dyscrasias, e.g. multiple myeloma. Experimental design Using LC-MS/MS data, Ig constant region peptides and transitions were selected for liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM). Quantitative assays were used to assess Igs in serum from 83 patients. Results LC-MRM assays quantify serum levels of Igs and their isoforms (IgG1–4, IgA1–2, IgM, IgD, and IgE, as well as kappa(κ) and lambda(λ) light chains). LC-MRM quantification has been applied to single samples from a patient cohort and a longitudinal study of an IgE patient undergoing treatment, to enable comparison with existing clinical methods. Proof-of-concept data for defining and monitoring variable region peptides are provided using the H929 multiple myeloma cell line and two MM patients. Conclusions and Clinical Relevance LC-MRM assays targeting constant region peptides determine the type and isoform of the involved immunoglobulin and quantify its expression; the LC-MRM approach has improved sensitivity compared with the current clinical method, but slightly higher interassay variability. Detection of variable region peptides is a promising way to improve Ig quantification, which could produce a dramatic increase in sensitivity over existing methods, and could further complement current clinical techniques. PMID:24723328

Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers.

PubMed

Chen, Yi-Ting; Chen, Hsiao-Wei; Wu, Chun-Feng; Chu, Lichieh Julie; Chiang, Wei-Fang; Wu, Chih-Ching; Yu, Jau-Song; Tsai, Cheng-Han; Liang, Kung-Hao; Chang, Yu-Sun; Wu, Maureen; Ou Yang, Wei-Ting

2017-05-01

Multiple (selected) reaction monitoring (MRM/SRM) of peptides is a growing technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplex-capable than antibody-based techniques. The technique has been applied clinically to the large-scale quantification of multiple target proteins in different types of fluids. However, previous MRM-based studies have placed less focus on sample-preparation workflow and analytical performance in the precise quantification of proteins in saliva, a noninvasively sampled body fluid. In this study, we evaluated the analytical performance of a simple and robust multiple reaction monitoring (MRM)-based targeted proteomics approach incorporating liquid chromatography with mass spectrometry detection (LC-MRM/MS). This platform was used to quantitatively assess the biomarker potential of a group of 56 salivary proteins that have previously been associated with human cancers. To further enhance the development of this technology for assay of salivary samples, we optimized the workflow for salivary protein digestion and evaluated quantification performance, robustness and technical limitations in analyzing clinical samples. Using a clinically well-characterized cohort of two independent clinical sample sets (total n = 119), we quantitatively characterized these protein biomarker candidates in saliva specimens from controls and oral squamous cell carcinoma (OSCC) patients. The results clearly showed a significant elevation of most targeted proteins in saliva samples from OSCC patients compared with controls. Overall, this platform was capable of assaying the most highly multiplexed panel of salivary protein biomarkers, highlighting the clinical utility of MRM in oral cancer biomarker research. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Efficient use of retention time for the analysis of 302 drugs in equine plasma by liquid chromatography-MS/MS with scheduled multiple reaction monitoring and instant library searching for doping control.

PubMed

Liu, Ying; Uboh, Cornelius E; Soma, Lawrence R; Li, Xiaoqing; Guan, Fuyu; You, Youwen; Chen, Jin-Wen

2011-09-01

Multiple drug target analysis (MDTA) used in doping control is more efficient than single drug target analysis (SDTA). The number of drugs with the potential for abuse is so extensive that full coverage is not possible with SDTA. To address this problem, a liquid chromatography tandem mass spectrometric method was developed for simultaneous analysis of 302 drugs using a scheduled multiple reaction monitoring (s-MRM) algorithm. With a known retention time of an analyte, the s-MRM algorithm monitors each MRM transition only around its expected retention time. Analytes were recovered from plasma by liquid-liquid extraction. Information-dependent acquisition (IDA) functionality was used to combine s-MRM with enhanced product ion (EPI) scans within the same chromatographic analysis. An EPI spectrum library was also generated for rapid identification of analytes. Analysis time for the 302 drugs was 7 min. Scheduled MRM improved the quality of the chromatograms, signal response, reproducibility, and enhanced signal-to-noise ratio (S/N), resulting in more data points. Reduction in total cycle time from 2.4 s in conventional MRM (c-MRM) to 1 s in s-MRM allowed completion of the EPI scan at the same time. The speed for screening and identification of multiple drugs in equine plasma for doping control analysis was greatly improved by this method.

Statistical characterization of multiple-reaction monitoring mass spectrometry (MRM-MS) assays for quantitative proteomics

PubMed Central

2012-01-01

Multiple reaction monitoring mass spectrometry (MRM-MS) with stable isotope dilution (SID) is increasingly becoming a widely accepted assay for the quantification of proteins and peptides. These assays have shown great promise in relatively high throughput verification of candidate biomarkers. While the use of MRM-MS assays is well established in the small molecule realm, their introduction and use in proteomics is relatively recent. As such, statistical and computational methods for the analysis of MRM-MS data from proteins and peptides are still being developed. Based on our extensive experience with analyzing a wide range of SID-MRM-MS data, we set forth a methodology for analysis that encompasses significant aspects ranging from data quality assessment, assay characterization including calibration curves, limits of detection (LOD) and quantification (LOQ), and measurement of intra- and interlaboratory precision. We draw upon publicly available seminal datasets to illustrate our methods and algorithms. PMID:23176545

Statistical characterization of multiple-reaction monitoring mass spectrometry (MRM-MS) assays for quantitative proteomics.

PubMed

Mani, D R; Abbatiello, Susan E; Carr, Steven A

2012-01-01

Multiple reaction monitoring mass spectrometry (MRM-MS) with stable isotope dilution (SID) is increasingly becoming a widely accepted assay for the quantification of proteins and peptides. These assays have shown great promise in relatively high throughput verification of candidate biomarkers. While the use of MRM-MS assays is well established in the small molecule realm, their introduction and use in proteomics is relatively recent. As such, statistical and computational methods for the analysis of MRM-MS data from proteins and peptides are still being developed. Based on our extensive experience with analyzing a wide range of SID-MRM-MS data, we set forth a methodology for analysis that encompasses significant aspects ranging from data quality assessment, assay characterization including calibration curves, limits of detection (LOD) and quantification (LOQ), and measurement of intra- and interlaboratory precision. We draw upon publicly available seminal datasets to illustrate our methods and algorithms.

MRMer, an interactive open source and cross-platform system for data extraction and visualization of multiple reaction monitoring experiments.

PubMed

Martin, Daniel B; Holzman, Ted; May, Damon; Peterson, Amelia; Eastham, Ashley; Eng, Jimmy; McIntosh, Martin

2008-11-01

Multiple reaction monitoring (MRM) mass spectrometry identifies and quantifies specific peptides in a complex mixture with very high sensitivity and speed and thus has promise for the high throughput screening of clinical samples for candidate biomarkers. We have developed an interactive software platform, called MRMer, for managing highly complex MRM-MS experiments, including quantitative analyses using heavy/light isotopic peptide pairs. MRMer parses and extracts information from MS files encoded in the platform-independent mzXML data format. It extracts and infers precursor-product ion transition pairings, computes integrated ion intensities, and permits rapid visual curation for analyses exceeding 1000 precursor-product pairs. Results can be easily output for quantitative comparison of consecutive runs. Additionally MRMer incorporates features that permit the quantitative analysis experiments including heavy and light isotopic peptide pairs. MRMer is open source and provided under the Apache 2.0 license.

Meat Authentication via Multiple Reaction Monitoring Mass Spectrometry of Myoglobin Peptides.

PubMed

Watson, Andrew D; Gunning, Yvonne; Rigby, Neil M; Philo, Mark; Kemsley, E Kate

2015-10-20

A rapid multiple reaction monitoring (MRM) mass spectrometric method for the detection and relative quantitation of the adulteration of meat with that of an undeclared species is presented. Our approach uses corresponding proteins from the different species under investigation and corresponding peptides from those proteins, or CPCP. Selected peptide markers can be used for species detection. The use of ratios of MRM transition peak areas for corresponding peptides is proposed for relative quantitation. The approach is introduced by use of myoglobin from four meats: beef, pork, horse and lamb. Focusing in the present work on species identification, by use of predictive tools, we determine peptide markers that allow the identification of all four meats and detection of one meat added to another at levels of 1% (w/w). Candidate corresponding peptide pairs to be used for the relative quantification of one meat added to another have been observed. Preliminary quantitation data presented here are encouraging.

A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS).

PubMed

Unwin, Richard D; Griffiths, John R; Whetton, Anthony D

2009-01-01

The application of a targeted mass spectrometric workflow to the sensitive identification of post-translational modifications is described. This protocol employs multiple reaction monitoring (MRM) to search for all putative peptides specifically modified in a target protein. Positive MRMs trigger an MS/MS experiment to confirm the nature and site of the modification. This approach, termed MIDAS (MRM-initiated detection and sequencing), is more sensitive than approaches using neutral loss scanning or precursor ion scanning methodologies, due to a more efficient use of duty cycle along with a decreased background signal associated with MRM. We describe the use of MIDAS for the identification of phosphorylation, with a typical experiment taking just a couple of hours from obtaining a peptide sample. With minor modifications, the MIDAS method can be applied to other protein modifications or unmodified peptides can be used as a MIDAS target.

Data Pre-Processing for Label-Free Multiple Reaction Monitoring (MRM) Experiments

PubMed Central

Chung, Lisa M.; Colangelo, Christopher M.; Zhao, Hongyu

2014-01-01

Multiple Reaction Monitoring (MRM) conducted on a triple quadrupole mass spectrometer allows researchers to quantify the expression levels of a set of target proteins. Each protein is often characterized by several unique peptides that can be detected by monitoring predetermined fragment ions, called transitions, for each peptide. Concatenating large numbers of MRM transitions into a single assay enables simultaneous quantification of hundreds of peptides and proteins. In recognition of the important role that MRM can play in hypothesis-driven research and its increasing impact on clinical proteomics, targeted proteomics such as MRM was recently selected as the Nature Method of the Year. However, there are many challenges in MRM applications, especially data pre‑processing where many steps still rely on manual inspection of each observation in practice. In this paper, we discuss an analysis pipeline to automate MRM data pre‑processing. This pipeline includes data quality assessment across replicated samples, outlier detection, identification of inaccurate transitions, and data normalization. We demonstrate the utility of our pipeline through its applications to several real MRM data sets. PMID:24905083

Data Pre-Processing for Label-Free Multiple Reaction Monitoring (MRM) Experiments.

PubMed

Chung, Lisa M; Colangelo, Christopher M; Zhao, Hongyu

2014-06-05

Multiple Reaction Monitoring (MRM) conducted on a triple quadrupole mass spectrometer allows researchers to quantify the expression levels of a set of target proteins. Each protein is often characterized by several unique peptides that can be detected by monitoring predetermined fragment ions, called transitions, for each peptide. Concatenating large numbers of MRM transitions into a single assay enables simultaneous quantification of hundreds of peptides and proteins. In recognition of the important role that MRM can play in hypothesis-driven research and its increasing impact on clinical proteomics, targeted proteomics such as MRM was recently selected as the Nature Method of the Year. However, there are many challenges in MRM applications, especially data pre‑processing where many steps still rely on manual inspection of each observation in practice. In this paper, we discuss an analysis pipeline to automate MRM data pre‑processing. This pipeline includes data quality assessment across replicated samples, outlier detection, identification of inaccurate transitions, and data normalization. We demonstrate the utility of our pipeline through its applications to several real MRM data sets.

Liquid chromatography-electrospray ionization tandem mass spectrometry and dynamic multiple reaction monitoring method for determining multiple pesticide residues in tomato.

PubMed

Andrade, G C R M; Monteiro, S H; Francisco, J G; Figueiredo, L A; Botelho, R G; Tornisielo, V L

2015-05-15

A quick and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry method, using dynamic multiple reaction monitoring and a 1.8-μm particle size analytical column, was developed to determine 57 pesticides in tomato in a 13-min run. QuEChERS (quick, easy, cheap, effective, rugged, and safe) method for samples preparations and validations was carried out in compliance with EU SANCO guidelines. The method was applied to 58 tomato samples. More than 84% of the compounds investigated showed limits of detection equal to or lower than 5 mg kg(-1). A mild (<20%), medium (20-50%), and strong (>50%) matrix effect was observed for 72%, 25%, and 3% of the pesticides studied, respectively. Eighty-one percent of the pesticides showed recoveries ranging between 70% and 120%. Twelve pesticides were detected in 35 samples, all below the maximum residue levels permitted in the Brazilian legislation; 15 samples exceeded the maximum residue levels established by the EU legislation for methamidophos; and 10 exceeded limits for acephate and four for bromuconazole. Copyright © 2014 Elsevier Ltd. All rights reserved.

Multiplexed immunosensing and kinetics monitoring in nanofluidic devices with highly enhanced target capture efficiency

PubMed Central

Lin, Yii-Lih; Huang, Yen-Jun; Teerapanich, Pattamon; Leïchlé, Thierry

2016-01-01

Nanofluidic devices promise high reaction efficiency and fast kinetic responses due to the spatial constriction of transported biomolecules with confined molecular diffusion. However, parallel detection of multiple biomolecules, particularly proteins, in highly confined space remains challenging. This study integrates extended nanofluidics with embedded protein microarray to achieve multiplexed real-time biosensing and kinetics monitoring. Implementation of embedded standard-sized antibody microarray is attained by epoxy-silane surface modification and a room-temperature low-aspect-ratio bonding technique. An effective sample transport is achieved by electrokinetic pumping via electroosmotic flow. Through the nanoslit-based spatial confinement, the antigen-antibody binding reaction is enhanced with ∼100% efficiency and may be directly observed with fluorescence microscopy without the requirement of intermediate washing steps. The image-based data provide numerous spatially distributed reaction kinetic curves and are collectively modeled using a simple one-dimensional convection-reaction model. This study represents an integrated nanofluidic solution for real-time multiplexed immunosensing and kinetics monitoring, starting from device fabrication, protein immobilization, device bonding, sample transport, to data analysis at Péclet number less than 1. PMID:27375819

Basic design of MRM assays for peptide quantification.

PubMed

James, Andrew; Jorgensen, Claus

2010-01-01

With the recent availability and accessibility of mass spectrometry for basic and clinical research, the requirement for stable, sensitive, and reproducible assays to specifically detect proteins of interest has increased. Multiple reaction monitoring (MRM) or selective reaction monitoring (SRM) is a highly selective, sensitive, and robust assay to monitor the presence and amount of biomolecules. Until recently, MRM was typically used for the detection of drugs and other biomolecules from body fluids. With increased focus on biomarkers and systems biology approaches, researchers in the proteomics field have taken advantage of this approach. In this chapter, we will introduce the reader to the basic principle of designing and optimizing an MRM workflow. We provide examples of MRM workflows for standard proteomic samples and provide suggestions for the reader who is interested in using MRM for quantification.

Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics

PubMed Central

Shi, Tujin; Su, Dian; Liu, Tao; Tang, Keqi; Camp, David G.; Qian, Wei-Jun; Smith, Richard D.

2012-01-01

Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—has emerged as a promising high-throughput targeted protein quantification technology for candidate biomarker verification and systems biology applications. A major bottleneck for current SRM technology, however, is insufficient sensitivity for e.g., detecting low-abundance biomarkers likely present at the low ng/mL to pg/mL range in human blood plasma or serum, or extremely low-abundance signaling proteins in cells or tissues. Herein we review recent advances in methods and technologies, including front-end immunoaffinity depletion, fractionation, selective enrichment of target proteins/peptides including posttranslational modifications (PTMs), as well as advances in MS instrumentation which have significantly enhanced the overall sensitivity of SRM assays and enabled the detection of low-abundance proteins at low to sub- ng/mL level in human blood plasma or serum. General perspectives on the potential of achieving sufficient sensitivity for detection of pg/mL level proteins in plasma are also discussed. PMID:22577010

Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Su, Dian; Liu, Tao

2012-04-01

Selected reaction monitoring (SRM)—also known as multiple reaction monitoring (MRM)—has emerged as a promising high-throughput targeted protein quantification technology for candidate biomarker verification and systems biology applications. A major bottleneck for current SRM technology, however, is insufficient sensitivity for e.g., detecting low-abundance biomarkers likely present at the pg/mL to low ng/mL range in human blood plasma or serum, or extremely low-abundance signaling proteins in the cells or tissues. Herein we review recent advances in methods and technologies, including front-end immunoaffinity depletion, fractionation, selective enrichment of target proteins/peptides or their posttranslational modifications (PTMs), as well as advances in MS instrumentation, whichmore » have significantly enhanced the overall sensitivity of SRM assays and enabled the detection of low-abundance proteins at low to sub- ng/mL level in human blood plasma or serum. General perspectives on the potential of achieving sufficient sensitivity for detection of pg/mL level proteins in plasma are also discussed.« less

Auditory decision aiding in supervisory control of multiple unmanned aerial vehicles.

PubMed

Donmez, Birsen; Cummings, M L; Graham, Hudson D

2009-10-01

This article is an investigation of the effectiveness of sonifications, which are continuous auditory alerts mapped to the state of a monitored task, in supporting unmanned aerial vehicle (UAV) supervisory control. UAV supervisory control requires monitoring a UAV across multiple tasks (e.g., course maintenance) via a predominantly visual display, which currently is supported with discrete auditory alerts. Sonification has been shown to enhance monitoring performance in domains such as anesthesiology by allowing an operator to immediately determine an entity's (e.g., patient) current and projected states, and is a promising alternative to discrete alerts in UAV control. However, minimal research compares sonification to discrete alerts, and no research assesses the effectiveness of sonification for monitoring multiple entities (e.g., multiple UAVs). The authors conducted an experiment with 39 military personnel, using a simulated setup. Participants controlled single and multiple UAVs and received sonifications or discrete alerts based on UAV course deviations and late target arrivals. Regardless of the number of UAVs supervised, the course deviation sonification resulted in reactions to course deviations that were 1.9 s faster, a 19% enhancement, compared with discrete alerts. However, course deviation sonifications interfered with the effectiveness of discrete late arrival alerts in general and with operator responses to late arrivals when supervising multiple vehicles. Sonifications can outperform discrete alerts when designed to aid operators to predict future states of monitored tasks. However, sonifications may mask other auditory alerts and interfere with other monitoring tasks that require divided attention. This research has implications for supervisory control display design.

Targeted quantitative analysis of Streptococcus pyogenes virulence factors by multiple reaction monitoring.

PubMed

Lange, Vinzenz; Malmström, Johan A; Didion, John; King, Nichole L; Johansson, Björn P; Schäfer, Juliane; Rameseder, Jonathan; Wong, Chee-Hong; Deutsch, Eric W; Brusniak, Mi-Youn; Bühlmann, Peter; Björck, Lars; Domon, Bruno; Aebersold, Ruedi

2008-08-01

In many studies, particularly in the field of systems biology, it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states. The high degree of reproducibility required for such experiments has not been achieved by classical mass spectrometry-based proteomics methods. In this study we describe the implementation of a targeted quantitative approach by which predetermined protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples. This approach consists of three steps. First, the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry, and the data generated are assembled in the PeptideAtlas database. Second, based on this proteome map, peptides uniquely identifying the proteins of interest, proteotypic peptides, are selected, and multiple reaction monitoring (MRM) transitions are established and validated by MS2 spectrum acquisition. This process of peptide selection, transition selection, and validation is supported by a suite of software tools, TIQAM (Targeted Identification for Quantitative Analysis by MRM), described in this study. Third, the selected target protein set is quantified in multiple samples by MRM. Applying this approach we were able to reliably quantify low abundance virulence factors from cultures of the human pathogen Streptococcus pyogenes exposed to increasing amounts of plasma. The resulting quantitative protein patterns enabled us to clearly define the subset of virulence proteins that is regulated upon plasma exposure.

Ethylene monitoring and control system

NASA Technical Reports Server (NTRS)

Nelson, Bruce N. (Inventor); Kanc, James A. (Inventor); Richard, II, Roy V. (Inventor)

2000-01-01

A system that can accurately monitor and control low concentrations of ethylene gas includes a test chamber configured to receive sample gas potentially containing an ethylene concentration and ozone, a detector configured to receive light produced during a reaction between the ethylene and ozone and to produce signals related thereto, and a computer connected to the detector to process the signals to determine therefrom a value of the concentration of ethylene in the sample gas. The supply for the system can include a four way valve configured to receive pressurized gas at one input and a test chamber. A piston is journaled in the test chamber with a drive end disposed in a drive chamber and a reaction end defining with walls of the test chamber a variable volume reaction chamber. The drive end of the piston is pneumatically connected to two ports of the four way valve to provide motive force to the piston. A manifold is connected to the variable volume reaction chamber, and is configured to receive sample gasses from at least one of a plurality of ports connectable to degreening rooms and to supply the sample gas to the reactive chamber for reaction with ozone. The apparatus can be used to monitor and control the ethylene concentration in multiple degreening rooms.

Ethylene monitoring and control system

NASA Technical Reports Server (NTRS)

Nelson, Bruce N. (Inventor); Kane, James A. (Inventor); Richard, II, Roy V. (Inventor)

2001-01-01

A system that can accurately monitor and control low concentrations of ethylene gas includes a test chamber configured to receive sample gas potentially containing an ethylene concentration and ozone, a detector configured to receive light produced during a reaction between the ethylene and ozone and to produce signals related thereto, and a computer connected to the detector to process the signals to determine therefrom a value of the concentration of ethylene in the sample gas. The supply for the system can include a four way valve configured to receive pressurized gas at one input and a test chamber. A piston is journaled in the test chamber with a drive end disposed in a drive chamber and a reaction end defining with walls of the test chamber a variable volume reaction chamber. The drive end of the piston is pneumatically connected to two ports of the four way valve to provide motive force to the piston. A manifold is connected to the variable volume reaction chamber, and is configured to receive sample gasses from at least one of a plurality of ports connectable to degreening rooms and to supply the sample gas to the reactive chamber for reaction with ozone. The apparatus can be used to monitor and control the ethylene concentration in multiple degreening rooms.

Recommendations for Optimizing Tuberculosis Treatment: Therapeutic Drug Monitoring, Pharmacogenetics, and Nutritional Status Considerations

PubMed Central

Choi, Rihwa; Jeong, Byeong-Ho

2017-01-01

Although tuberculosis is largely a curable disease, it remains a major cause of morbidity and mortality worldwide. Although the standard 6-month treatment regimen is highly effective for drug-susceptible tuberculosis, the use of multiple drugs over long periods of time can cause frequent adverse drug reactions. In addition, some patients with drug-susceptible tuberculosis do not respond adequately to treatment and develop treatment failure and drug resistance. Response to tuberculosis treatment could be affected by multiple factors associated with the host-pathogen interaction including genetic factors and the nutritional status of the host. These factors should be considered for effective tuberculosis control. Therefore, therapeutic drug monitoring (TDM), which is individualized drug dosing guided by serum drug concentrations during treatment, and pharmacogenetics-based personalized dosing guidelines of anti-tuberculosis drugs could reduce the incidence of adverse drug reactions and increase the likelihood of successful treatment outcomes. Moreover, assessment and management of comorbid conditions including nutritional status could improve anti-tuberculosis treatment response. PMID:28028995

Simultaneous screening for 238 drugs in blood by liquid chromatography-ion spray tandem mass spectrometry with multiple-reaction monitoring.

PubMed

Gergov, M; Ojanperä, I; Vuori, E

2003-09-25

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method is presented for the qualitative screening for 238 drugs in blood samples, which is considerably more than in previous methods. After a two-step liquid-liquid extraction and C(18) chromatography, the compounds were introduced into a triple quadrupole mass spectrometer equipped with a turbo ion spray ion source operating in the positive ionization mode. Identification was based on the compound's absolute retention time, protonated molecular ion, and one representative fragment ion obtained by multiple reaction monitoring (MRM) at an individually selected collision energy of 20, 35, or 50 eV. The limit of detection (LOD) for the majority of the compounds (80%) was < or = 0.05 mg/l, ranging from 0.002 mg/l (e.g., antihistamines) to 5 mg/l (acidic compounds), and for malathion it was 10 mg/l. The LOD values were sufficiently low to allow the majority of compounds to be detected at therapeutic concentrations in the blood.

Recommendations for Optimizing Tuberculosis Treatment: Therapeutic Drug Monitoring, Pharmacogenetics, and Nutritional Status Considerations.

PubMed

Choi, Rihwa; Jeong, Byeong Ho; Koh, Won Jung; Lee, Soo Youn

2017-03-01

Although tuberculosis is largely a curable disease, it remains a major cause of morbidity and mortality worldwide. Although the standard 6-month treatment regimen is highly effective for drug-susceptible tuberculosis, the use of multiple drugs over long periods of time can cause frequent adverse drug reactions. In addition, some patients with drug-susceptible tuberculosis do not respond adequately to treatment and develop treatment failure and drug resistance. Response to tuberculosis treatment could be affected by multiple factors associated with the host-pathogen interaction including genetic factors and the nutritional status of the host. These factors should be considered for effective tuberculosis control. Therefore, therapeutic drug monitoring (TDM), which is individualized drug dosing guided by serum drug concentrations during treatment, and pharmacogenetics-based personalized dosing guidelines of anti-tuberculosis drugs could reduce the incidence of adverse drug reactions and increase the likelihood of successful treatment outcomes. Moreover, assessment and management of comorbid conditions including nutritional status could improve anti-tuberculosis treatment response.

Hydrophilic interaction chromatography-multiple reaction monitoring mass spectrometry method for basic building block analysis of low molecular weight heparins prepared through nitrous acid depolymerization.

PubMed

Sun, Xiaojun; Guo, Zhimou; Yu, Mengqi; Lin, Chao; Sheng, Anran; Wang, Zhiyu; Linhardt, Robert J; Chi, Lianli

2017-01-06

Low molecular weight heparins (LMWHs) are important anticoagulant drugs that are prepared through depolymerization of unfractionated heparin. Based on the types of processing reactions and the structures of the products, LMWHs can be divided into different classifications. Enoxaparin is prepared by benzyl esterification and alkaline depolymerization, while dalteparin and nadroparin are prepared through nitrous acid depolymerization followed by borohydride reduction. Compositional analysis of their basic building blocks is an effective way to provide structural information on heparin and LMWHs. However, most current compositional analysis methods have been limited to heparin and enoxaparin. A sensitive and comprehensive approach is needed for detailed investigation of the structure of LMWHs prepared through nitrous acid depolymerization, especially their characteristic saturated non-reducing end (NRE) and 2,5-anhydro-d-mannitol reducing end (RE). A maltose modified hydrophilic interaction column offers improved separation of complicated mixtures of acidic disaccharides and oligosaccharides. A total of 36 basic building blocks were unambiguously identified by high-resolution tandem mass spectrometry (MS). Multiple reaction monitoring (MRM) MS/MS quantification was developed and validated in the analysis of dalteparin and nadroparin samples. Each group of building blocks revealed different aspects of the properties of LMWHs, such as functional motifs required for anticoagulant activity, the structure of heparin starting materials, cleavage sites in the depolymerization reaction, and undesired structural modifications resulting from side reactions. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography

PubMed Central

Kim, Jun Seok; Lee, Cheolju

2015-01-01

Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins. PMID:26544075

A reliable and rapid tool for plasma quantification of 18 psychotropic drugs by ESI tandem mass spectrometry.

PubMed

Vecchione, Gennaro; Casetta, Bruno; Chiapparino, Antonella; Bertolino, Alessandro; Tomaiuolo, Michela; Cappucci, Filomena; Gatta, Raffaella; Margaglione, Maurizio; Grandone, Elvira

2012-01-01

A simple liquid chromatographic tandem mass spectrometry (LC-MS/MS) method has been developed for simultaneous analysis of 17 basic and one acid psychotropic drugs in human plasma. The method relies on a protein precipitation step for sample preparation and offers high sensitivity, wide linearity without interferences from endogenous matrix components. Chromatography was run on a reversed-phase column with an acetonitrile-H₂O mixture. The quantification of target compounds was performed in multiple reaction monitoring (MRM) and by switching the ionization polarity within the analytical run. A further sensitivity increase was obtained by implementing the functionality "scheduled multiple reaction monitoring" (sMRM) offered by the recent version of the software package managing the instrument. The overall injection interval was less than 5.5 min. Regression coefficients of the calibration curves and limits of quantification (LOQ) showed a good coverage of over-therapeutic, therapeutic and sub-therapeutic ranges. Recovery rates, measured as percentage of recovery of spiked plasma samples, were ≥ 94%. Precision and accuracy data have been satisfactory for a therapeutic drug monitoring (TDM) service as for managing plasma samples from patients receiving psycho-pharmacological treatment. Copyright © 2012 Elsevier B.V. All rights reserved.

Range of protein detection by selected/multiple reaction monitoring mass spectrometry in an unfractionated human cell culture lysate.

PubMed

Ebhardt, H Alexander; Sabidó, Eduard; Hüttenhain, Ruth; Collins, Ben; Aebersold, Ruedi

2012-04-01

Selected or multiple reaction monitoring is a targeted mass spectrometry method (S/MRM-MS), in which many peptides are simultaneously and consistently analyzed during a single liquid chromatography-mass spectrometry (LC-S/MRM-MS) measurement. These capabilities make S/MRM-MS an attractive method to monitor a consistent set of proteins over various experimental conditions. To increase throughput for S/MRM-MS it is advantageous to use scheduled methods and unfractionated protein extracts. Here, we established the practically measurable dynamic range of proteins reliably detectable and quantifiable in an unfractionated protein extract from a human cell line using LC-S/MRM-MS. Initially, we analyzed S/MRM transition peak groups in terms of interfering signals and compared S/MRM transition peak groups to MS1-triggered MS2 spectra using dot-product analysis. Finally, using unfractionated protein extract from human cell lysate, we quantified the upper boundary of copies per cell to be 35 million copies per cell, while 7500 copies per cell represents a lower boundary using a single 35 min linear gradient LC-S/MRM-MS measurement on a current, standard commercial instrument. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography.

PubMed

Park, Seong-Jun; Ahn, Hee-Sung; Kim, Jun Seok; Lee, Cheolju

2015-01-01

Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins.

Evaluation of Direct Infusion-Multiple Reaction Monitoring Mass Spectrometry for Quantification of Heat Shock Proteins

PubMed Central

Xiang, Yun; Koomen, John M.

2012-01-01

Protein quantification with liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) has emerged as a powerful platform for assessing panels of biomarkers. In this study, direct infusion, using automated, chip-based nanoelectrospray ionization, coupled with MRM (DI-MRM) is used for protein quantification. Removal of the LC separation step increases the importance of evaluating the ratios between the transitions. Therefore, the effects of solvent composition, analyte concentration, spray voltage, and quadrupole resolution settings on fragmentation patterns have been studied using peptide and protein standards. After DI-MRM quantification was evaluated for standards, quantitative assays for the expression of heat shock proteins (HSPs) were translated from LC-MRM to DI-MRM for implementation in cell line models of multiple myeloma. Requirements for DI-MRM assay development are described. Then, the two methods are compared; criteria for effective DI-MRM analysis are reported based on the analysis of HSP expression in digests of whole cell lysates. The increased throughput of DI-MRM analysis is useful for rapid analysis of large batches of similar samples, such as time course measurements of cellular responses to therapy. PMID:22293045

Increased Depth and Breadth of Plasma Protein Quantitation via Two-Dimensional Liquid Chromatography/Multiple Reaction Monitoring-Mass Spectrometry with Labeled Peptide Standards.

PubMed

Percy, Andrew J; Yang, Juncong; Chambers, Andrew G; Borchers, Christoph H

2016-01-01

Absolute quantitative strategies are emerging as a powerful and preferable means of deriving concentrations in biological samples for systems biology applications. Method development is driven by the need to establish new-and validate current-protein biomarkers of high-to-low abundance for clinical utility. In this chapter, we describe a methodology involving two-dimensional (2D) reversed-phase liquid chromatography (RPLC), operated under alkaline and acidic pH conditions, combined with multiple reaction monitoring (MRM)-mass spectrometry (MS) (also called selected reaction monitoring (SRM)-MS) and a complex mixture of stable isotope-labeled standard (SIS) peptides, to quantify a broad and diverse panel of 253 proteins in human blood plasma. The quantitation range spans 8 orders of magnitude-from 15 mg/mL (for vitamin D-binding protein) to 450 pg/mL (for protein S100-B)-and includes 31 low-abundance proteins (defined as being <10 ng/mL) of potential disease relevance. The method is designed to assess candidates at the discovery and/or verification phases of the biomarker pipeline and can be adapted to examine smaller or alternate panels of proteins for higher sample throughput. Also detailed here is the application of our recently developed software tool-Qualis-SIS-for protein quantitation (via regression analysis of standard curves) and quality assessment of the resulting data. Overall, this chapter provides the blueprint for the replication of this quantitative proteomic method by proteomic scientists of all skill levels.

Comparative quantification of human intestinal bacteria based on cPCR and LDR/LCR

PubMed Central

Tang, Zhou-Rui; Li, Kai; Zhou, Yu-Xun; Xiao, Zhen-Xian; Xiao, Jun-Hua; Huang, Rui; Gu, Guo-Hao

2012-01-01

AIM: To establish a multiple detection method based on comparative polymerase chain reaction (cPCR) and ligase detection reaction (LDR)/ligase chain reaction (LCR) to quantify the intestinal bacterial components. METHODS: Comparative quantification of 16S rDNAs from different intestinal bacterial components was used to quantify multiple intestinal bacteria. The 16S rDNAs of different bacteria were amplified simultaneously by cPCR. The LDR/LCR was examined to actualize the genotyping and quantification. Two beneficial (Bifidobacterium, Lactobacillus) and three conditionally pathogenic bacteria (Enterococcus, Enterobacterium and Eubacterium) were used in this detection. With cloned standard bacterial 16S rDNAs, standard curves were prepared to validate the quantitative relations between the ratio of original concentrations of two templates and the ratio of the fluorescence signals of their final ligation products. The internal controls were added to monitor the whole detection flow. The quantity ratio between two bacteria was tested. RESULTS: cPCR and LDR revealed obvious linear correlations with standard DNAs, but cPCR and LCR did not. In the sample test, the distributions of the quantity ratio between each two bacterial species were obtained. There were significant differences among these distributions in the total samples. But these distributions of quantity ratio of each two bacteria remained stable among groups divided by age or sex. CONCLUSION: The detection method in this study can be used to conduct multiple intestinal bacteria genotyping and quantification, and to monitor the human intestinal health status as well. PMID:22294830

Comparative quantification of human intestinal bacteria based on cPCR and LDR/LCR.

PubMed

Tang, Zhou-Rui; Li, Kai; Zhou, Yu-Xun; Xiao, Zhen-Xian; Xiao, Jun-Hua; Huang, Rui; Gu, Guo-Hao

2012-01-21

To establish a multiple detection method based on comparative polymerase chain reaction (cPCR) and ligase detection reaction (LDR)/ligase chain reaction (LCR) to quantify the intestinal bacterial components. Comparative quantification of 16S rDNAs from different intestinal bacterial components was used to quantify multiple intestinal bacteria. The 16S rDNAs of different bacteria were amplified simultaneously by cPCR. The LDR/LCR was examined to actualize the genotyping and quantification. Two beneficial (Bifidobacterium, Lactobacillus) and three conditionally pathogenic bacteria (Enterococcus, Enterobacterium and Eubacterium) were used in this detection. With cloned standard bacterial 16S rDNAs, standard curves were prepared to validate the quantitative relations between the ratio of original concentrations of two templates and the ratio of the fluorescence signals of their final ligation products. The internal controls were added to monitor the whole detection flow. The quantity ratio between two bacteria was tested. cPCR and LDR revealed obvious linear correlations with standard DNAs, but cPCR and LCR did not. In the sample test, the distributions of the quantity ratio between each two bacterial species were obtained. There were significant differences among these distributions in the total samples. But these distributions of quantity ratio of each two bacteria remained stable among groups divided by age or sex. The detection method in this study can be used to conduct multiple intestinal bacteria genotyping and quantification, and to monitor the human intestinal health status as well.

A New Approach To Selectively Identify And Quantify Perfluorochemicals In Complex Matrices Using Ion Trap Time-Of-Flight Mass Spectrometry

EPA Science Inventory

Most of the published studies focusing on the environmental and biological distribution of perfluorinated alkyl compounds (PFCs) have used triple quadrupole mass spectrometers for compound identification and quantitation. Multiple-reaction-monitoring (MRM) is a sensitive mass sp...

EPA CRL MS014: Analysis of Aldicarb, Bromadiolone, Carbofuran, Oxamyl and Methomyl in Water by Multiple Reaction Monitoring Liquid Chromatography / Tandem Mass Spectrometry (LC/MS/MS)

EPA Pesticide Factsheets

Method MS014 describes procedures for solvent extraction of aldicarb, bromadiolone, carbofuran, oxamyl and methomyl from water samples, followed by analysis using liquid chromatography tandem mass spectrometry (LC-MS-MS).

MRM for the verification of cancer biomarker proteins: recent applications to human plasma and serum.

PubMed

Chambers, Andrew G; Percy, Andrew J; Simon, Romain; Borchers, Christoph H

2014-04-01

Accurate cancer biomarkers are needed for early detection, disease classification, prediction of therapeutic response and monitoring treatment. While there appears to be no shortage of candidate biomarker proteins, a major bottleneck in the biomarker pipeline continues to be their verification by enzyme linked immunosorbent assays. Multiple reaction monitoring (MRM), also known as selected reaction monitoring, is a targeted mass spectrometry approach to protein quantitation and is emerging to bridge the gap between biomarker discovery and clinical validation. Highly multiplexed MRM assays are readily configured and enable simultaneous verification of large numbers of candidates facilitating the development of biomarker panels which can increase specificity. This review focuses on recent applications of MRM to the analysis of plasma and serum from cancer patients for biomarker verification. The current status of this approach is discussed along with future directions for targeted mass spectrometry in clinical biomarker validation.

Visual detection of multiple genetically modified organisms in a capillary array.

PubMed

Shao, Ning; Chen, Jianwei; Hu, Jiaying; Li, Rong; Zhang, Dabing; Guo, Shujuan; Hui, Junhou; Liu, Peng; Yang, Litao; Tao, Sheng-Ce

2017-01-31

There is an urgent need for rapid, low-cost multiplex methodologies for the monitoring of genetically modified organisms (GMOs). Here, we report a C[combining low line]apillary A[combining low line]rray-based L[combining low line]oop-mediated isothermal amplification for M[combining low line]ultiplex visual detection of nucleic acids (CALM) platform for the simple and rapid monitoring of GMOs. In CALM, loop-mediated isothermal amplification (LAMP) primer sets are pre-fixed to the inner surface of capillaries. The surface of the capillary array is hydrophobic while the capillaries are hydrophilic, enabling the simultaneous loading and separation of the LAMP reaction mixtures into each capillary by capillary forces. LAMP reactions in the capillaries are then performed in parallel, and the results are visually detected by illumination with a hand-held UV device. Using CALM, we successfully detected seven frequently used transgenic genes/elements and five plant endogenous reference genes with high specificity and sensitivity. Moreover, we found that measurements of real-world blind samples by CALM are consistent with results obtained by independent real-time PCRs. Thus, with an ability to detect multiple nucleic acids in a single easy-to-operate test, we believe that CALM will become a widely applied technology in GMO monitoring.

Mining Missing Membrane Proteins by High-pH Reverse-Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry.

PubMed

Kitata, Reta Birhanu; Dimayacyac-Esleta, Baby Rorielyn T; Choong, Wai-Kok; Tsai, Chia-Feng; Lin, Tai-Du; Tsou, Chih-Chiang; Weng, Shao-Hsing; Chen, Yi-Ju; Yang, Pan-Chyr; Arco, Susan D; Nesvizhskii, Alexey I; Sung, Ting-Yi; Chen, Yu-Ju

2015-09-04

Despite significant efforts in the past decade toward complete mapping of the human proteome, 3564 proteins (neXtProt, 09-2014) are still "missing proteins". Over one-third of these missing proteins are annotated as membrane proteins, owing to their relatively challenging accessibility with standard shotgun proteomics. Using nonsmall cell lung cancer (NSCLC) as a model study, we aim to mine missing proteins from disease-associated membrane proteome, which may be still largely under-represented. To increase identification coverage, we employed Hp-RP StageTip prefractionation of membrane-enriched samples from 11 NSCLC cell lines. Analysis of membrane samples from 20 pairs of tumor and adjacent normal lung tissue was incorporated to include physiologically expressed membrane proteins. Using multiple search engines (X!Tandem, Comet, and Mascot) and stringent evaluation of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66% membrane proteins) and 178 missing proteins (74 membrane proteins) with PSM-, peptide-, and protein-level FDR of 1%. Through multiple reaction monitoring using synthetic peptides, we provided additional evidence of eight missing proteins including seven with transmembrane helix domains. This study demonstrates that mining missing proteins focused on cancer membrane subproteome can greatly contribute to map the whole human proteome. All data were deposited into ProteomeXchange with the identifier PXD002224.

Simultaneous Profiling of Lysoglycerophospholipids in Rice (Oryza sativa L.) Using Direct Infusion-Tandem Mass Spectrometry with Multiple Reaction Monitoring.

PubMed

Lim, Dong Kyu; Mo, Changyeun; Long, Nguyen Phuoc; Kim, Giyoung; Kwon, Sung Won

2017-03-29

White rice is the final product after the hull and bran layers have been removed during the milling process. Although lysoglycerophospholipids (lysoGPLs) only occupy a small proportion in white rice, they are essential for evaluating rice authenticity and quality. In this study, we developed a high-throughput and targeted lipidomics approach that involved direct infusion-tandem mass spectrometry with multiple reaction monitoring to simultaneously profile lysoGPLs in white rice. The method is capable of characterizing 17 lysoGPLs within 1 min. In addition, unsupervised and supervised analyses exhibited a considerably large diversity of lysoGPL concentrations in white rice from different origins. In particular, a classification model was built using identified lysoGPLs that can differentiate white rice from Korea, China, and Japan. Among the discriminatory lysoGPLs, for the lysoPE(16:0) and lysoPE(18:2) compositions, there were relatively small within-group variations, and they were considerably different among the three countries. In conclusion, our proposed method provides a rapid, high-throughput, and comprehensive format for profiling lysoGPLs in rice samples.

Flavanol Quantification of Grapes via Multiple Reaction Monitoring Mass Spectrometry. Application to Differentiation among Clones of Vitis vinifera L. cv. Rufete Grapes.

PubMed

García-Estévez, Ignacio; Alcalde-Eon, Cristina; Escribano-Bailón, M Teresa

2017-08-09

The determination of the detailed flavanol composition in food matrices is not a simple task because of the structural similarities of monomers and, consequently, oligomers and polymers. The aim of this study was the development and validation of an HPLC-MS/MS-multiple reaction monitoring (MRM) method that would allow the accurate and precise quantification of catechins, gallocatechins, and oligomeric proanthocyanidins. The high correlation coefficients of the calibration curves (>0.993), the recoveries not statistically different from 100%, the good intra- and interday precisions (<5%), and the LOD and LOQ values, low enough to quantify flavanols in grapes, are good results from the method validation procedure. Its usefulness has also been tested by determining the detailed composition of Vitis vinifera L. cv. Rufete grapes. Seventy-two (38 nongalloylated and 34 galloylated) and 53 (24 procyanidins and 29 prodelphinidins) flavanols have been identified and quantified in grape seed and grape skin, respectively. The use of HCA and PCA on the detailed flavanol composition has allowed differentiation among Rufete clones.

Linezolid desensitization for a patient with multiple medication hypersensitivity reactions.

PubMed

Bagwell, Autumn D; Stollings, Joanna L; White, Katie D; Fadugba, Olajumoke O; Choi, Jane J

2013-01-01

To describe a case in which a linezolid desensitization protocol was successfully used for a polymicrobial surgical wound infection in a patient with multiple drug hypersensitivity reactions. A 24-year-old woman with vocal cord dysfunction requiring tracheostomy was admitted for a surgical wound infection following a tracheostomy fistula closure procedure. The patient reported multiple antibiotic allergies including penicillins (rash), sulfonamides (rash), vancomycin (anaphylaxis), azithromycin (rash), cephalosporins (anaphylaxis), levofloxacin (unspecified), clindamycin (unspecified), and carbapenems (unspecified). Gram stain of the purulent wound drainage demonstrated mixed gram-negative and gram-positive flora, and bacterial cultures were overgrown with Proteus mirabilis, which precluded identification of other pathogens. Following failed test doses of linezolid, tigecycline, and daptomycin, all of which resulted in hypersensitivity reactions, a 16-step linezolid desensitization protocol was developed and successfully implemented without adverse reactions. The patient completed a 2-week course of antibiotic therapy that included linezolid upon finishing the desensitization protocol. Linezolid is useful in treating complicated and uncomplicated skin and soft tissue infections caused by gram-positive bacteria. With precautions, including premedication, a monitored nursing unit, and immediate availability of an emergency anaphylaxis kit, drug desensitization allows patients the ability to safely use medications to which they may have an immediate hypersensitivity reaction. Minimal data exist on linezolid desensitization protocols. Linezolid desensitization can be a viable option in patients requiring antimicrobial therapy for complicated gram-positive skin infections.

Establishing a reliable multiple reaction monitoring-based method for the quantification of obesity-associated comorbidities in serum and adipose tissue requires intensive clinical validation.

PubMed

Oberbach, Andreas; Schlichting, Nadine; Neuhaus, Jochen; Kullnick, Yvonne; Lehmann, Stefanie; Heinrich, Marco; Dietrich, Arne; Mohr, Friedrich Wilhelm; von Bergen, Martin; Baumann, Sven

2014-12-05

Multiple reaction monitoring (MRM)-based mass spectrometric quantification of peptides and their corresponding proteins has been successfully applied for biomarker validation in serum. The option of multiplexing offers the chance to analyze various proteins in parallel, which is especially important in obesity research. Here, biomarkers that reflect multiple comorbidities and allow monitoring of therapy outcomes are required. Besides the suitability of established MRM assays for serum protein quantification, it is also feasible for analysis of tissues secreting the markers of interest. Surprisingly, studies comparing MRM data sets with established methods are rare, and therefore the biological and clinical value of most analytes remains questionable. A MRM method using nano-UPLC-MS/MS for the quantification of obesity related surrogate markers for several comorbidities in serum, plasma, visceral and subcutaneous adipose tissue was established. Proteotypic peptides for complement C3, adiponectin, angiotensinogen, and plasma retinol binding protein (RBP4) were quantified using isotopic dilution analysis and compared to the standard ELISA method. MRM method variabilities were mainly below 10%. The comparison with other MS-based approaches showed a good correlation. However, large differences in absolute quantification for complement C3 and adiponectin were obtained compared to ELISA, while less marked differences were observed for angiotensinogen and RBP4. The verification of MRM in obesity was performed to discriminate first lean and obese phenotype and second to monitor excessive weight loss after gastric bypass surgery in a seven-month follow-up. The presented MRM assay was able to discriminate obese phenotype from lean and monitor weight loss related changes of surrogate markers. However, inclusion of additional biomarkers was necessary to interpret the MRM data on obesity phenotype properly. In summary, the development of disease-related MRMs should include a step of matching the MRM data with clinically approved standard methods and defining reference values in well-sized representative age, gender, and disease-matched cohorts.

Mass spectrometric directed system for the continuous-flow synthesis and purification of diphenhydramine.

PubMed

Loren, Bradley P; Wleklinski, Michael; Koswara, Andy; Yammine, Kathryn; Hu, Yanyang; Nagy, Zoltan K; Thompson, David H; Cooks, R Graham

2017-06-01

A highly integrated approach to the development of a process for the continuous synthesis and purification of diphenhydramine is reported. Mass spectrometry (MS) is utilized throughout the system for on-line reaction monitoring, off-line yield quantitation, and as a reaction screening module that exploits reaction acceleration in charged microdroplets for high throughput route screening. This effort has enabled the discovery and optimization of multiple routes to diphenhydramine in glass microreactors using MS as a process analytical tool (PAT). The ability to rapidly screen conditions in charged microdroplets was used to guide optimization of the process in a microfluidic reactor. A quantitative MS method was developed and used to measure the reaction kinetics. Integration of the continuous-flow reactor/on-line MS methodology with a miniaturized crystallization platform for continuous reaction monitoring and controlled crystallization of diphenhydramine was also achieved. Our findings suggest a robust approach for the continuous manufacture of pharmaceutical drug products, exemplified in the particular case of diphenhydramine, and optimized for efficiency and crystal size, and guided by real-time analytics to produce the agent in a form that is readily adapted to continuous synthesis.

Multiple reaction monitoring targeted LC-MS analysis of potential cell death marker proteins for increased bioprocess control.

PubMed

Albrecht, Simone; Kaisermayer, Christian; Reinhart, David; Ambrose, Monica; Kunert, Renate; Lindeberg, Anna; Bones, Jonathan

2018-05-01

The monitoring of protein biomarkers for the early prediction of cell stress and death is a valuable tool for process characterization and efficient biomanufacturing control. A representative set of six proteins, namely GPDH, PRDX1, LGALS1, CFL1, TAGLN2 and MDH, which were identified in a previous CHO-K1 cell death model using discovery LC-MS E was translated into a targeted liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) platform and verified. The universality of the markers was confirmed in a cell growth model for which three Chinese hamster ovary host cell lines (CHO-K1, CHO-S, CHO-DG44) were grown in batch culture in two different types of basal media. LC-MRM-MS was also applied to spent media (n = 39) from four perfusion biomanufacturing series. Stable isotope-labelled peptide analogues and a stable isotope-labelled monoclonal antibody were used for improved protein quantitation and simultaneous monitoring of the workflow reproducibility. Significant increases in protein concentrations were observed for all viability marker proteins upon increased dead cell numbers and allowed for discrimination of spent media with dead cell densities below and above 1 × 10 6  dead cells/mL which highlights the potential of the selected viability marker proteins in bioprocess control. Graphical abstract Overview of the LC-MRM-MS workflow for the determination of proteomic markers in conditioned media from the bioreactor that correlate with CHO cell death.

A Generic Multiple Reaction Monitoring Based Approach for Plant Flavonoids Profiling Using a Triple Quadrupole Linear Ion Trap Mass Spectrometry

NASA Astrophysics Data System (ADS)

Yan, Zhixiang; Lin, Ge; Ye, Yang; Wang, Yitao; Yan, Ru

2014-06-01

Flavonoids are one of the largest classes of plant secondary metabolites serving a variety of functions in plants and associating with a number of health benefits for humans. Typically, they are co-identified with many other secondary metabolites using untargeted metabolomics. The limited data quality of untargeted workflow calls for a shift from the breadth-first to the depth-first screening strategy when a specific biosynthetic pathway is focused on. Here we introduce a generic multiple reaction monitoring (MRM)-based approach for flavonoids profiling in plants using a hybrid triple quadrupole linear ion trap (QTrap) mass spectrometer. The approach includes four steps: (1) preliminary profiling of major aglycones by multiple ion monitoring triggered enhanced product ion scan (MIM-EPI); (2) glycones profiling by precursor ion triggered EPI scan (PI-EPI) of major aglycones; (3) comprehensive aglycones profiling by combining MIM-EPI and neutral loss triggered EPI scan (NL-EPI) of major glycone; (4) in-depth flavonoids profiling by MRM-EPI with elaborated MRM transitions. Particularly, incorporation of the NH3 loss and sugar elimination proved to be very informative and confirmative for flavonoids screening. This approach was applied for profiling flavonoids in Astragali radix ( Huangqi), a famous herb widely used for medicinal and nutritional purposes in China. In total, 421 flavonoids were tentatively characterized, among which less than 40 have been previously reported in this medicinal plant. This MRM-based approach provides versatility and sensitivity that required for flavonoids profiling in plants and serves as a useful tool for plant metabolomics.

The Next-Generation PCR-Based Quantification Method for Ambient Waters: Digital PCR.

PubMed

Cao, Yiping; Griffith, John F; Weisberg, Stephen B

2016-01-01

Real-time quantitative PCR (qPCR) is increasingly being used for ambient water monitoring, but development of digital polymerase chain reaction (digital PCR) has the potential to further advance the use of molecular techniques in such applications. Digital PCR refines qPCR by partitioning the sample into thousands to millions of miniature reactions that are examined individually for binary endpoint results, with DNA density calculated from the fraction of positives using Poisson statistics. This direct quantification removes the need for standard curves, eliminating the labor and materials associated with creating and running standards with each batch, and removing biases associated with standard variability and mismatching amplification efficiency between standards and samples. Confining reactions and binary endpoint measurements to small partitions also leads to other performance advantages, including reduced susceptibility to inhibition, increased repeatability and reproducibility, and increased capacity to measure multiple targets in one analysis. As such, digital PCR is well suited for ambient water monitoring applications and is particularly advantageous as molecular methods move toward autonomous field application.

Real-time monitoring of surface-initiated atom transfer radical polymerization using silicon photonic microring resonators: implications for combinatorial screening of polymer brush growth conditions.

PubMed

Limpoco, F Ted; Bailey, Ryan C

2011-09-28

We directly monitor in parallel and in real time the temporal profiles of polymer brushes simultaneously grown via multiple ATRP reaction conditions on a single substrate using arrays of silicon photonic microring resonators. In addition to probing relative polymerization rates, we show the ability to evaluate the dynamic properties of the in situ grown polymers. This presents a powerful new platform for studying modified interfaces that may allow for the combinatorial optimization of surface-initiated polymerization conditions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yung, M. M.; Cheah, S.; Kuhn, J. N.

Sulfur K-edge XANES was used to monitor sulfur species transforming from sulfides to sulfates during steam + air regeneration on a Ni/Mg/K/Al{sub 2}O{sub 3} catalyst used to condition biomass-derived syngas. The catalyst was tested for multiple reaction/regeneration cycles. Post-reaction samples showed the presence of sulfides on the H{sub 2}S-poisoned nickel catalyst. Although some gaseous sulfur species were observed to leave the catalyst bed during regeneration, sulfur remained on the catalyst and a transformation from sulfides to sulfates was observed. The subsequent H{sub 2} reduction led to a partial reduction of sulfates back to sulfides. A proposed reaction sequence is presentedmore » and recommended regeneration strategies are discussed.« less

Method development aspects for the quantitation of pharmaceutical compounds in human plasma with a matrix-assisted laser desorption/ionization source in the multiple reaction monitoring mode.

PubMed

Kovarik, Peter; Grivet, Chantal; Bourgogne, Emmanuel; Hopfgartner, Gérard

2007-01-01

The present work investigates various method development aspects for the quantitative analysis of pharmaceutical compounds in human plasma using matrix-assisted laser desorption/ionization and multiple reaction monitoring (MALDI-MRM). Talinolol was selected as a model analyte. Liquid-liquid extraction (LLE) and protein precipitation were evaluated regarding sensitivity and throughput for the MALDI-MRM technique and its applicability without and with chromatographic separation. Compared to classical electrospray liquid chromatography/mass spectrometry (LC/ESI-MS) method development, with MALDI-MRM the tuning of the analyte in single MS mode is more challenging due to interfering matrix background ions. An approach is proposed using background subtraction. With LLE and using a 200 microL human plasma aliquot acceptable precision and accuracy could be obtained in the range of 1 to 1000 ng/mL without any LC separation. Approximately 3 s were required for one analysis. A full calibration curve and its quality control samples (20 samples) can be analyzed within 1 min. Combining LC with the MALDI analysis allowed improving the linearity down to 50 pg/mL, while reducing the throughput potential only by two-fold. Matrix effects are still a significant issue with MALDI but can be monitored in a similar way to that used for LC/ESI-MS analysis.

Autonomous Multi-sensor Coordination: The Science Goal Monitor

NASA Technical Reports Server (NTRS)

Koratkar, Anuradha; Jung, John; Geiger, Jenny; Grosvenor, Sandy

2004-01-01

Next-generation science and exploration systems will employ new observation strategies that will use multiple sensors in a dynamic environment to provide high quality monitoring, self-consistent analyses and informed decision making. The Science Goal Monitor (SGM) is a prototype software tool being developed to explore the nature of automation necessary to enable dynamic observing of earth phenomenon. The tools being developed in SGM improve our ability to autonomously monitor multiple independent sensors and coordinate reactions to better observe the dynamic phenomena. The SGM system enables users to specify events of interest and how to react when an event is detected. The system monitors streams of data to identify occurrences of the key events previously specified by the scientist/user. When an event occurs, the system autonomously coordinates the execution of the users desired reactions between different sensors. The information can be used to rapidly respond to a variety of fast temporal events. Investigators will no longer have to rely on after-the-fact data analysis to determine what happened. Our paper describes a series of prototype demonstrations that we have developed using SGM and NASA's Earth Observing-1 (EO-1) satellite and Earth Observing Systems Aqua/Terra spacecrafts MODIS instrument. Our demonstrations show the promise of coordinating data from different sources, analyzing the data for a relevant event, autonomously updating and rapidly obtaining a follow-on relevant image. SGM is being used to investigate forest fires, floods and volcanic eruptions. We are now identifying new earth science scenarios that will have more complex SGM reasoning. By developing and testing a prototype in an operational environment, we are also establishing and gathering metrics to gauge the success of automating science campaigns.

Dinosterane and other steroidal hydrocarbons of dinoflagellate origin in sediments and petroleum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Summons, R.E.; Volkman, J.K.; Boreham, C.J.

1987-11-01

The steroidal alkanes of a selection of sediments and oils have been examined by GC-MS with multiple metastable reaction monitoring. Specific 4-methyl sterane isomers have been identified by comparison with isomers synthesized from sterols isolated from dinoflagellates. An immature marine oil shale and two mature marine oils of Triassic to early Cretaceous age contained high concentrations of C{sub 30} steranes comprising desmethyl, 24-ethyl-4{alpha}-methylcholestane and 4{alpha},23,24-trimethylcholestane (dinosterane) isomers. An immature non-marine oil shale and two non-marine oils of Cretaceous to Eocene age contained stereoisomers of 24-ethyl-4{alpha}-methylcholestane as the dominant C{sub 30} steranes. Reaction monitoring analyses in GC-MS are particularly suited tomore » unravelling complex distributions of homologous and stereoisomeric steroids encountered in oils and their source rocks.« less

Metabolic networks in motion: 13C-based flux analysis

PubMed Central

Sauer, Uwe

2006-01-01

Many properties of complex networks cannot be understood from monitoring the components—not even when comprehensively monitoring all protein or metabolite concentrations—unless such information is connected and integrated through mathematical models. The reason is that static component concentrations, albeit extremely informative, do not contain functional information per se. The functional behavior of a network emerges only through the nonlinear gene, protein, and metabolite interactions across multiple metabolic and regulatory layers. I argue here that intracellular reaction rates are the functional end points of these interactions in metabolic networks, hence are highly relevant for systems biology. Methods for experimental determination of metabolic fluxes differ fundamentally from component concentration measurements; that is, intracellular reaction rates cannot be detected directly, but must be estimated through computer model-based interpretation of stable isotope patterns in products of metabolism. PMID:17102807

UV-Visible Absorption Spectroscopy Enhanced X-ray Crystallography at Synchrotron and X-ray Free Electron Laser Sources.

PubMed

Cohen, Aina E; Doukov, Tzanko; Soltis, Michael S

2016-01-01

This review describes the use of single crystal UV-Visible Absorption micro-Spectrophotometry (UV-Vis AS) to enhance the design and execution of X-ray crystallography experiments for structural investigations of reaction intermediates of redox active and photosensitive proteins. Considerations for UV-Vis AS measurements at the synchrotron and associated instrumentation are described. UV-Vis AS is useful to verify the intermediate state of an enzyme and to monitor the progression of reactions within crystals. Radiation induced redox changes within protein crystals may be monitored to devise effective diffraction data collection strategies. An overview of the specific effects of radiation damage on macromolecular crystals is presented along with data collection strategies that minimize these effects by combining data from multiple crystals used at the synchrotron and with the X-ray free electron laser.

Advances in targeted proteomics and applications to biomedical research

PubMed Central

Shi, Tujin; Song, Ehwang; Nie, Song; Rodland, Karin D.; Liu, Tao; Qian, Wei-Jun; Smith, Richard D.

2016-01-01

Targeted proteomics technique has emerged as a powerful protein quantification tool in systems biology, biomedical research, and increasing for clinical applications. The most widely used targeted proteomics approach, selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), can be used for quantification of cellular signaling networks and preclinical verification of candidate protein biomarkers. As an extension to our previous review on advances in SRM sensitivity herein we review recent advances in the method and technology for further enhancing SRM sensitivity (from 2012 to present), and highlighting its broad biomedical applications in human bodily fluids, tissue and cell lines. Furthermore, we also review two recently introduced targeted proteomics approaches, parallel reaction monitoring (PRM) and data-independent acquisition (DIA) with targeted data extraction on fast scanning high-resolution accurate-mass (HR/AM) instruments. Such HR/AM targeted quantification with monitoring all target product ions addresses SRM limitations effectively in specificity and multiplexing; whereas when compared to SRM, PRM and DIA are still in the infancy with a limited number of applications. Thus, for HR/AM targeted quantification we focus our discussion on method development, data processing and analysis, and its advantages and limitations in targeted proteomics. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large-scale quantification of hundreds of target proteins are discussed. PMID:27302376

Advances in targeted proteomics and applications to biomedical research

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Song, Ehwang; Nie, Song

Targeted proteomics technique has emerged as a powerful protein quantification tool in systems biology, biomedical research, and increasing for clinical applications. The most widely used targeted proteomics approach, selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), can be used for quantification of cellular signaling networks and preclinical verification of candidate protein biomarkers. As an extension to our previous review on advances in SRM sensitivity (Shi et al., Proteomics, 12, 1074–1092, 2012) herein we review recent advances in the method and technology for further enhancing SRM sensitivity (from 2012 to present), and highlighting its broad biomedical applications inmore » human bodily fluids, tissue and cell lines. Furthermore, we also review two recently introduced targeted proteomics approaches, parallel reaction monitoring (PRM) and data-independent acquisition (DIA) with targeted data extraction on fast scanning high-resolution accurate-mass (HR/AM) instruments. Such HR/AM targeted quantification with monitoring all target product ions addresses SRM limitations effectively in specificity and multiplexing; whereas when compared to SRM, PRM and DIA are still in the infancy with a limited number of applications. Thus, for HR/AM targeted quantification we focus our discussion on method development, data processing and analysis, and its advantages and limitations in targeted proteomics. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large-scale quantification of hundreds of target proteins are discussed.« less

Mining Missing Membrane Proteins by High-pH Reverse Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry

PubMed Central

Kitata, Reta Birhanu; Dimayacyac-Esleta, Baby Rorielyn T.; Choong, Wai-Kok; Tsai, Chia-Feng; Lin, Tai-Du; Tsou, Chih-Chiang; Weng, Shao-Hsing; Chen, Yi-Ju; Yang, Pan-Chyr; Arco, Susan D.; Nesvizhskii, Alexey I.; Sung, Ting-Yi; Chen, Yu-Ju

2016-01-01

Despite significant efforts in the past decade towards complete mapping of the human proteome, 3564 proteins (neXtProt, 09-2014) are still “missing proteins”. Over one-third of these missing proteins are annotated as membrane proteins, owing to their relatively challenging accessibility with standard shotgun proteomics. Using non-small cell lung cancer (NSCLC) as a model study, we aim to mine missing proteins from disease-associated membrane proteome, which may be still largely under-represented. To increase identification coverage, we employed Hp-RP StageTip pre-fractionation of membrane-enriched samples from 11 NSCLC cell lines. Analysis of membrane samples from 20 pairs of tumor and adjacent normal lung tissue were incorporated to include physiologically expressed membrane proteins. Using multiple search engines (X!Tandem, Comet and Mascot) and stringent evaluation of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66% membrane proteins) and 178 missing proteins (74 membrane proteins) with PSM-, peptide-, and protein-level FDR of 1%. Through multiple reaction monitoring (MRM) using synthetic peptides, we provided additional evidences for 8 missing proteins including 7 with transmembrane helix domains (TMH). This study demonstrates that mining missing proteins focused on cancer membrane sub-proteome can greatly contribute to map the whole human proteome. All data were deposited into ProteomeXchange with the identifier PXD002224. PMID:26202522

Integrated pipeline for mass spectrometry-based discovery and confirmation of biomarkers demonstrated in a mouse model of breast cancer.

PubMed

Whiteaker, Jeffrey R; Zhang, Heidi; Zhao, Lei; Wang, Pei; Kelly-Spratt, Karen S; Ivey, Richard G; Piening, Brian D; Feng, Li-Chia; Kasarda, Erik; Gurley, Kay E; Eng, Jimmy K; Chodosh, Lewis A; Kemp, Christopher J; McIntosh, Martin W; Paulovich, Amanda G

2007-10-01

Despite their potential to impact diagnosis and treatment of cancer, few protein biomarkers are in clinical use. Biomarker discovery is plagued with difficulties ranging from technological (inability to globally interrogate proteomes) to biological (genetic and environmental differences among patients and their tumors). We urgently need paradigms for biomarker discovery. To minimize biological variation and facilitate testing of proteomic approaches, we employed a mouse model of breast cancer. Specifically, we performed LC-MS/MS of tumor and normal mammary tissue from a conditional HER2/Neu-driven mouse model of breast cancer, identifying 6758 peptides representing >700 proteins. We developed a novel statistical approach (SASPECT) for prioritizing proteins differentially represented in LC-MS/MS datasets and identified proteins over- or under-represented in tumors. Using a combination of antibody-based approaches and multiple reaction monitoring-mass spectrometry (MRM-MS), we confirmed the overproduction of multiple proteins at the tissue level, identified fibulin-2 as a plasma biomarker, and extensively characterized osteopontin as a plasma biomarker capable of early disease detection in the mouse. Our results show that a staged pipeline employing shotgun-based comparative proteomics for biomarker discovery and multiple reaction monitoring for confirmation of biomarker candidates is capable of finding novel tissue and plasma biomarkers in a mouse model of breast cancer. Furthermore, the approach can be extended to find biomarkers relevant to human disease.

Selected reaction monitoring as an effective method for reliable quantification of disease-associated proteins in maple syrup urine disease.

PubMed

Fernández-Guerra, Paula; Birkler, Rune I D; Merinero, Begoña; Ugarte, Magdalena; Gregersen, Niels; Rodríguez-Pombo, Pilar; Bross, Peter; Palmfeldt, Johan

2014-09-01

Selected reaction monitoring (SRM) mass spectrometry can quantitatively measure proteins by specific targeting of peptide sequences, and allows the determination of multiple proteins in one single analysis. Here, we show the feasibility of simultaneous measurements of multiple proteins in mitochondria-enriched samples from cultured fibroblasts from healthy individuals and patients with mutations in branched-chain α-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial multienzyme complex and its defective activity causes maple syrup urine disease (MSUD), a rare but severe inherited metabolic disorder. Four different genes encode the catalytic subunits of BCKDH: E1α (BCKDHA), E1β (BCKDHB), E2 (DBT), and E3 (DLD). All four proteins were successfully quantified in healthy individuals. However, the E1α and E1β proteins were not detected in patients carrying mutations in one of those genes, whereas mRNA levels were almost unaltered, indicating instability of E1α and E1β monomers. Using SRM we elucidated the protein effects of mutations generating premature termination codons or misfolded proteins. SRM is a complement to transcript level measurements and a valuable tool to shed light on molecular mechanisms and on effects of pharmacological therapies at protein level. SRM is particularly effective for inherited disorders caused by multiple proteins such as defects in multienzyme complexes.

Selected reaction monitoring as an effective method for reliable quantification of disease-associated proteins in maple syrup urine disease

PubMed Central

Fernández-Guerra, Paula; Birkler, Rune I D; Merinero, Begoña; Ugarte, Magdalena; Gregersen, Niels; Rodríguez-Pombo, Pilar; Bross, Peter; Palmfeldt, Johan

2014-01-01

Selected reaction monitoring (SRM) mass spectrometry can quantitatively measure proteins by specific targeting of peptide sequences, and allows the determination of multiple proteins in one single analysis. Here, we show the feasibility of simultaneous measurements of multiple proteins in mitochondria-enriched samples from cultured fibroblasts from healthy individuals and patients with mutations in branched-chain α-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial multienzyme complex and its defective activity causes maple syrup urine disease (MSUD), a rare but severe inherited metabolic disorder. Four different genes encode the catalytic subunits of BCKDH: E1α (BCKDHA), E1β (BCKDHB), E2 (DBT), and E3 (DLD). All four proteins were successfully quantified in healthy individuals. However, the E1α and E1β proteins were not detected in patients carrying mutations in one of those genes, whereas mRNA levels were almost unaltered, indicating instability of E1α and E1β monomers. Using SRM we elucidated the protein effects of mutations generating premature termination codons or misfolded proteins. SRM is a complement to transcript level measurements and a valuable tool to shed light on molecular mechanisms and on effects of pharmacological therapies at protein level. SRM is particularly effective for inherited disorders caused by multiple proteins such as defects in multienzyme complexes. PMID:25333063

Liquid Marble Coalescence and Triggered Microreaction Driven by Acoustic Levitation.

PubMed

Chen, Zhen; Zang, Duyang; Zhao, Liang; Qu, Mengfei; Li, Xu; Li, Xiaoguang; Li, Lixin; Geng, Xingguo

2017-06-27

Liquid marbles show promising potential for application in the microreactor field. Control of the coalescence between two or among multiple liquid marbles is critical; however, the successful merging of two isolated marbles is difficult because of their mechanically robust particle shells. In this work, the coalescence of multiple liquid marbles was achieved via acoustic levitation. The dynamic behaviors of the liquid marbles were monitored by a high-speed camera. Driven by the sound field, the liquid marbles moved toward each other, collided, and eventually coalesced into a larger single marble. The underlying mechanisms of this process were probed via sound field simulation and acoustic radiation pressure calculation. The results indicated that the pressure gradient on the liquid marble surface favors the formation of a liquid bridge between the liquid marbles, resulting in their coalescence. A preliminary indicator reaction was induced by the coalescence of dual liquid marbles, which suggests that expected chemical reactions can be successfully triggered with multiple reagents contained in isolated liquid marbles via acoustic levitation.

Ultra-performance liquid chromatography tandem mass-spectrometry (uplc-ms/ms) for the rapid, simultaneous analysis of thiamin, riboflavin, flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk

USDA-ARS?s Scientific Manuscript database

A novel, rapid and sensitive Ultra Performance Liquid-Chromatography tandem Mass-Spectrometry (UPLC-MS/MS) method for the simultaneous determination of several B-vitamins in human milk was developed. Resolution by retention time or multiple reaction monitoring (MRM) for thiamin, riboflavin, flavin a...

Development of Diagnostic Biomarkers for Detecting Diabetic Retinopathy at Early Stages Using Quantitative Proteomics

PubMed Central

Min, Hophil; Kim, Sang Jin; Oh, Sohee; Kim, Kyunggon; Yu, Hyeong Gon; Park, Taesung; Kim, Youngsoo

2016-01-01

Diabetic retinopathy (DR) is a common microvascular complication caused by diabetes mellitus (DM) and is a leading cause of vision impairment and loss among adults. Here, we performed a comprehensive proteomic analysis to discover biomarkers for DR. First, to identify biomarker candidates that are specifically expressed in human vitreous, we performed data-mining on both previously published DR-related studies and our experimental data; 96 proteins were then selected. To confirm and validate the selected biomarker candidates, candidates were selected, confirmed, and validated using plasma from diabetic patients without DR (No DR) and diabetics with mild or moderate nonproliferative diabetic retinopathy (Mi or Mo NPDR) using semiquantitative multiple reaction monitoring (SQ-MRM) and stable-isotope dilution multiple reaction monitoring (SID-MRM). Additionally, we performed a multiplex assay using 15 biomarker candidates identified in the SID-MRM analysis, which resulted in merged AUC values of 0.99 (No DR versus Mo NPDR) and 0.93 (No DR versus Mi and Mo NPDR). Although further validation with a larger sample size is needed, the 4-protein marker panel (APO4, C7, CLU, and ITIH2) could represent a useful multibiomarker model for detecting the early stages of DR. PMID:26665153

Automatic identification approach for high-performance liquid chromatography-multiple reaction monitoring fatty acid global profiling.

PubMed

Tie, Cai; Hu, Ting; Jia, Zhi-Xin; Zhang, Jin-Lan

2015-08-18

Fatty acids (FAs) are a group of lipid molecules that are essential to organisms. As potential biomarkers for different diseases, FAs have attracted increasing attention from both biological researchers and the pharmaceutical industry. A sensitive and accurate method for globally profiling and identifying FAs is required for biomarker discovery. The high selectivity and sensitivity of high-performance liquid chromatography-multiple reaction monitoring (HPLC-MRM) gives it great potential to fulfill the need to identify FAs from complicated matrices. This paper developed a new approach for global FA profiling and identification for HPLC-MRM FA data mining. Mathematical models for identifying FAs were simulated using the isotope-induced retention time (RT) shift (IRS) and peak area ratios between parallel isotope peaks for a series of FA standards. The FA structures were predicated using another model based on the RT and molecular weight. Fully automated FA identification software was coded using the Qt platform based on these mathematical models. Different samples were used to verify the software. A high identification efficiency (greater than 75%) was observed when 96 FA species were identified in plasma. This FAs identification strategy promises to accelerate FA research and applications.

Development of a liquid chromatography-multiple reaction monitoring procedure for concurrent verification of exposure to different forms of mustard agents.

PubMed

Yeo, Thong-Hiang; Ho, Mer-Lin; Loke, Weng-Keong

2008-01-01

A novel liquid chromatography-multiple reaction monitoring (LC-MRM) procedure has been developed for retrospective diagnosis of exposure to different forms of mustard agents. This concise method is able to validate prior exposure to nitrogen mustards (HN-1, HN-2, and HN-3) or sulfur mustard (HD) in a single run, which significantly reduces analysis time compared to separate runs to screen for different mustards' biomarkers based on tandem mass spectrometry. Belonging to one of the more toxic classes of chemical warfare agents, these potent vesicants bind covalently to the cysteine-34 residue of human serum albumin. This results in the formation of stable adducts whose identities were confirmed by a de novo sequencing bioinformatics software package. Our developed technique tracks these albumin-derived adduct biomarkers in blood samples which persist in vitro following exposure, enabling a detection limit of 200 nM of HN-1, 100 nM of HN-2, 200 nM of HN-3, or 50 nM of HD in human blood. The CWA-adducts formed in blood samples can be conveniently and sensitively analyzed by this MRM technique to allow rapid and reliable screening.

MRMPROBS: a data assessment and metabolite identification tool for large-scale multiple reaction monitoring based widely targeted metabolomics.

PubMed

Tsugawa, Hiroshi; Arita, Masanori; Kanazawa, Mitsuhiro; Ogiwara, Atsushi; Bamba, Takeshi; Fukusaki, Eiichiro

2013-05-21

We developed a new software program, MRMPROBS, for widely targeted metabolomics by using the large-scale multiple reaction monitoring (MRM) mode. The strategy became increasingly popular for the simultaneous analysis of up to several hundred metabolites at high sensitivity, selectivity, and quantitative capability. However, the traditional method of assessing measured metabolomics data without probabilistic criteria is not only time-consuming but is often subjective and makeshift work. Our program overcomes these problems by detecting and identifying metabolites automatically, by separating isomeric metabolites, and by removing background noise using a probabilistic score defined as the odds ratio from an optimized multivariate logistic regression model. Our software program also provides a user-friendly graphical interface to curate and organize data matrices and to apply principal component analyses and statistical tests. For a demonstration, we conducted a widely targeted metabolome analysis (152 metabolites) of propagating Saccharomyces cerevisiae measured at 15 time points by gas and liquid chromatography coupled to triple quadrupole mass spectrometry. MRMPROBS is a useful and practical tool for the assessment of large-scale MRM data available to any instrument or any experimental condition.

Biomarker Development for Intraductal Papillary Mucinous Neoplasms Using Multiple Reaction Monitoring Mass Spectrometry.

PubMed

Kim, Yikwon; Kang, MeeJoo; Han, Dohyun; Kim, Hyunsoo; Lee, KyoungBun; Kim, Sun-Whe; Kim, Yongkang; Park, Taesung; Jang, Jin-Young; Kim, Youngsoo

2016-01-04

Intraductal papillary mucinous neoplasm (IPMN) is a common precursor of pancreatic cancer (PC). Much clinical attention has been directed toward IPMNs due to the increase in the prevalence of PC. The diagnosis of IPMN depends primarily on a radiological examination, but the diagnostic accuracy of this tool is not satisfactory, necessitating the development of accurate diagnostic biomarkers for IPMN to prevent PC. Recently, high-throughput targeted proteomic quantification methods have accelerated the discovery of biomarkers, rendering them powerful platforms for the evolution of IPMN diagnostic biomarkers. In this study, a robust multiple reaction monitoring (MRM) pipeline was applied to discovery and verify IPMN biomarker candidates in a large cohort of plasma samples. Through highly reproducible MRM assays and a stringent statistical analysis, 11 proteins were selected as IPMN marker candidates with high confidence in 184 plasma samples, comprising a training (n = 84) and test set (n = 100). To improve the discriminatory power, we constructed a six-protein panel by combining marker candidates. The multimarker panel had high discriminatory power in distinguishing between IPMN and controls, including other benign diseases. Consequently, the diagnostic accuracy of IPMN can be improved dramatically with this novel plasma-based panel in combination with a radiological examination.

Chromatographic peak deconvolution of constitutional isomers by multiple-reaction-monitoring mass spectrometry.

PubMed

Trapp, Oliver

2010-02-12

Highly efficient and sophisticated separation techniques are available to analyze complex compound mixtures with superior sensitivities and selectivities often enhanced by a 2nd dimension, e.g. a separation technique or spectroscopic and spectrometric techniques. For enantioselective separations numerous chiral stationary phases (CSPs) exist to cover a broad range of chiral compounds. Despite these advances enantioselective separations can become very challenging for mixtures of stereolabile constitutional isomers, because the on-column interconversion can lead to completely overlapping peak profiles. Typically, multidimensional separation techniques, e.g. multidimensional GC (MDGC), using an achiral 1st separation dimension and transferring selected analytes to a chiral 2nd separation are the method of choice to approach such problems. However, this procedure is very time consuming and only predefined sections of peaks can be transferred by column switching to the second dimension. Here we demonstrate for stereolabile 1,2-dialkylated diaziridines a technique to experimentally deconvolute overlapping gas chromatographic elution profiles of constitutional isomers based on multiple-reaction-monitoring MS (MRM-MS). The here presented technique takes advantage of different fragmentation probabilities and pathways to isolate the elution profile of configurational isomers. Copyright 2009 Elsevier B.V. All rights reserved.

Comparison of pharmacokinetic behavior of two iridoid glycosides in rat plasma after oral administration of crude Cornus officinals and its jiuzhipin by high performance liquid chromatography triple quadrupole mass spectrometry combined with multiple reactions monitoring mode

PubMed Central

Chen, Xiaocheng; Cao, Gang; Jiang, Jianping

2014-01-01

Objective: The present study examined the pharmacokinetic profiles of two iridoid glycosides named morroniside and loganin in rat plasma after oral administration of crude and processed Cornus officinals. Materials and Methods: A rapid, selective and specific high-performance liquid chromatography/electrospray ionization tandem mass spectrometry with multiple reactions monitoring mode was developed to simultaneously investigate the pharmacokinetic profiles of morroniside and loganin in rat plasma after oral administration of crude C. officinals and its jiuzhipin. Results: The morroniside and loganin in crude and processed C. officinals could be simultaneously determined within 7.4 min. Linear calibration curves were obtained over the concentration ranges of 45.45-4800 ng/mL for all the analytes. The intra-and inter-day precisions relative standard deviation was lesser than 2.84% and 4.12%, respectively. Conclusion: The pharmacokinetic parameters of two iridoid glucosides were also compared systematically between crude and processed C. officinals. This paper provides the theoretical proofs for further explaining the processing mechanism of Traditional Chinese Medicines. PMID:24914290

In vivo bioluminescence imaging of cell differentiation in biomaterials: a platform for scaffold development.

PubMed

Bagó, Juli R; Aguilar, Elisabeth; Alieva, Maria; Soler-Botija, Carolina; Vila, Olaia F; Claros, Silvia; Andrades, José A; Becerra, José; Rubio, Nuria; Blanco, Jerónimo

2013-03-01

In vivo testing is a mandatory last step in scaffold development. Agile longitudinal noninvasive real-time monitoring of stem cell behavior in biomaterials implanted in live animals should facilitate the development of scaffolds for tissue engineering. We report on a noninvasive bioluminescence imaging (BLI) procedure for simultaneous monitoring of changes in the expression of multiple genes to evaluate scaffold performance in vivo. Adipose tissue-derived stromal mensenchymal cells were dually labeled with Renilla red fluorescent protein and firefly green fluorescent protein chimeric reporters regulated by cytomegalovirus and tissue-specific promoters, respectively. Labeled cells were induced to differentiate in vitro and in vivo, by seeding in demineralized bone matrices (DBMs) and monitored by BLI. Imaging results were validated by RT-polymerase chain reaction and histological procedures. The proposed approach improves molecular imaging and measurement of changes in gene expression of cells implanted in live animals. This procedure, applicable to the simultaneous analysis of multiple genes from cells seeded in DBMs, should facilitate engineering of scaffolds for tissue repair.

In Vivo Bioluminescence Imaging of Cell Differentiation in Biomaterials: A Platform for Scaffold Development

PubMed Central

Bagó, Juli R.; Aguilar, Elisabeth; Alieva, Maria; Soler-Botija, Carolina; Vila, Olaia F.; Claros, Silvia; Andrades, José A.; Becerra, José; Rubio, Nuria

2013-01-01

In vivo testing is a mandatory last step in scaffold development. Agile longitudinal noninvasive real-time monitoring of stem cell behavior in biomaterials implanted in live animals should facilitate the development of scaffolds for tissue engineering. We report on a noninvasive bioluminescence imaging (BLI) procedure for simultaneous monitoring of changes in the expression of multiple genes to evaluate scaffold performance in vivo. Adipose tissue-derived stromal mensenchymal cells were dually labeled with Renilla red fluorescent protein and firefly green fluorescent protein chimeric reporters regulated by cytomegalovirus and tissue-specific promoters, respectively. Labeled cells were induced to differentiate in vitro and in vivo, by seeding in demineralized bone matrices (DBMs) and monitored by BLI. Imaging results were validated by RT-polymerase chain reaction and histological procedures. The proposed approach improves molecular imaging and measurement of changes in gene expression of cells implanted in live animals. This procedure, applicable to the simultaneous analysis of multiple genes from cells seeded in DBMs, should facilitate engineering of scaffolds for tissue repair. PMID:23013334

Multiplexed Quantitation of Endogenous Proteins in Dried Blood Spots by Multiple Reaction Monitoring - Mass Spectrometry

PubMed Central

Chambers, Andrew G.; Percy, Andrew J.; Yang, Juncong; Camenzind, Alexander G.; Borchers, Christoph H.

2013-01-01

Dried blood spot (DBS) sampling, coupled with multiple reaction monitoring mass spectrometry (MRM-MS), is a well-established approach for quantifying a wide range of small molecule biomarkers and drugs. This sampling procedure is simpler and less-invasive than those required for traditional plasma or serum samples enabling collection by minimally trained personnel. Many analytes are stable in the DBS format without refrigeration, which reduces the cost and logistical challenges of sample collection in remote locations. These advantages make DBS sample collection desirable for advancing personalized medicine through population-wide biomarker screening. Here we expand this technology by demonstrating the first multiplexed method for the quantitation of endogenous proteins in DBS samples. A panel of 60 abundant proteins in human blood was targeted by monitoring proteotypic tryptic peptides and their stable isotope-labeled analogs by MRM. Linear calibration curves were obtained for 40 of the 65 peptide targets demonstrating multiple proteins can be quantitatively extracted from DBS collection cards. The method was also highly reproducible with a coefficient of variation of <15% for all 40 peptides. Overall, this assay quantified 37 proteins spanning a range of more than four orders of magnitude in concentration within a single 25 min LC/MRM-MS analysis. The protein abundances of the 33 proteins quantified in matching DBS and whole blood samples showed an excellent correlation, with a slope of 0.96 and an R2 value of 0.97. Furthermore, the measured concentrations for 80% of the proteins were stable for at least 10 days when stored at −20 °C, 4 °C and 37 °C. This work represents an important first step in evaluating the integration of DBS sampling with highly-multiplexed MRM for quantitation of endogenous proteins. PMID:23221968

Negative reactions to monitoring: Do they undermine the ability of monitoring to protect adolescents?

PubMed

Laird, Robert D; Zeringue, Megan M; Lambert, Emily S

2018-02-01

This study focused on adolescents' negative reactions to parental monitoring to determine whether parents should avoid excessive monitoring because adolescents find monitoring behaviors to be over-controlling and privacy invasive. Adolescents (n = 242, M age = 15.4 years; 51% female) reported monitoring, negative reactions, warmth, antisocial behavior, depressive symptoms, and disclosure. Adolescents additionally reported antisocial behavior, depressive symptoms, and disclosure one to two years later. In cross-sectional analyses, less monitoring but more negative reactions were linked with less disclosure, suggesting that negative reactions can undermine parents' ability to obtain information. Although monitoring behaviors were not related to depressive symptoms, more negative reactions were linked with more depressive symptoms, suggesting that negative reactions also may increase depressive symptoms as a side effect of monitoring behavior. Negative reactions were not linked to antisocial behavior. There were no longitudinal links between negative reactions and changes in disclosure, antisocial behavior, or depressive symptoms. Copyright © 2017. Published by Elsevier Ltd.

Effect of collision energy optimization on the measurement of peptides by selected reaction monitoring (SRM) mass spectrometry.

PubMed

Maclean, Brendan; Tomazela, Daniela M; Abbatiello, Susan E; Zhang, Shucha; Whiteaker, Jeffrey R; Paulovich, Amanda G; Carr, Steven A; Maccoss, Michael J

2010-12-15

Proteomics experiments based on Selected Reaction Monitoring (SRM, also referred to as Multiple Reaction Monitoring or MRM) are being used to target large numbers of protein candidates in complex mixtures. At present, instrument parameters are often optimized for each peptide, a time and resource intensive process. Large SRM experiments are greatly facilitated by having the ability to predict MS instrument parameters that work well with the broad diversity of peptides they target. For this reason, we investigated the impact of using simple linear equations to predict the collision energy (CE) on peptide signal intensity and compared it with the empirical optimization of the CE for each peptide and transition individually. Using optimized linear equations, the difference between predicted and empirically derived CE values was found to be an average gain of only 7.8% of total peak area. We also found that existing commonly used linear equations fall short of their potential, and should be recalculated for each charge state and when introducing new instrument platforms. We provide a fully automated pipeline for calculating these equations and individually optimizing CE of each transition on SRM instruments from Agilent, Applied Biosystems, Thermo-Scientific and Waters in the open source Skyline software tool ( http://proteome.gs.washington.edu/software/skyline ).

MRM assay for quantitation of complement components in human blood plasma - a feasibility study on multiple sclerosis.

PubMed

Rezeli, Melinda; Végvári, Akos; Ottervald, Jan; Olsson, Tomas; Laurell, Thomas; Marko-Varga, György

2011-12-10

As a proof-of-principle study, a multiple reaction monitoring (MRM) assay was developed for quantitation of proteotypic peptides, representing seven plasma proteins associated with inflammation (complement components and C-reactive protein). The assay development and the sample analysis were performed on a linear ion trap mass spectrometer. We were able to quantify 5 of the 7 target proteins in depleted plasma digests with reasonable reproducibility over a 2 orders of magnitude linear range (RSD≤25%). The assay panel was utilized for the analysis of a small multiple sclerosis sample cohort with 10 diseased and 8 control patients. Copyright © 2011 Elsevier B.V. All rights reserved.

Rapid detection and identification of N-acetyl-L-cysteine thioethers using constant neutral loss and theoretical multiple reaction monitoring combined with enhanced product-ion scans on a linear ion trap mass spectrometer.

PubMed

Scholz, Karoline; Dekant, Wolfgang; Völkel, Wolfgang; Pähler, Axel

2005-12-01

A sensitive and specific liquid chromatography-mass spectrometry (LC-MS) method based on the combination of constant neutral loss scans (CNL) with product ion scans was developed on a linear ion trap. The method is applicable for the detection and identification of analytes with identical chemical substructures (such as conjugates of xenobiotics formed in biological systems) which give common CNLs. A specific CNL was observed for thioethers of N-acetyl-L-cysteine (mercapturic acids, MA) by LC-MS/MS. MS and HPLC parameters were optimized with 16 MAs available as reference compounds. All of these provided a CNL of 129 Da in the negative-ion mode. To assess sensitivity, a multiple reaction monitoring (MRM) mode with 251 theoretical transitions using the CNL of 129 Da combined with a product ion scan (IDA thMRM) was compared with CNL combined with a product ion scan (IDA CNL). An information-dependent acquisition (IDA) uses a survey scan such as MRM (multiple reaction monitoring) to generate "informations" and starting a second acquisition experiment such as a product ion scan using these "informations." Th-MRM means calculated transitions and not transitions generated from an available standard in the tuning mode. The product ion spectra provide additional information on the chemical structure of the unknown analytes. All MA standards were spiked in low concentrations to rat urines and were detected with both methods with LODs ranging from 60 pmol/mL to 1.63 nmol/mL with IDA thMRM. The expected product ion spectra were observed in urine. Application of this screening method to biological samples indicated the presence of a number of MAs in urine of unexposed rats, and resulted in the identification of 1,4-dihydroxynonene mercapturic acid as one of these MAs by negative and positive product ion spectra. These results show that the developed methods have a high potential to serve as both a prescreen to detect unknown MAs and to identify these analytes in complex matrix.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matz, Dallas L.; Schalnat, Matthew C.; Pemberton, Jeanne E.

The reaction between small organic molecules and low work function metals is of interest in organometallic, astronomical, and optoelectronic device chemistry. Here, thin, solid-state, amorphous benzene and pyridine films are reacted with Ca at 30 K under ultrahigh vacuum with the reaction progress monitored by Raman spectroscopy. Although both films react with Ca to produce product species identifiable by their vibrational spectroscopic signatures, benzene is less reactive with Ca than pyridine. Benzene reacts by electron transfer from Ca to benzene producing multiple species including the phenyl radical anion, the phenyl radical, and the benzyne diradical. Pyridine initially reacts along amore » similar electron transfer pathway as indicated by the presence of the corresponding pyridyl radical and pyridyne diradical species, but these pyridyl radicals are less stable and subject to further ring-opening reactions that lead to a complex array of smaller molecule reaction products and ultimately amorphous carbon. The elucidation of this reaction pathway provides insight into the reactions of aromatics with Ca that are relevant in the areas of catalysis, astrochemistry, and organic optoelectronics.« less

A rapid and practical technique for real-time monitoring of biomolecular interactions using mechanical responses of macromolecules

NASA Astrophysics Data System (ADS)

Tarhan, Mehmet C.; Lafitte, Nicolas; Tauran, Yannick; Jalabert, Laurent; Kumemura, Momoko; Perret, Grégoire; Kim, Beomjoon; Coleman, Anthony W.; Fujita, Hiroyuki; Collard, Dominique

2016-06-01

Monitoring biological reactions using the mechanical response of macromolecules is an alternative approach to immunoassays for providing real-time information about the underlying molecular mechanisms. Although force spectroscopy techniques, e.g. AFM and optical tweezers, perform precise molecular measurements at the single molecule level, sophisticated operation prevent their intensive use for systematic biosensing. Exploiting the biomechanical assay concept, we used micro-electro mechanical systems (MEMS) to develop a rapid platform for monitoring bio/chemical interactions of bio macromolecules, e.g. DNA, using their mechanical properties. The MEMS device provided real-time monitoring of reaction dynamics without any surface or molecular modifications. A microfluidic device with a side opening was fabricated for the optimal performance of the MEMS device to operate at the air-liquid interface for performing bioassays in liquid while actuating/sensing in air. The minimal immersion of the MEMS device in the channel provided long-term measurement stability (>10 h). Importantly, the method allowed monitoring effects of multiple solutions on the same macromolecule bundle (demonstrated with DNA bundles) without compromising the reproducibility. We monitored two different types of effects on the mechanical responses of DNA bundles (stiffness and viscous losses) exposed to pH changes (2.1 to 4.8) and different Ag+ concentrations (1 μM to 0.1 M).

Accurate hybrid stochastic simulation of a system of coupled chemical or biochemical reactions.

PubMed

Salis, Howard; Kaznessis, Yiannis

2005-02-01

The dynamical solution of a well-mixed, nonlinear stochastic chemical kinetic system, described by the Master equation, may be exactly computed using the stochastic simulation algorithm. However, because the computational cost scales with the number of reaction occurrences, systems with one or more "fast" reactions become costly to simulate. This paper describes a hybrid stochastic method that partitions the system into subsets of fast and slow reactions, approximates the fast reactions as a continuous Markov process, using a chemical Langevin equation, and accurately describes the slow dynamics using the integral form of the "Next Reaction" variant of the stochastic simulation algorithm. The key innovation of this method is its mechanism of efficiently monitoring the occurrences of slow, discrete events while simultaneously simulating the dynamics of a continuous, stochastic or deterministic process. In addition, by introducing an approximation in which multiple slow reactions may occur within a time step of the numerical integration of the chemical Langevin equation, the hybrid stochastic method performs much faster with only a marginal decrease in accuracy. Multiple examples, including a biological pulse generator and a large-scale system benchmark, are simulated using the exact and proposed hybrid methods as well as, for comparison, a previous hybrid stochastic method. Probability distributions of the solutions are compared and the weak errors of the first two moments are computed. In general, these hybrid methods may be applied to the simulation of the dynamics of a system described by stochastic differential, ordinary differential, and Master equations.

Nonthermal microwave effects revisited: on the importance of internal temperature monitoring and agitation in microwave chemistry.

PubMed

Herrero, M Antonia; Kremsner, Jennifer M; Kappe, C Oliver

2008-01-04

The concept of nonthermal microwave effects has received considerable attention in recent years and is the subject of intense debate in the scientific community. Nonthermal microwave effects have been postulated to result from a direct stabilizing interaction of the electric field with specific (polar) molecules in the reaction medium that is not related to a macroscopic temperature effect. In order to probe the existence of nonthermal microwave effects, four synthetic transformations (Diels-Alder cycloaddition, alkylation of triphenylphosphine and 1,2,4-triazole, direct amide bond formation) were reevaluated under both microwave dielectric heating and conventional thermal heating. In all four cases, previous studies have claimed the existence of nonthermal microwave effects in these reactions. Experimentally, significant differences in conversion and/or product distribution comparing the conventionally and microwave-heated experiments performed at the same measured reaction temperature were found. The current reevaluation of these reactions was performed in a dedicated reactor setup that allowed accurate internal reaction temperature measurements using a multiple fiber-optic probe system. Using this technology, the importance of efficient stirring and internal temperature measurement in microwave-heated reactions was made evident. Inefficient agitation leads to temperature gradients within the reaction mixture due to field inhomogeneities in the microwave cavity. Using external infrared temperature sensors in some cases results in significant inaccuracies in the temperature measurement. Applying the fiber-optic probe temperature monitoring device, a critical reevaluation of all four reactions has provided no evidence for the existence of nonthermal microwave effects. Ensuring efficient agitation of the reaction mixture via magnetic stirring, no significant differences in terms of conversion and selectivity between experiments performed under microwave or oil bath conditions at the same internally measured reaction temperatures were experienced. The observed effects were purely thermal and not related to the microwave field.

Catalytic Reduction of 4-Nitrophenol: A Quantitative Assessment of the Role of Dissolved Oxygen in Determining the Induction Time.

PubMed

Menumerov, Eredzhep; Hughes, Robert A; Neretina, Svetlana

2016-12-14

The reduction of 4-nitrophenol to 4-aminophenol by borohydride is one of the foremost model catalytic reactions because it allows for a straightforward assessment of catalysts using the kinetic parameters extracted from the real-time spectroscopic monitoring of an aqueous solution. Crucial to its standing as a model reaction is a comprehensive mechanistic framework able to explain the entire time evolution of the reaction. While much of this framework is in place, there is still much debate over the cause of the induction period, an initial time interval where no reaction seemingly occurs. Here, we report on the simultaneous monitoring of the spectroscopic signal and the dissolved oxygen content within the aqueous solution. It reveals that the induction period is the time interval required for the level of dissolved oxygen to fall below a critical value that is dependent upon whether Au, Ag, or Pd nanoparticles are used as the catalyst. With this understanding, we are able to exert complete control over the induction period, being able to eliminate it, extend it indefinitely, or even induce multiple induction periods over the course of a single reaction. Moreover, we have determined that the reaction product, 4-aminophenol, in the presence of the same catalyst reacts with dissolved oxygen to form 4-nitrophenolate. The implication of these results is that the induction period relates, not to some activation of the catalyst, but to a time interval where the reaction product is being rapidly transformed back into a reactant by a side reaction.

Unravelling RNA-substrate interactions in a ribozyme-catalysed reaction using fluorescent turn-on probes.

PubMed

Gaffarogullari, Ece Cazibe; Greulich, Peter; Kobitski, Andrei Yu; Nierth, Alexander; Nienhaus, G Ulrich; Jäschke, Andres

2015-04-07

The Diels-Alder reaction is one of the most important C-C bond-forming reactions in organic chemistry, and much effort has been devoted to controlling its enantio- and diastereoselectivity. The Diels-Alderase ribozyme (DAse) catalyses the reaction between anthracene dienes and maleimide dienophiles with multiple-turnover, stereoselectivity, and up to 1100-fold rate acceleration. Here, a new generation of anthracene-BODIPY-based fluorescent probes was developed to monitor catalysis by the DAse. The brightness of these probes increases up to 93-fold upon reaction with N-pentylmaleimide (NPM), making these useful tools for investigating the stereochemistry of the ribozyme-catalysed reaction. With these probes, we observed that the DAse catalyses the reaction with >91% de and >99% ee. The stereochemistry of the major product was determined unambiguously by rotating-frame nuclear Overhauser NMR spectroscopy (ROESY-NMR) and is in agreement with crystallographic structure information. The pronounced fluorescence change of the probes furthermore allowed a complete kinetic analysis, which revealed an ordered bi uni type reaction mechanism, with the dienophile binding first. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sub-Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single-Molecule FRET.

PubMed

Baltierra-Jasso, Laura E; Morten, Michael J; Magennis, Steven W

2018-03-05

Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here, we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constant of 10 m -1  s -1 . We also followed the displacement from a DNA three-way junction (3WJ) by ssDNA. The presence of three internal mismatched bases in the middle of the invading strand did not prevent displacement from the 3WJ, but reduced the second-order rate constant by about 50 %. We attribute strand exchange in the dsDNA and 3WJ to a zero-toehold pathway from the blunt-ended duplex arms. The single-molecule approach demonstrated here will be useful for studying complex DNA networks. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Improved Long-term Outcomes of Patients With Inflammatory Bowel Disease Receiving Proactive Compared With Reactive Monitoring of Serum Concentrations of Infliximab.

PubMed

Papamichael, Konstantinos; Chachu, Karen A; Vajravelu, Ravy K; Vaughn, Byron P; Ni, Josephine; Osterman, Mark T; Cheifetz, Adam S

2017-10-01

Monitoring serum concentrations of tumor necrosis factor antagonists in patients receiving these drugs as treatment for inflammatory bowel disease (IBD), also called therapeutic drug monitoring, is performed either after patient loss of response (reactive drug monitoring) or in patients in clinical remission in which the drug is titrated to a target concentration (proactive drug monitoring). We compared long-term outcomes of patients with IBD undergoing proactive vs reactive monitoring of serum concentrations of infliximab. We performed a multicenter, retrospective study of 264 consecutive patients with IBD (167 with Crohn's disease) receiving infliximab maintenance therapy. The subjects received proactive (n = 130) or reactive (n = 134) drug monitoring, based on measurements of first infliximab concentration and antibodies to infliximab, from September 2006 to January 2015; they were followed through December 2015 (median time of 2.4 years). We analyzed time to treatment failure, first IBD-related surgery or hospitalization, serious infusion reaction, and detection of antibodies to infliximab. Treatment failure was defined as drug discontinuation for loss of response or serious adverse event, or need for surgery. Multiple Cox regression analysis independently associated proactive drug monitoring, compared with reactive monitoring, with reduced risk for treatment failure (hazard ratio [HR], 0.16; 95% confidence interval [CI], 0.09-0.27; P < .001), IBD-related surgery (HR, 0.30; 95% CI, 0.11-0.80; P = .017), IBD-related hospitalization (HR, 0.16; 95% CI, 0.07-0.33; P < .001), antibodies to infliximab (HR, 0.25; 95% CI, 0.07-0.84; P = .025), and serious infusion reaction (HR, 0.17; 95% CI, 0.04-0.78; P = .023). In a retrospective analysis of patients with IBD receiving proactive vs reactive monitoring of serum concentration of infliximab, proactive monitoring was associated with better clinical outcomes, including greater drug durability, less need for IBD-related surgery or hospitalization, and lower risk of antibodies to infliximab or serious infusion reactions. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Mass spectrometric directed system for the continuous-flow synthesis and purification of diphenhydramine† †Electronic supplementary information (ESI) available: NMR spectra of selected product, mass spectra of selected products, crystallization information, and experimental procedures are supplied. See DOI: 10.1039/c7sc00905d Click here for additional data file.

PubMed Central

Loren, Bradley P.; Wleklinski, Michael; Koswara, Andy; Yammine, Kathryn; Hu, Yanyang

2017-01-01

A highly integrated approach to the development of a process for the continuous synthesis and purification of diphenhydramine is reported. Mass spectrometry (MS) is utilized throughout the system for on-line reaction monitoring, off-line yield quantitation, and as a reaction screening module that exploits reaction acceleration in charged microdroplets for high throughput route screening. This effort has enabled the discovery and optimization of multiple routes to diphenhydramine in glass microreactors using MS as a process analytical tool (PAT). The ability to rapidly screen conditions in charged microdroplets was used to guide optimization of the process in a microfluidic reactor. A quantitative MS method was developed and used to measure the reaction kinetics. Integration of the continuous-flow reactor/on-line MS methodology with a miniaturized crystallization platform for continuous reaction monitoring and controlled crystallization of diphenhydramine was also achieved. Our findings suggest a robust approach for the continuous manufacture of pharmaceutical drug products, exemplified in the particular case of diphenhydramine, and optimized for efficiency and crystal size, and guided by real-time analytics to produce the agent in a form that is readily adapted to continuous synthesis. PMID:28979759

Metabolism and bioactivation of the tricyclic antidepressant amitriptyline in human liver microsomes and human urine.

PubMed

Zhou, Xin; Chen, Chang; Zhang, Fangrong; Zhang, Yang; Feng, Yuling; Ouyang, Hui; Xu, Yong; Jiang, Hongliang

2016-07-01

Amitriptyline is a widely used tricyclic antidepressant, but the metabolic studies were conducted almost 20 years ago using high-performance liquid chromatography coupled with ultraviolet detector or radiolabeled methods. First, multiple ion monitoring (MIM)- enhanced product ion (EPI) scan was used to obtain the diagnostic ions or neutral losses in human liver microsome incubations with amitriptyline. Subsequently, predicted multiple reaction monitoring (MRM)-EPI scan was used to identify the metabolites in human urine with the diagnostic ions or neutral losses. Finally, product ion filtering and neutral loss filtering were used as the data mining tools to screen metabolites. Consequently, a total of 28 metabolites were identified in human urine after an oral administration using LC-MS/MS. An integrated workflow using LC-MS/MS was developed to comprehensively profile the metabolites of amitriptyline in human urine, in which five N-acetyl-l-cysteine conjugates were characterized as tentative biomarkers for idiosyncratic toxicity.

Variation and quantification among a target set of phosphopeptides in human plasma by multiple reaction monitoring (MRM) and SWATH MS2 data-independent acquisition

PubMed Central

Zawadzka, Anna M.; Schilling, Birgit; Held, Jason M.; Sahu, Alexandria K.; Cusack, Michael P.; Drake, Penelope M.; Fisher, Susan J.; Gibson, Bradford W.

2015-01-01

Human plasma contains proteins that reflect overall health and represents a rich source of proteins for identifying and understanding disease pathophysiology. However, few studies have investigated changes in plasma phosphoproteins. In addition, little is known about the normal variations in these phosphoproteins, especially with respect to specific sites of modification. To address these questions, we evaluated variability in plasma protein phosphorylation in healthy individuals using multiple reaction monitoring (MRM) and SWATH MS2 data-independent acquisition. First, we developed a discovery workflow for phosphopeptide enrichment from plasma and identified targets for MRM assays. Next, we analyzed plasma from healthy donors using an analytical workflow consisting of MRM and SWATH MS2 that targeted phosphopeptides from 58 and 68 phosphoproteins, respectively. These two methods produced similar results showing low variability in 13 phosphosites from 10 phosphoproteins (CVinter <30%) and high interpersonal variation of 16 phosphosites from 14 phosphoproteins (CVinter >30%). Moreover, these phosphopeptides originate from phosphoproteins involved in cellular processes governing homeostasis, immune response, cell-extracellular matrix interactions, lipid and sugar metabolism, and cell signaling. This limited assessment of technical and biological variability in phosphopeptides generated from plasma phosphoproteins among healthy volunteers constitutes a reference for future studies that target protein phosphorylation as biomarkers. PMID:24853916

Mass spectrometry-based targeted quantitative proteomics: achieving sensitive and reproducible detection of proteins.

PubMed

Boja, Emily S; Rodriguez, Henry

2012-04-01

Traditional shotgun proteomics used to detect a mixture of hundreds to thousands of proteins through mass spectrometric analysis, has been the standard approach in research to profile protein content in a biological sample which could lead to the discovery of new (and all) protein candidates with diagnostic, prognostic, and therapeutic values. In practice, this approach requires significant resources and time, and does not necessarily represent the goal of the researcher who would rather study a subset of such discovered proteins (including their variations or posttranslational modifications) under different biological conditions. In this context, targeted proteomics is playing an increasingly important role in the accurate measurement of protein targets in biological samples in the hope of elucidating the molecular mechanism of cellular function via the understanding of intricate protein networks and pathways. One such (targeted) approach, selected reaction monitoring (or multiple reaction monitoring) mass spectrometry (MRM-MS), offers the capability of measuring multiple proteins with higher sensitivity and throughput than shotgun proteomics. Developing and validating MRM-MS-based assays, however, is an extensive and iterative process, requiring a coordinated and collaborative effort by the scientific community through the sharing of publicly accessible data and datasets, bioinformatic tools, standard operating procedures, and well characterized reagents. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid determination of alkaloids in Macleaya cordata using ionic liquid extraction followed by multiple reaction monitoring UPLC-MS/MS analysis.

PubMed

Li, Linqiu; Huang, Mingyuan; Shao, Junli; Lin, Bokun; Shen, Qing

2017-02-20

The ultrasonic-assisted extraction (UAE) and ionic liquid based dispersive liquid-liquid microextraction (IL-DLLME) have been successfully applied in extracting of six alkaloids from M. cordata. 1-hexyl-3-methylimidazolium tetrafluoroborate ([C 6 MIM][BF 4 ]) aqueous solution was used as extraction solvent. The target analytes in raw material were deposited into a single drop of 1-hexyl-3-methylimidazolium hexafluorophosphate ([C 6 MIM][PF 6 ]), which was in situ formed by mixing [C 6 MIM][BF 4 ] and potassium hexafluorophosphate ([K][PF 6 ]. Afterwards, the extract was analyzed by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) in multiple-reaction monitoring (MRM) mode. The proposed method was fully validated in terms of linearity (0.9983-0.9992), LOD (0.080ngmL -1 ), LOQ (0.25ngmL -1 ), intra-day precision (<5.46%), inter-day precision (<6.36%), and recovery (86.42-112.48%). The results indicate that the approach of combining IL-DLLME with UPLC-MS/MS is powerful and practical for analyzing alkaloids in M. cordata., and it also has great potential for comprehensive quality control of other herbal medicines. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification and determination of fat-soluble vitamins and metabolites in human serum by liquid chromatography/triple quadrupole mass spectrometry with multiple reaction monitoring.

PubMed

Priego Capote, Feliciano; Jiménez, José Ruiz; Granados, José María Mata; de Castro, María Dolores Luque

2007-01-01

A method for determination of fat-soluble vitamins K(1), K(3), A, D(2), D(3) and E (as alpha- and delta-tocopherol) and metabolites 25-hydroxyvitamin D(2) and D(3) and 1,25-dihydroxyvitamin D(3) in human serum by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in positive mode is proposed. Highly selective identification of the target compounds in serum was confirmed by the most representative transitions from precursor ion to product ion. Quantitative MS/MS analysis was carried out by multiple reaction monitoring optimizing the most sensitive transition for each analyte in order to achieve low detection limits (from 0.012 to 0.3 ng/mL estimated with serum). The analysis was performed with 1 mL of serum, which was subjected to protein precipitation, liquid-liquid extraction to an organic phase, evaporation to dryness and reconstitution with methanol. The precision of the overall method ranged from 3.17-6.76% as intra-day variability and from 5.07-11.53% as inter-day variability. The method, validated by the standard addition method, provides complete information on the fat-soluble vitamins profile, which is of interest in clinical and metabolomics studies.

What is in a beer? Proteomic characterization and relative quantification of hordein (gluten) in beer.

PubMed

Colgrave, Michelle L; Goswami, Hareshwar; Howitt, Crispin A; Tanner, Gregory J

2012-01-01

The suite of prolamin proteins present in barley flour was characterized in this study, in which we provide spectral evidence for 3 previously characterized prolamins, 8 prolamins with only transcript evidence, and 19 genome-derived predicted prolamins. An additional 9 prolamins were identified by searching the complete spectral set against an unannotated translated EST database. Analyses of wort, the liquid extracted from the mashing process during beer production, and beer were undertaken and a similar suite of prolamins were identified. We have demonstrated by using tandem mass spectrometry that hordeins are indeed present in beer despite speculation to the contrary. Multiple reaction monitoring (MRM) mass spectrometry was used for the rapid analyses of hordein in barley (Hordeum vulgare L.) beer. A selection of international beers were analyzed and compared to the results obtained with hordein deletion beers. The hordein deletion beers were brewed from grains carrying mutations that prevented the accumulation of either B-hordeins (Risø 56) or C-hordeins (Risø 1508). No intact C-hordeins were detected in beer, although fragments of C-hordeins were present in wort. Multiple reaction monitoring analysis of non-barley based gluten (hordein)-free beers targeting the major hordein protein families was performed and confirmed the absence of hordein in several gluten-free commercial beers.

Monitoring mechanistic details in the synthesis of pyrimidines via real-time, ultrafast multidimensional NMR spectroscopy

PubMed Central

Pardo, Zulay D.; Olsen, Greg; Fernández-Valle, María Encarnación; Frydman, Lucio; Martínez-Álvarez, Roberto; Herrera, Antonio

2016-01-01

Recent years have witnessed unprecedented advances in the development of fast multidimensional NMR acquisition techniques. This progress could open valuable new opportunities for the elucidation of chemical and biochemical processes. This study demonstrates one such capability, with the first real-time 2D dynamic analysis of a complex organic reaction relying on unlabeled substrates. Implementing such measurements required the development of new ultrafast 2D methods, capable of monitoring multiple spectral regions of interest as the reaction progressed. The alternate application of these acquisitions in an interleaved, excitation-optimized fashion, allowed us to extract new structural and dynamic insight concerning the reaction between aliphatic ketones and triflic anhydride in the presence of nitriles to yield alkylpyrimidines. Up to 2500 2D NMR data sets were thus collected over the course of this nearly 100 min long reaction, in an approach resembling that used in functional magnetic resonance imaging. With the aid of these new frequency-selective low-gradient-strength experiments, supplemented by chemical shift calculations of the spectral coordinates observed in the 2D heteronuclear correlations, previously postulated intermediates involved in the alkylpyrimidine formation process could be confirmed, and hitherto undetected ones were revealed. The potential and limitations of the resulting methods are discussed. PMID:22283498

Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification.

PubMed

Wang, Yi; Wang, Yan; Ma, Ai-Jing; Li, Dong-Xun; Luo, Li-Juan; Liu, Dong-Xin; Jin, Dong; Liu, Kai; Ye, Chang-Yun

2015-07-08

We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61-65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique.

A Demonstration of Concrete Structural Health Monitoring Framework for Degradation due to Alkali-Silica Reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mahadevan, Sankaran; Agarwal, Vivek; Neal, Kyle

Assessment and management of aging concrete structures in nuclear power plants require a more systematic approach than simple reliance on existing code margins of safety. Structural health monitoring of concrete structures aims to understand the current health condition of a structure based on heterogeneous measurements to produce high-confidence actionable information regarding structural integrity that supports operational and maintenance decisions. This ongoing research project is seeking to develop a probabilistic framework for health diagnosis and prognosis of aging concrete structures in a nuclear power plant that is subjected to physical, chemical, environment, and mechanical degradation. The proposed framework consists of fourmore » elements: monitoring, data analytics, uncertainty quantification and prognosis. This report focuses on degradation caused by ASR (alkali-silica reaction). Controlled specimens were prepared to develop accelerated ASR degradation. Different monitoring techniques – thermography, digital image correlation (DIC), mechanical deformation measurements, nonlinear impact resonance acoustic spectroscopy (NIRAS), and vibro-acoustic modulation (VAM) -- were used to detect the damage caused by ASR. Heterogeneous data from the multiple techniques was used for damage diagnosis and prognosis, and quantification of the associated uncertainty using a Bayesian network approach. Additionally, MapReduce technique has been demonstrated with synthetic data. This technique can be used in future to handle large amounts of observation data obtained from the online monitoring of realistic structures.« less

Monitoring and control of spacecraft systems using procedural reasoning

NASA Technical Reports Server (NTRS)

Georgeff, Michael P.; Ingrand, Francois Felix

1990-01-01

Research concerned with automating the monitoring and control of spacecraft systems is discussed. In particular, the application of SRI's Procedural Reasoning System (PRS) to the handling of malfunctions in the Reaction Control System (RCS) of NASA's Space Shuttle is examined. Unlike traditional monitoring and control systems, PRS is able to reason about and perform complex tasks in a very flexible and robust manner, somewhat in the manner of a human assistant. Using various RCS malfunctions as examples (including sensor faults, leaking components, multiple alarms, and regulator and jet failures), it is shown how PRS manages to combine both goal-directed reasoning and the ability to react rapidly to unanticipated changes in its environment. In conclusion, some important issues in the design of PRS are reviewed and future enhancements are indicated.

NCI Launches Proteomics Assay Portal | Office of Cancer Clinical Proteomics Research

Cancer.gov

In a paper recently published by the journal Nature Methods, Investigators from the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (NCI-CPTAC) announced the launch of a proteomics Assay Portal for multiple reaction monitoring-mass spectrometry (MRM-MS) assays.  This community web-based repository for well-characterized quantitative proteomic assays currently consists of 456 unique peptide assays to 282 unique proteins and ser

Molecular Profiles for Lung Cancer Pathogenesis and Detection in US Veterans

DTIC Science & Technology

2012-10-01

will be further strengthened via Multiple Reaction Monitoring ( MRM ) performed on the remaining samples by the Vanderbilt group. MRM using mass...proteomics detects all protein changes in the sample in an unfocused fashion, MRM is targeted and highly selective, allowing us to specifically look for...proteins of interest. To this end, we have generated a list of candidate proteins for MRM utilizing shotgun proteomic, mRNA array, and miRNA array

Intact protein analysis of ubiquitin in cerebrospinal fluid by multiple reaction monitoring reveals differences in Alzheimer's disease and frontotemporal lobar degeneration.

PubMed

Oeckl, Patrick; Steinacker, Petra; von Arnim, Christine A F; Straub, Sarah; Nagl, Magdalena; Feneberg, Emily; Weishaupt, Jochen H; Ludolph, Albert C; Otto, Markus

2014-11-07

The impairment of the ubiquitin-proteasome system (UPS) is thought to be an early event in neurodegeneration, and monitoring UPS alterations might serve as a disease biomarker. Our aim was to establish an alternate method to antibody-based assays for the selective measurement of free monoubiquitin in cerebrospinal fluid (CSF). Free monoubiquitin was measured with liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MS/MS) in CSF of patients with Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), behavioral variant of frontotemporal dementia (bvFTD), Creutzfeldt-Jakob disease (CJD), Parkinson's disease (PD), primary progressive aphasia (PPA), and progressive supranuclear palsy (PSP). The LC-MS/MS method showed excellent intra- and interassay precision (4.4-7.4% and 4.9-10.3%) and accuracy (100-107% and 100-106%). CSF ubiquitin concentration was increased compared with that of controls (33.0 ± 9.7 ng/mL) in AD (47.5 ± 13.1 ng/mL, p < 0.05) and CJD patients (171.5 ± 103.5 ng/mL, p < 0.001) but not in other neurodegenerative diseases. Receiver operating characteristic curve (ROC) analysis of AD vs control patients revealed an area under the curve (AUC) of 0.832, and the specificity and sensitivity were 75 and 75%, respectively. ROC analysis of AD and FTLD patients yielded an AUC of 0.776, and the specificity and sensitivity were 53 and 100%, respectively. In conclusion, our LC-MS/MS method may facilitate ubiquitin determination to a broader community and might help to discriminate AD, CJD, and FTLD patients.

Mass Spectrometric Immunoassay and Multiple Reaction Monitoring as Targeted MS-based Quantitative Approaches in Biomarker Development: Potential Applications to Cardiovascular Disease and Diabetes

PubMed Central

Yassine, Hussein; Borges, Chad R.; Schaab, Matthew R.; Billheimer, Dean; Stump, Craig; Reaven, Peter; Lau, Serrine S.; Nelson, Randall

2014-01-01

Type 2 diabetes (T2DM) is an important risk factor for cardiovascular disease (CVD)—the leading cause of death in the US. Yet not all subjects with T2DM are at equal risk for CVD complications; the challenge lies in identifying those at greatest risk. Therapies directed towards treating conventional risk factors have failed to significantly reduce this residual risk in T2DM patients. Thus newer targets and markers are needed for the development and testing of novel therapies. Herein we review two complementary mass spectrometry-based approaches—Mass Spectrometric Immunoassay (MSIA) and tandem mass spectrometry as multiple reaction monitoring (MRM)—for the analysis of plasma proteins and post translational modifications (PTMs) of relevance to T2DM and CVD. Together, these complementary approaches allow for high-throughput monitoring of many PTMs and the absolute quantification of proteins near the low picomolar range. In this review article, we discuss the clinical relevance of the HDL proteome and Apolipoprotein A-I PTMs to T2DM and CVD as well as provide illustrative MSIA and MRM data on high density lipoprotein (HDL) proteins from T2DM patients to provide examples of how these mass spectrometry approaches can be applied to gain new insight regarding cardiovascular risk factors. Also discussed are the reproducibility, interpretation and limitations of each technique with an emphasis on their capacities to facilitate the translation of new biomarkers into clinical practice. PMID:23696124

A Sensitive Dilute-and-Shoot Approach for the Simultaneous Screening of 71 Stimulants and 7 Metabolites in Human Urine by LC-MS-MS with Dynamic MRM.

PubMed

Dong, Ying; Yan, Kuan; Ma, Yanhua; Wang, Shan; He, Genye; Deng, Jing; Yang, Zhiyong

2015-10-01

A novel, reliable and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed with dynamic multiple reaction monitoring (dMRM) mode for the simultaneous screening of 71 stimulants and 7 metabolites in human urine using unsophisticated MS instruments (Agilent triple-quadruple 6410 B mass spectrometer). With a known retention time of an analyte, dMRM algorithm monitors each MRM transition only around its expected retention time. Therefore, dMRM enables the maximization of dwell times and provides much higher sensitivity and reproducibility than the conventional multiple reaction monitoring mode (cMRM). After precipitation of protein, the urine sample was injected into LC-MS-MS system directly without sample pre-concentration. For comparison, cMRM and dMRM acquisitions were performed under the same chromatographic conditions. The result showed that the signal response and quality of the chromatograms for each stimulant improved significantly with dMRM over cMRM. The method has been fully validated giving limits of detection (0.1-25 ng/mL) satisfactory for its application to anti-doping analysis. The repeatability of the concentrations and the retention times are good both for intra- and for inter-day experiments (%CV of concentrations always <20 and %CV of retention times <0.5). The method also afforded satisfactory results in terms of accuracy, matrix effect and specificity. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Latex Allergies: A Review of Recognition, Evaluation, Management, Prevention, Education, and Alternative Product Use

PubMed Central

Schroyer, Traci; Catalfano, Jennifer

2003-01-01

Objectives: To provide information about latex allergies; to determine how to recognize, evaluate, and manage emergencies related to latex allergies; and how to identify those at risk for latex allergies. Additionally, ways to prevent latex exposure, to educate health care workers and athletes about latex allergy, and to provide safe alternatives to latex are investigated. Data Sources: We searched MEDLINE and SPORT Discus for the years 1998–2002 using the key words latex, allergies, rubber, anaphylaxis, gloves, cross-reaction, IgE (immunoglobulin G) proteins, and radioallergosorbent test (RAST). Background: Latex, a sap from the rubber tree, is found in many products used in everyday life. Latex is composed of compounds that may cause an allergic reaction, whose severity can range from irritant dermatitis to type IV dermatitis to type I systemic reaction. Recognition of the signs and symptoms associated with these reactions by the health care professional may help to prevent a more severe reaction from occurring. Reactions can be complicated by contact with other substances, thus causing a cross-reaction. Some individuals are more at risk of latex allergies due to repetitive exposure to latex through their career paths, multiple surgeries, other allergies, or respiratory conditions. Management of an acute reaction involves removal of the irritant, cleansing of the affected area, monitoring vital signs for changes, and seeking additional medical assistance as warranted. Recommendations: Those at risk may be identified through a thorough medical history and allergy testing. Prevention techniques and guidelines are examined, with an emphasis on education at multiple levels. Product information for nonlatex equipment and supplies for the athletic training room is offered, with additional resource information provided. PMID:16558678

RNA–protein binding kinetics in an automated microfluidic reactor

PubMed Central

Ridgeway, William K.; Seitaridou, Effrosyni; Phillips, Rob; Williamson, James R.

2009-01-01

Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic ‘Riboreactor’ has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome. PMID:19759214

Detecting molecular forms of antithrombin by LC-MRM-MS: defining the measurands.

PubMed

Ruhaak, L Renee; Romijn, Fred P H T M; Smit, Nico P M; van der Laarse, Arnoud; Pieterse, Mervin M; de Maat, Moniek P M; Haas, Fred J L M; Kluft, Cornelis; Amiral, Jean; Meijer, Piet; Cobbaert, Christa M

2018-05-01

Antithrombin (AT) is a critical regulator of coagulation, and its overall activity is typically measured using functional tests. A large number of molecular forms of AT have been identified and each individual carries multiple molecular proteoforms representing variable activities. Conventional functional tests are completely blind for these proteoforms. A method that ensures properly defined measurands for AT is therefore needed. We here assess whether mass spectrometry technology, in particular multiple reaction monitoring (MRM), is suitable for the quantification of AT and the qualitative detection of its molecular proteoforms. Plasma proteins were denatured, reduced and alkylated prior to enzymatic digestion. MRM transitions were developed towards tryptic peptides and glycopeptides using AT purified from human plasma. For each peptide, three transitions were measured, and stable isotope-labeled peptides were used for quantitation. Completeness of digestion was assessed using digestion time curves. MRM transitions were developed for 19 tryptic peptides and 4 glycopeptides. Two peptides, FDTISEK and FATTFYQHLADSK, were used for quantitation, and using a calibration curve of isolated AT in 40 g/L human serum albumin, CVs below 3.5% were obtained for FDTISEK, whereas CVs below 8% were obtained for FATTFYQHLADSK. Of the 26 important AT mutations, 20 can be identified using this method, while altered glycosylation profiles can also be detected. We here show the feasibility of the liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) technique for the quantitation of AT and the qualitative analysis of most of its molecular proteoforms. Knowing the measurands will enable standardization of AT tests by providing in-depth information on the molecular proteoforms of AT.

Automated detection of inaccurate and imprecise transitions in peptide quantification by multiple reaction monitoring mass spectrometry.

PubMed

Abbatiello, Susan E; Mani, D R; Keshishian, Hasmik; Carr, Steven A

2010-02-01

Multiple reaction monitoring mass spectrometry (MRM-MS) of peptides with stable isotope-labeled internal standards (SISs) is increasingly being used to develop quantitative assays for proteins in complex biological matrices. These assays can be highly precise and quantitative, but the frequent occurrence of interferences requires that MRM-MS data be manually reviewed, a time-intensive process subject to human error. We developed an algorithm that identifies inaccurate transition data based on the presence of interfering signal or inconsistent recovery among replicate samples. The algorithm objectively evaluates MRM-MS data with 2 orthogonal approaches. First, it compares the relative product ion intensities of the analyte peptide to those of the SIS peptide and uses a t-test to determine if they are significantly different. A CV is then calculated from the ratio of the analyte peak area to the SIS peak area from the sample replicates. The algorithm identified problematic transitions and achieved accuracies of 94%-100%, with a sensitivity and specificity of 83%-100% for correct identification of errant transitions. The algorithm was robust when challenged with multiple types of interferences and problematic transitions. This algorithm for automated detection of inaccurate and imprecise transitions (AuDIT) in MRM-MS data reduces the time required for manual and subjective inspection of data, improves the overall accuracy of data analysis, and is easily implemented into the standard data-analysis work flow. AuDIT currently works with results exported from MRM-MS data-processing software packages and may be implemented as an analysis tool within such software.

Automated Detection of Inaccurate and Imprecise Transitions in Peptide Quantification by Multiple Reaction Monitoring Mass Spectrometry

PubMed Central

Abbatiello, Susan E.; Mani, D. R.; Keshishian, Hasmik; Carr, Steven A.

2010-01-01

BACKGROUND Multiple reaction monitoring mass spectrometry (MRM-MS) of peptides with stable isotope–labeled internal standards (SISs) is increasingly being used to develop quantitative assays for proteins in complex biological matrices. These assays can be highly precise and quantitative, but the frequent occurrence of interferences requires that MRM-MS data be manually reviewed, a time-intensive process subject to human error. We developed an algorithm that identifies inaccurate transition data based on the presence of interfering signal or inconsistent recovery among replicate samples. METHODS The algorithm objectively evaluates MRM-MS data with 2 orthogonal approaches. First, it compares the relative product ion intensities of the analyte peptide to those of the SIS peptide and uses a t-test to determine if they are significantly different. A CV is then calculated from the ratio of the analyte peak area to the SIS peak area from the sample replicates. RESULTS The algorithm identified problematic transitions and achieved accuracies of 94%–100%, with a sensitivity and specificity of 83%–100% for correct identification of errant transitions. The algorithm was robust when challenged with multiple types of interferences and problematic transitions. CONCLUSIONS This algorithm for automated detection of inaccurate and imprecise transitions (AuDIT) in MRM-MS data reduces the time required for manual and subjective inspection of data, improves the overall accuracy of data analysis, and is easily implemented into the standard data-analysis work flow. AuDIT currently works with results exported from MRM-MS data-processing software packages and may be implemented as an analysis tool within such software. PMID:20022980

Warmth and legitimacy beliefs contextualize adolescents' negative reactions to parental monitoring.

PubMed

LaFleur, Laura K; Zhao, Yinan; Zeringue, Megan M; Laird, Robert D

2016-08-01

This study sought to identify conditions under which parents' monitoring behaviors are most strongly linked to adolescents' negative reactions (i.e., feelings of being controlled and invaded). 242 adolescents (49.2% male; M age = 15.4 years) residing in the United States of America reported parental monitoring and warmth, and their own feelings of being controlled and invaded and beliefs in the legitimacy of parental authority. Analyses tested whether warmth and legitimacy beliefs moderate and/or suppress the link between parents' monitoring behaviors and adolescents' negative reactions. Monitoring was associated with more negative reactions, controlling for legitimacy beliefs and warmth. More monitoring was associated with more negative reactions only at weaker levels of legitimacy beliefs, and at lower levels of warmth. The link between monitoring and negative reactions is sensitive to the context within which monitoring occurs with the strongest negative reactions found in contexts characterized by low warmth and weak legitimacy beliefs. Copyright © 2016 The Foundation for Professionals in Services for Adolescents. Published by Elsevier Ltd. All rights reserved.

Ketamine-Based Anesthetic Protocols and Evoked Potential Monitoring: A Risk/Benefit Overview.

PubMed

Stoicea, Nicoleta; Versteeg, Gregory; Florescu, Diana; Joseph, Nicholas; Fiorda-Diaz, Juan; Navarrete, Víctor; Bergese, Sergio D

2016-01-01

Since its discovery, ketamine, a non-competitive N-methyl D-aspartate (NMDA) receptor antagonist related to phencyclidine, has been linked to multiple adverse reactions sometimes described as "out of body" and "near death experiences," including emergence phenomena, delusions, hallucinations, delirium, and confusion. Due to these effects, ketamine has been withdrawn from mainstream anesthetic use in adult patients. Evoked potentials (EPs) are utilized to monitor neural pathways during surgery, detect intraoperative stress or damage, detect and define the level of neural lesions, and define abnormalities. Unfortunately, many of the volatile anesthetics commonly used during spinal and neurologic procedures suppress EP amplitude and monitoring. Ketamine has been found in several preclinical and clinical studies to actually increase EP amplitude and thus has been used as an analgesic adjunct in procedures where EP monitoring is critical. Once the gap in our knowledge of ketamine's risks has been sufficiently addressed in animal models, informed clinical trials should be conducted in order to properly incorporate ketamine-based anesthetic regimens during EP-monitored neurosurgeries.

Multi-site assessment of the precision and reproducibility of multiple reaction monitoring–based measurements of proteins in plasma

PubMed Central

Addona, Terri A; Abbatiello, Susan E; Schilling, Birgit; Skates, Steven J; Mani, D R; Bunk, David M; Spiegelman, Clifford H; Zimmerman, Lisa J; Ham, Amy-Joan L; Keshishian, Hasmik; Hall, Steven C; Allen, Simon; Blackman, Ronald K; Borchers, Christoph H; Buck, Charles; Cardasis, Helene L; Cusack, Michael P; Dodder, Nathan G; Gibson, Bradford W; Held, Jason M; Hiltke, Tara; Jackson, Angela; Johansen, Eric B; Kinsinger, Christopher R; Li, Jing; Mesri, Mehdi; Neubert, Thomas A; Niles, Richard K; Pulsipher, Trenton C; Ransohoff, David; Rodriguez, Henry; Rudnick, Paul A; Smith, Derek; Tabb, David L; Tegeler, Tony J; Variyath, Asokan M; Vega-Montoto, Lorenzo J; Wahlander, Åsa; Waldemarson, Sofia; Wang, Mu; Whiteaker, Jeffrey R; Zhao, Lei; Anderson, N Leigh; Fisher, Susan J; Liebler, Daniel C; Paulovich, Amanda G; Regnier, Fred E; Tempst, Paul; Carr, Steven A

2010-01-01

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low µg/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma. PMID:19561596

Rapid determination of eight bioactive alkaloids in Portulaca oleracea L. by the optimal microwave extraction combined with positive-negative conversion multiple reaction monitor (+/-MRM) technology.

PubMed

Liang, Xiao; Tian, Jinlong; Li, Lingzhi; Gao, Jun; Zhang, Qingyi; Gao, Pinyi; Song, Shaojiang

2014-03-01

A rapid and reliable microwave extraction and the triple quadrupole-linear ion trap mass spectrometry method was developed and validated for the determination of eight alkaloids in Portulaca oleracea L. The optimal microwave extraction (MWE) condition was performed at 60 °C for 12 min with ethanol-water (70:30, v/v) as the extracting solvent, and the solvent to solid ratio was 30:1. The alkaloids were first detected simultaneously by electrospray ionization tandem mass spectrometry under positive-negative conversion multiple reaction monitor ((+/-)MRM) technique. With investigating three different columns, samples were separated in only 8 min on a Waters ACQUITY UPLC HSS T3 (50 × 2.1 mm(2), 1.8 μm) column using acetonitrile and formic acid-water solution as a mobile phase with a flow rate at 0.2 mL/min. All calibration curves showed good linearity (r>0.999) within the test ranges. The method developed was validated with acceptable sensitivity, intra- and inter-day precision, reproducibility, and extraction recoveries. It was successfully applied to the determination of eight alkaloids in Portulaca oleracea L. from different sources and different harvest periods. The method also provide a reference for extraction and determination of alkaloids in other complex systems. © 2013 Elsevier B.V. All rights reserved.

Rapid Verification of Candidate Serological Biomarkers Using Gel-based, Label-free Multiple Reaction Monitoring

PubMed Central

Tang, Hsin-Yao; Beer, Lynn A.; Barnhart, Kurt T.; Speicher, David W.

2011-01-01

Stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) has emerged as a promising platform for verification of serological candidate biomarkers. However, cost and time needed to synthesize and evaluate stable isotope peptides, optimize spike-in assays, and generate standard curves, quickly becomes unattractive when testing many candidate biomarkers. In this study, we demonstrate that label-free multiplexed MRM-MS coupled with major protein depletion and 1-D gel separation is a time-efficient, cost-effective initial biomarker verification strategy requiring less than 100 μl serum. Furthermore, SDS gel fractionation can resolve different molecular weight forms of targeted proteins with potential diagnostic value. Because fractionation is at the protein level, consistency of peptide quantitation profiles across fractions permits rapid detection of quantitation problems for specific peptides from a given protein. Despite the lack of internal standards, the entire workflow can be highly reproducible, and long-term reproducibility of relative protein abundance can be obtained using different mass spectrometers and LC methods with external reference standards. Quantitation down to ~200 pg/mL could be achieved using this workflow. Hence, the label-free GeLC-MRM workflow enables rapid, sensitive, and economical initial screening of large numbers of candidate biomarkers prior to setting up SID-MRM assays or immunoassays for the most promising candidate biomarkers. PMID:21726088

Optimized protocol for quantitative multiple reaction monitoring-based proteomic analysis of formalin-fixed, paraffin embedded tissues

PubMed Central

Kennedy, Jacob J.; Whiteaker, Jeffrey R.; Schoenherr, Regine M.; Yan, Ping; Allison, Kimberly; Shipley, Melissa; Lerch, Melissa; Hoofnagle, Andrew N.; Baird, Geoffrey Stuart; Paulovich, Amanda G.

2016-01-01

Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin embedded (FFPE) tissues. While the feasibility of using targeted, multiple reaction monitoring-mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e. 9 processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R2 = 0.94) and immuno-MRM (R2 = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens. PMID:27462933

Comparison of Protein Immunoprecipitation-Multiple Reaction Monitoring with ELISA for Assay of Biomarker Candidates in Plasma

PubMed Central

2013-01-01

Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the analysis of six colon cancer biomarker candidates (metalloproteinase inhibitor 1 (TIMP1), cartilage oligomeric matrix protein (COMP), thrombospondin-2 (THBS2), endoglin (ENG), mesothelin (MSLN) and matrix metalloproteinase-9 (MMP9)) in plasma from colon cancer patients and noncancer controls. Proteins were analyzed by multiplex immunoprecipitation from plasma with the ELISA capture antibodies, further purified by SDS-PAGE, digested and analyzed by stable isotope dilution MRM. IP-MRM provided linear responses (r = 0.978–0.995) between 10 and 640 ng/mL for the target proteins spiked into a “mock plasma” matrix consisting of 60 mg/mL bovine serum albumin. Measurement variation (coefficient of variation at the limit of detection) for IP-MRM assays ranged from 2.3 to 19%, which was similar to variation for ELISAs of the same samples. IP-MRM and ELISA measurements for all target proteins except ENG were highly correlated (r = 0.67–0.97). IP-MRM with high-quality capture antibodies thus provides an effective alternative method to ELISA for protein quantitation in biological fluids. PMID:24224610

A multiple reaction monitoring (MRM) method to detect Bcr-Abl kinase activity in CML using a peptide biosensor.

PubMed

Yang, Tzu-Yi; Eissler, Christie L; Hall, Mark C; Parker, Laurie L

2013-01-01

The protein kinase Bcr-Abl plays a major role in the pathogenesis of chronic myelogenous leukemia (CML), and is the target of the breakthrough drug imatinib (Gleevec™). While most patients respond well to imatinib, approximately 30% never achieve remission or develop resistance within 1-5 years of starting imatinib treatment. Evidence from clinical studies suggests that achieving at least 50% inhibition of a patient's Bcr-Abl kinase activity (relative to their level at diagnosis) is associated with improved patient outcomes, including reduced occurrence of resistance and longer maintenance of remission. Accordingly, sensitive assays for detecting Bcr-Abl kinase activity compatible with small amounts of patient material are desirable as potential companion diagnostics for imatinib. Here we report the detection of Bcr-Abl activity and inhibition by imatinib in the human CML cell line K562 using a cell-penetrating peptide biosensor and multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. MRM enabled reproducible, selective detection of the peptide biosensor at fmol levels from aliquots of cell lysate equivalent to ~15,000 cells. This degree of sensitivity will facilitate the miniaturization of the entire assay procedure down to cell numbers approaching 15,000, making it practical for translational applications in patient cells in which the limited amount of available patient material often presents a major challenge.

Quantification of seminolipid by LC-ESI-MS/MS-multiple reaction monitoring: compensatory levels in Cgt(+/⁻) mice.

PubMed

Kongmanas, Kessiri; Xu, Hongbin; Yaghoubian, Arman; Franchini, Laura; Panza, Luigi; Ronchetti, Fiamma; Faull, Kym; Tanphaichitr, Nongnuj

2010-12-01

Seminolipid, also known as sulfogalactosylglycerolipid (SGG), plays important roles in male reproduction. Therefore, an accurate and sensitive method for SGG quantification in testes and sperm is needed. Here we compare SGG quantitation by the traditional colorimetric Azure A assay with LC-ESI-MS/MS using multiple reaction monitoring (MRM). Inclusion of deuterated SGG as the internal standard endowed accuracy to the MRM method. The results showed reasonable agreement between the two procedures for purified samples, but for crude lipid extracts, the colorimetric assay significantly overestimated the SGG content. Using ESI-MS/MS MRM, C16:0-alkyl/C16:0-acyl SGG of Cgt(+/⁻) mice was quantified to be 406.06 ± 23.63 μg/g testis and 0.13 ± 0.02 μg/million sperm, corresponding to 78% and 87% of the wild-type values, respectively. CGT (ceramide galactosyltransferase) is a critical enzyme in the SGG biosynthesis pathway. Cgt⁻/⁻ males depleted of SGG are infertile due to spermatogenesis arrest. However, Cgt(+/⁻) males sire offspring. The higher than 50% expression level of SGG in Cgt(+/⁻) animals, compared with the wild-type expression, might be partly due to compensatory translation of the active CGT enzyme. The results also indicated that 78% of SGG levels in Cgt(+/⁻) mice were sufficient for normal spermatogenesis.

Rapid verification of candidate serological biomarkers using gel-based, label-free multiple reaction monitoring.

PubMed

Tang, Hsin-Yao; Beer, Lynn A; Barnhart, Kurt T; Speicher, David W

2011-09-02

Stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) has emerged as a promising platform for verification of serological candidate biomarkers. However, cost and time needed to synthesize and evaluate stable isotope peptides, optimize spike-in assays, and generate standard curves quickly becomes unattractive when testing many candidate biomarkers. In this study, we demonstrate that label-free multiplexed MRM-MS coupled with major protein depletion and 1D gel separation is a time-efficient, cost-effective initial biomarker verification strategy requiring less than 100 μL of serum. Furthermore, SDS gel fractionation can resolve different molecular weight forms of targeted proteins with potential diagnostic value. Because fractionation is at the protein level, consistency of peptide quantitation profiles across fractions permits rapid detection of quantitation problems for specific peptides from a given protein. Despite the lack of internal standards, the entire workflow can be highly reproducible, and long-term reproducibility of relative protein abundance can be obtained using different mass spectrometers and LC methods with external reference standards. Quantitation down to ~200 pg/mL could be achieved using this workflow. Hence, the label-free GeLC-MRM workflow enables rapid, sensitive, and economical initial screening of large numbers of candidate biomarkers prior to setting up SID-MRM assays or immunoassays for the most promising candidate biomarkers.

Large-scale isotype-specific quantification of Serum amyloid A 1/2 by multiple reaction monitoring in crude sera.

PubMed

Sung, Hye-Jin; Jeon, Seon-Ae; Ahn, Jung-Mo; Seul, Kyung-Jo; Kim, Jin Young; Lee, Ju Yeon; Yoo, Jong Shin; Lee, Soo-Youn; Kim, Hojoong; Cho, Je-Yoel

2012-04-03

Quantification is an essential step in biomarker development. Multiple reaction monitoring (MRM) is a new modified mass spectrometry-based quantification technology that does not require antibody development. Serum amyloid A (SAA) is a positive acute-phase protein identified as a lung cancer biomarker in our previous study. Acute SAA exists in two isoforms with highly similar (92%) amino acid sequences. Until now, studies of SAA have been unable to distinguish between SAA1 and SAA2. To overcome the unavailability of a SAA2-specific antibody, we developed MRM methodology for the verification of SAA1 and SAA2 in clinical crude serum samples from 99 healthy controls and 100 lung adenocarcinoma patients. Differential measurement of SAA1 and SAA2 was made possible for the first time with the developed isotype-specific MRM method. Most healthy control samples had small or no MS/MS peaks of the targeted peptides otherwise, higher peak areas with 10- to 34-fold increase over controls were detected in lung cancer samples. In addition, our SAA1 MRM data demonstrated good agreement with the SAA1 enzyme-linked immunosorbent assay (ELISA) data. Finally, successful quantification of SAA2 in crude serum by MRM, for the first time, shows that SAA2 can be a good biomarker for the detection of lung cancers. Copyright © 2012 Elsevier B.V. All rights reserved.

A Multiple Reaction Monitoring (MRM) Method to Detect Bcr-Abl Kinase Activity in CML Using a Peptide Biosensor

PubMed Central

Yang, Tzu-Yi; Eissler, Christie L.; Hall, Mark C.; Parker, Laurie L.

2013-01-01

The protein kinase Bcr-Abl plays a major role in the pathogenesis of chronic myelogenous leukemia (CML), and is the target of the breakthrough drug imatinib (Gleevec™). While most patients respond well to imatinib, approximately 30% never achieve remission or develop resistance within 1–5 years of starting imatinib treatment. Evidence from clinical studies suggests that achieving at least 50% inhibition of a patient’s Bcr-Abl kinase activity (relative to their level at diagnosis) is associated with improved patient outcomes, including reduced occurrence of resistance and longer maintenance of remission. Accordingly, sensitive assays for detecting Bcr-Abl kinase activity compatible with small amounts of patient material are desirable as potential companion diagnostics for imatinib. Here we report the detection of Bcr-Abl activity and inhibition by imatinib in the human CML cell line K562 using a cell-penetrating peptide biosensor and multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. MRM enabled reproducible, selective detection of the peptide biosensor at fmol levels from aliquots of cell lysate equivalent to ∼15,000 cells. This degree of sensitivity will facilitate the miniaturization of the entire assay procedure down to cell numbers approaching 15,000, making it practical for translational applications in patient cells in which the limited amount of available patient material often presents a major challenge. PMID:23437189

YPED: An Integrated Bioinformatics Suite and Database for Mass Spectrometry-based Proteomics Research

PubMed Central

Colangelo, Christopher M.; Shifman, Mark; Cheung, Kei-Hoi; Stone, Kathryn L.; Carriero, Nicholas J.; Gulcicek, Erol E.; Lam, TuKiet T.; Wu, Terence; Bjornson, Robert D.; Bruce, Can; Nairn, Angus C.; Rinehart, Jesse; Miller, Perry L.; Williams, Kenneth R.

2015-01-01

We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database (YPED) that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a single laboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography–tandem mass spectrometry (LC–MS/MS) database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring (MRM)/selective reaction monitoring (SRM) assay development. We have linked YPED’s database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results. PMID:25712262

Quantification of free fatty acids in human stratum corneum using tandem mass spectrometry and surrogate analyte approach.

PubMed

Dapic, Irena; Kobetic, Renata; Brkljacic, Lidija; Kezic, Sanja; Jakasa, Ivone

2018-02-01

The free fatty acids (FFAs) are one of the major components of the lipids in the stratum corneum (SC), the uppermost layer of the skin. Relative composition of FFAs has been proposed as a biomarker of the skin barrier status in patients with atopic dermatitis (AD). Here, we developed an LC-ESI-MS/MS method for simultaneous quantification of a range of FFAs with long and very long chain length in the SC collected by adhesive tape (D-Squame). The method, based on derivatization with 2-bromo-1-methylpyridinium iodide and 3-carbinol-1-methylpyridinium iodide, allowed highly sensitive detection and quantification of FFAs using multiple reaction monitoring. For the quantification, we applied a surrogate analyte approach and internal standardization using isotope labeled derivatives of FFAs. Adhesive tapes showed the presence of several FFAs, which are also present in the SC, a problem encountered in previous studies. Therefore, the levels of FFAs in the SC were corrected using C12:0, which was present on the adhesive tape, but not detected in the SC. The method was applied to SC samples from patients with atopic dermatitis and healthy subjects. Quantification using multiple reaction monitoring allowed sufficient sensitivity to analyze FFAs of chain lengths C16-C28 in the SC collected on only one tape strip. Copyright © 2017 John Wiley & Sons, Ltd.

YPED: an integrated bioinformatics suite and database for mass spectrometry-based proteomics research.

PubMed

Colangelo, Christopher M; Shifman, Mark; Cheung, Kei-Hoi; Stone, Kathryn L; Carriero, Nicholas J; Gulcicek, Erol E; Lam, TuKiet T; Wu, Terence; Bjornson, Robert D; Bruce, Can; Nairn, Angus C; Rinehart, Jesse; Miller, Perry L; Williams, Kenneth R

2015-02-01

We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database (YPED) that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a single laboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography-tandem mass spectrometry (LC-MS/MS) database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring (MRM)/selective reaction monitoring (SRM) assay development. We have linked YPED's database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

Quantification of methionine and selenomethionine in biological samples using multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS).

PubMed

Vu, Dai Long; Ranglová, Karolína; Hájek, Jan; Hrouzek, Pavel

2018-05-01

Quantification of selenated amino-acids currently relies on methods employing inductively coupled plasma mass spectrometry (ICP-MS). Although very accurate, these methods do not allow the simultaneous determination of standard amino-acids, hampering the comparison of the content of selenated versus non-selenated species such as methionine (Met) and selenomethionine (SeMet). This paper reports two approaches for the simultaneous quantification of Met and SeMet. In the first approach, standard enzymatic hydrolysis employing Protease XIV was applied for the preparation of samples. The second approach utilized methanesulfonic acid (MA) for the hydrolysis of samples, either in a reflux system or in a microwave oven, followed by derivatization with diethyl ethoxymethylenemalonate. The prepared samples were then analyzed by multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS). Both approaches provided platforms for the accurate determination of selenium/sulfur substitution rate in Met. Moreover the second approach also provided accurate simultaneous quantification of Met and SeMet with a low limit of detection, low limit of quantification and wide linearity range, comparable to the commonly used gas chromatography mass spectrometry (GC-MS) method or ICP-MS. The novel method was validated using certified reference material in conjunction with the GC-MS reference method. Copyright © 2018. Published by Elsevier B.V.

Fining of Red Wine Monitored by Multiple Light Scattering.

PubMed

Ferrentino, Giovanna; Ramezani, Mohsen; Morozova, Ksenia; Hafner, Daniela; Pedri, Ulrich; Pixner, Konrad; Scampicchio, Matteo

2017-07-12

This work describes a new approach based on multiple light scattering to study red wine clarification processes. The whole spectral signal (1933 backscattering points along the length of each sample vial) were fitted by a multivariate kinetic model that was built with a three-step mechanism, implying (1) adsorption of wine colloids to fining agents, (2) aggregation into larger particles, and (3) sedimentation. Each step is characterized by a reaction rate constant. According to the first reaction, the results showed that gelatin was the most efficient fining agent, concerning the main objective, which was the clarification of the wine, and consequently the increase in its limpidity. Such a trend was also discussed in relation to the results achieved by nephelometry, total phenols, ζ-potential, color, sensory, and electronic nose analyses. Also, higher concentrations of the fining agent (from 5 to 30 g/100 L) or higher temperatures (from 10 to 20 °C) sped up the process. Finally, the advantage of using the whole spectral signal vs classical univariate approaches was demonstrated by comparing the uncertainty associated with the rate constants of the proposed kinetic model. Overall, multiple light scattering technique showed a great potential for studying fining processes compared to classical univariate approaches.

Monitoring bacterial resistance to chloramphenicol and other antibiotics by liquid chromatography electrospray ionization tandem mass spectrometry using selected reaction monitoring.

PubMed

Haag, Anthony M; Medina, Audrie M; Royall, Ariel E; Herzog, Norbert K; Niesel, David W

2013-06-01

Antibiotic resistance is a growing problem worldwide. For this reason, clinical laboratories often determine the susceptibility of the bacterial isolate to a number of different antibiotics in order to establish the most effective antibiotic for treatment. Unfortunately, current susceptibility assays are time consuming. Antibiotic resistance often involves the chemical modification of an antibiotic to an inactive form by an enzyme expressed by the bacterium. Selected reaction monitoring (SRM) has the ability to quickly monitor and identify these chemical changes in an unprecedented time scale. In this work, we used SRM as a technique to determine the susceptibility of several different antibiotics to the chemically modifying enzymes β-lactamase and chloramphenicol acetyltransferase, enzymes used by bacteria to confer resistance to major classes of commonly used antibiotics. We also used this technique to directly monitor the effects of resistant bacteria grown in a broth containing a specific antibiotic. Because SRM is highly selective and can also identify chemical changes in a multitude of antibiotics in a single assay, SRM has the ability to detect organisms that are resistant to multiple antibiotics in a single assay. For these reasons, the use of SRM greatly reduces the time it takes to determine the susceptibility or resistance of an organism to a multitude of antibiotics by eliminating the time-consuming process found in other currently used methods. Copyright © 2013 John Wiley & Sons, Ltd.

A Database of Reaction Monitoring Mass Spectrometry Assays for Elucidating Therapeutic Response in Cancer

PubMed Central

Remily-Wood, Elizabeth R.; Liu, Richard Z.; Xiang, Yun; Chen, Yi; Thomas, C. Eric; Rajyaguru, Neal; Kaufman, Laura M.; Ochoa, Joana E.; Hazlehurst, Lori; Pinilla-Ibarz, Javier; Lancet, Jeffrey; Zhang, Guolin; Haura, Eric; Shibata, David; Yeatman, Timothy; Smalley, Keiran S.M.; Dalton, William S.; Huang, Emina; Scott, Ed; Bloom, Gregory C.; Eschrich, Steven A.; Koomen, John M.

2012-01-01

Purpose The Quantitative Assay Database (QuAD), http://proteome.moffitt.org/QUAD/, facilitates widespread implementation of quantitative mass spectrometry in cancer biology and clinical research through sharing of methods and reagents for monitoring protein expression and modification. Experimental Design Liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM) assays are developed using SDS-PAGE fractionated lysates from cancer cell lines. Pathway maps created using GeneGO Metacore provide the biological relationships between proteins and illustrate concepts for multiplexed analysis; each protein can be selected to examine assay development at the protein and peptide level. Results The coupling of SDS-PAGE and LC-MRM screening has been used to detect 876 peptides from 218 cancer-related proteins in model systems including colon, lung, melanoma, leukemias, and myeloma, which has led to the development of 95 quantitative assays including stable-isotope labeled peptide standards. Methods are published online and peptide standards are made available to the research community. Protein expression measurements for heat shock proteins, including a comparison with ELISA and monitoring response to the HSP90 inhibitor, 17-DMAG, are used to illustrate the components of the QuAD and its potential utility. Conclusions and Clinical Relevance This resource enables quantitative assessment of protein components of signaling pathways and biological processes and holds promise for systematic investigation of treatment responses in cancer. PMID:21656910

Influence of vegetable oils fatty acid composition on reaction temperature and glycerides conversion to biodiesel during transesterification.

PubMed

Pinzi, S; Gandía, L M; Arzamendi, G; Ruiz, J J; Dorado, M P

2011-01-01

Presence of unreacted glycerides in biodiesel may reduce drastically its quality. This is why conversion of raw material in biodiesel through transesterification needs to readjust reaction parameter values to complete. In the present work, monitoring of glycerides transformation in biodiesel during the transesterification of vegetable oils was carried out. To check the influence of the chemical composition on glycerides conversion, selected vegetable oils covered a wide range of fatty acid composition. Reactions were carried out under alkali-transesterification in the presence of methanol. In addition, a multiple regression model was proposed. Results showed that kinetics depends on chemical and physical properties of the oils. It was found that the optimal reaction temperature depends on both length and unsaturation degree of vegetable oils fatty acid chains. Vegetable oils with higher degree of unsaturation exhibit faster monoglycerides conversion to biodiesel. It can be concluded that fatty acid composition influences reaction parameters and glycerides conversion, hence biodiesel yield and economic viability. Copyright © 2010 Elsevier Ltd. All rights reserved.

Combining in situ characterization methods in one set-up: looking with more eyes into the intricate chemistry of the synthesis and working of heterogeneous catalysts.

PubMed

Bentrup, Ursula

2010-12-01

Several in situ techniques are known which allow investigations of catalysts and catalytic reactions under real reaction conditions using different spectroscopic and X-ray methods. In recent years, specific set-ups have been established which combine two or more in situ methods in order to get a more detailed understanding of catalytic systems. This tutorial review will give a summary of currently available set-ups equipped with multiple techniques for in situ catalyst characterization, catalyst preparation, and reaction monitoring. Besides experimental and technical aspects of method coupling including X-ray techniques, spectroscopic methods (Raman, UV-vis, FTIR), and magnetic resonance spectroscopies (NMR, EPR), essential results will be presented to demonstrate the added value of multitechnique in situ approaches. A special section is focussed on selected examples of use which show new developments and application fields.

Four reversible and reconfigurable structures for three-phase emulsions: extended morphologies and applications

NASA Astrophysics Data System (ADS)

Ge, Xue-Hui; Geng, Yu-Hao; Zhang, Qiao-Chu; Shao, Meng; Chen, Jian; Luo, Guang-Sheng; Xu, Jian-Hong

2017-02-01

Here in this article, we classify and conclude the four morphologies of three-phase emulsions. Remarkably, we achieve the reversible transformations between every shape. Through theoretical analysis, we choose four liquid systems to form these four morphologies. Then monodispersed droplets with these four morphologies are formed through a microfluidic device and captured in a petri-dish. By replacing their ambient solution of the captured emulsions, in-situ morphology transformations between each shape are achieved. The process is well recorded through photographs and videos and they are systematical and reversible. Finally, we use the droplets structure to form an on-off switch to start and shut off the evaporation of one volatile phase to achieve the process monitoring. This could be used to initiate and quench a reaction, which offers a novel idea to achieve the switchable and reversible reaction control in multiple-phase reactions.

Modular radiochemistry synthesis system

DOEpatents

Satyamurthy, Nagichettiar; Barrio, Jorge R.; Amarasekera, Bernard; Van Dam, Michael R.; Olma, Sebastian; Williams, Dirk; Eddings, Mark; Shen, Clifton Kwang-Fu

2016-11-01

A modular chemical production system includes multiple modules for performing a chemical reaction, particularly of radiochemical compounds, from a remote location. One embodiment comprises a reaction vessel including a moveable heat source with the position thereof relative to the reaction vessel being controllable from a remote position. Alternatively the heat source may be fixed in location and the reaction vial is moveable into and out of the heat source. The reaction vessel has one or more sealing plugs, the positioning of which in relationship to the reaction vessel is controllable from a remote position. Also the one or more reaction vessel sealing plugs can include one or more conduits there through for delivery of reactants, gases at atmospheric or an elevated pressure, inert gases, drawing a vacuum and removal of reaction end products to and from the reaction vial, the reaction vial with sealing plug in position being operable at elevated pressures. The modular chemical production system is assembled from modules which can each include operating condition sensors and controllers configured for monitoring and controlling the individual modules and the assembled system from a remote position. Other modules include, but are not limited to a Reagent Storage and Delivery Module, a Cartridge Purification Module, a Microwave Reaction Module, an External QC/Analysis/Purification Interface Module, an Aliquotting Module, an F-18 Drying Module, a Concentration Module, a Radiation Counting Module, and a Capillary Reactor Module.

Modular radiochemistry synthesis system

DOEpatents

Satyamurthy, Nagichettiar; Barrio, Jorge R.; Amarasekera, Bernard; Van Dam, R. Michael; Olma, Sebastian; Williams, Dirk; Eddings, Mark; Shen, Clifton Kwang-Fu

2015-12-15

A modular chemical production system includes multiple modules for performing a chemical reaction, particularly of radiochemical compounds, from a remote location. One embodiment comprises a reaction vessel including a moveable heat source with the position thereof relative to the reaction vessel being controllable from a remote position. Alternatively the heat source may be fixed in location and the reaction vial is moveable into and out of the heat source. The reaction vessel has one or more sealing plugs, the positioning of which in relationship to the reaction vessel is controllable from a remote position. Also the one or more reaction vessel sealing plugs can include one or more conduits there through for delivery of reactants, gases at atmospheric or an elevated pressure, inert gases, drawing a vacuum and removal of reaction end products to and from the reaction vial, the reaction vial with sealing plug in position being operable at elevated pressures. The modular chemical production system is assembled from modules which can each include operating condition sensors and controllers configured for monitoring and controlling the individual modules and the assembled system from a remote position. Other modules include, but are not limited to a Reagent Storage and Delivery Module, a Cartridge Purification Module, a Microwave Reaction Module, an External QC/Analysis/Purification Interface Module, an Aliquotting Module, an F-18 Drying Module, a Concentration Module, a Radiation Counting Module, and a Capillary Reactor Module.

Modular radiochemistry synthesis system

DOEpatents

Satyamurthy, Nagichettiar; Barrio, Jorge R; Amarasekera, Bernard; Van Dam, R. Michael; Olma, Sebastian; Williams, Dirk; Eddings, Mark A; Shen, Clifton Kwang-Fu

2015-02-10

A modular chemical production system includes multiple modules for performing a chemical reaction, particularly of radiochemical compounds, from a remote location. One embodiment comprises a reaction vessel including a moveable heat source with the position thereof relative to the reaction vessel being controllable from a remote position. Alternatively the heat source may be fixed in location and the reaction vial is moveable into and out of the heat source. The reaction vessel has one or more sealing plugs, the positioning of which in relationship to the reaction vessel is controllable from a remote position. Also the one or more reaction vessel sealing plugs can include one or more conduits there through for delivery of reactants, gases at atmospheric or an elevated pressure, inert gases, drawing a vacuum and removal of reaction end products to and from the reaction vial, the reaction vial with sealing plug in position being operable at elevated pressures. The modular chemical production system is assembled from modules which can each include operating condition sensors and controllers configured for monitoring and controlling the individual modules and the assembled system from a remote position. Other modules include, but are not limited to a Reagent Storage and Delivery Module, a Cartridge Purification Module, a Microwave Reaction Module, an External QC/Analysis/Purification Interface Module, an Aliquotting Module, an F-18 Drying Module, a Concentration Module, a Radiation Counting Module, and a Capillary Reactor Module.

Quantitative Analysis of Tissue Samples by Combining iTRAQ Isobaric Labeling with Selected/Multiple Reaction Monitoring (SRM/MRM).

PubMed

Narumi, Ryohei; Tomonaga, Takeshi

2016-01-01

Mass spectrometry-based phosphoproteomics is an indispensible technique used in the discovery and quantification of phosphorylation events on proteins in biological samples. The application of this technique to tissue samples is especially useful for the discovery of biomarkers as well as biological studies. We herein describe the application of a large-scale phosphoproteome analysis and SRM/MRM-based quantitation to develop a strategy for the systematic discovery and validation of biomarkers using tissue samples.

Preprocessing and Analysis of LC-MS-Based Proteomic Data

PubMed Central

Tsai, Tsung-Heng; Wang, Minkun; Ressom, Habtom W.

2016-01-01

Liquid chromatography coupled with mass spectrometry (LC-MS) has been widely used for profiling protein expression levels. This chapter is focused on LC-MS data preprocessing, which is a crucial step in the analysis of LC-MS based proteomics. We provide a high-level overview, highlight associated challenges, and present a step-by-step example for analysis of data from LC-MS based untargeted proteomic study. Furthermore, key procedures and relevant issues with the subsequent analysis by multiple reaction monitoring (MRM) are discussed. PMID:26519169

Verification of multimarkers for detection of early stage diabetic retinopathy using multiple reaction monitoring.

PubMed

Kim, Kyunggon; Kim, Sang Jin; Han, Dohyun; Jin, Jonghwa; Yu, Jiyoung; Park, Kyong Soo; Yu, Hyeong Gon; Kim, Youngsoo

2013-03-01

Diabetic retinopathy (DR) is a complication of diabetes and 80% of diabetes mellitus (DM) patients whose DM duration is over 10 years can be expected to suffer with DR. The diagnosis of DR depends on an ophthalmological examination, and no molecular methods of screening DR status exist. Nonproliferative diabetic retinopathy (NPDR) is the early DR which is hard to be noticed in early NPDR, showing significant cause of adult blindness in type 2 diabetes patients. Protein biomarkers have been valuable in the diagnosis of disease and the use of multiple biomarkers has been suggested to overcome the low specificity of single ones. For biomarker development, multiple reaction monitoring (MRM) has been spotlighted as an alternative method to quantify target proteins with no need for immunoassay. In this study, 54 candidate DR marker proteins from a previous study were verified by MRM in plasma samples from NPDR patients in 3 stages (mild, moderate and severe; 15 cases each) and diabetic patients without retinopathy (15 cases) as a control. Notably, 27 candidate markers distinguished moderate NPDR from type 2 diabetic patients with no diabetic retinopathy, generating AUC values (>0.7). Specifically, 28 candidate proteins underwent changes in expression as type 2 diabetic patients with no diabetic retinopathy progressed to mild and moderate NPDR. Further, a combination of 4 markers from these 28 candidates had the improved specificity in distinguishing moderate NPDR from type 2 diabetic patients with no diabetic retinopathy, yielding a merged AUC value of nearly 1.0. We concluded that MRM is a fast, robust approach of multimarker panel determination and an assay platform that provides improved specificity compared with single biomarker assay systems.

Stepped MS(All) Relied Transition (SMART): An approach to rapidly determine optimal multiple reaction monitoring mass spectrometry parameters for small molecules.

PubMed

Ye, Hui; Zhu, Lin; Wang, Lin; Liu, Huiying; Zhang, Jun; Wu, Mengqiu; Wang, Guangji; Hao, Haiping

2016-02-11

Multiple reaction monitoring (MRM) is a universal approach for quantitative analysis because of its high specificity and sensitivity. Nevertheless, optimization of MRM parameters remains as a time and labor-intensive task particularly in multiplexed quantitative analysis of small molecules in complex mixtures. In this study, we have developed an approach named Stepped MS(All) Relied Transition (SMART) to predict the optimal MRM parameters of small molecules. SMART requires firstly a rapid and high-throughput analysis of samples using a Stepped MS(All) technique (sMS(All)) on a Q-TOF, which consists of serial MS(All) events acquired from low CE to gradually stepped-up CE values in a cycle. The optimal CE values can then be determined by comparing the extracted ion chromatograms for the ion pairs of interest among serial scans. The SMART-predicted parameters were found to agree well with the parameters optimized on a triple quadrupole from the same vendor using a mixture of standards. The parameters optimized on a triple quadrupole from a different vendor was also employed for comparison, and found to be linearly correlated with the SMART-predicted parameters, suggesting the potential applications of the SMART approach among different instrumental platforms. This approach was further validated by applying to simultaneous quantification of 31 herbal components in the plasma of rats treated with a herbal prescription. Because the sMS(All) acquisition can be accomplished in a single run for multiple components independent of standards, the SMART approach are expected to find its wide application in the multiplexed quantitative analysis of complex mixtures. Copyright © 2015 Elsevier B.V. All rights reserved.

Reference Cross Sections for Charged-particle Monitor Reactions

NASA Astrophysics Data System (ADS)

Hermanne, A.; Ignatyuk, A. V.; Capote, R.; Carlson, B. V.; Engle, J. W.; Kellett, M. A.; Kibédi, T.; Kim, G.; Kondev, F. G.; Hussain, M.; Lebeda, O.; Luca, A.; Nagai, Y.; Naik, H.; Nichols, A. L.; Nortier, F. M.; Suryanarayana, S. V.; Takács, S.; Tárkányi, F. T.; Verpelli, M.

2018-02-01

Evaluated cross sections of beam-monitor reactions are expected to become the de-facto standard for cross-section measurements that are performed over a very broad energy range in accelerators in order to produce particular radionuclides for industrial and medical applications. The requirements for such data need to be addressed in a timely manner, and therefore an IAEA coordinated research project was launched in December 2012 to establish or improve the nuclear data required to characterise charged-particle monitor reactions. An international team was assembled to recommend more accurate cross-section data over a wide range of targets and projectiles, undertaken in conjunction with a limited number of measurements and more extensive evaluations of the decay data of specific radionuclides. Least-square evaluations of monitor-reaction cross sections including uncertainty quantification have been undertaken for charged-particle beams of protons, deuterons, 3He- and 4He-particles. Recommended beam monitor reaction data with their uncertainties are available at the IAEA-NDS medical portal http://www-nds.iaea.org/medical/monitor_reactions.html.

Understanding the positive benefit:risk profile of alemtuzumab in relapsing multiple sclerosis: perspectives from the Alemtuzumab Clinical Development Program.

PubMed

Havrdova, Eva; Cohen, Jeffrey A; Horakova, Dana; Kovarova, Ivana; Meluzinova, Eva

2017-01-01

The introduction of high-efficacy therapies for relapsing-remitting multiple sclerosis has driven re-evaluation of treatment goals and benefit:risk considerations in treatment choice. In the alemtuzumab Phase II and III clinical trials, patients treated with alemtuzumab 12 mg versus subcutaneous interferon beta-1a demonstrated significantly reduced annualized relapse rates and improved magnetic resonance imaging outcomes, and were significantly more likely to achieve no evidence of disease activity and reduction in brain volume loss. In two of the studies, alemtuzumab-treated patients had a significantly reduced risk of 6-month confirmed disease worsening, compared with subcutaneous interferon beta-1a. Benefits were maintained throughout 5 years, with a majority of patients receiving no alemtuzumab retreatment or other disease-modifying therapy. Trial results support alemtuzumab's manageable, consistent safety profile in relapsing-remitting multiple sclerosis. Infusion-associated reactions, the most frequent adverse events (AEs), can be minimized by corticosteroid pretreatment, monitoring, and symptomatic management. Other AEs include infections and autoimmune events. Oral anti-herpes prophylaxis should be initiated on the first day of each alemtuzumab treatment course and continued according to local guidelines. Overall cancer risk was lower in the alemtuzumab clinical trials than in a reference population; however, continuing surveillance will determine if alemtuzumab may be associated with certain malignancies such as thyroid papillary carcinoma and melanoma, which are currently identified as potential risks. The post-approval risk management strategy includes a safety monitoring program. Autoimmune AEs (thyroid events, immune thrombocytopenia, nephropathies) can be detected in a timely manner with the monitoring program, which includes physician and patient education about the signs and symptoms, monthly renal and hematologic monitoring, and quarterly thyroid function monitoring for 48 months after the last alemtuzumab course. Education, vigilance by physicians and patients, and monthly laboratory monitoring are recommended to maintain alemtuzumab's positive benefit:risk profile.

Determination of human-use pharmaceuticals in filtered water by direct aqueous injection: high-performance liquid chromatography/tandem mass spectrometry

USGS Publications Warehouse

Furlong, Edward T.; Noriega, Mary C.; Kanagy, Christopher J.; Kanagy, Leslie K.; Coffey, Laura J.; Burkhardt, Mark R.

2014-01-01

This report describes a method for the determination of 110 human-use pharmaceuticals using a 100-microliter aliquot of a filtered water sample directly injected into a high-performance liquid chromatograph coupled to a triple-quadrupole tandem mass spectrometer using an electrospray ionization source operated in the positive ion mode. The pharmaceuticals were separated by using a reversed-phase gradient of formic acid/ammonium formate-modified water and methanol. Multiple reaction monitoring of two fragmentations of the protonated molecular ion of each pharmaceutical to two unique product ions was used to identify each pharmaceutical qualitatively. The primary multiple reaction monitoring precursor-product ion transition was quantified for each pharmaceutical relative to the primary multiple reaction monitoring precursor-product transition of one of 19 isotope-dilution standard pharmaceuticals or the pesticide atrazine, using an exact stable isotope analogue where possible. Each isotope-dilution standard was selected, when possible, for its chemical similarity to the unlabeled pharmaceutical of interest, and added to the sample after filtration but prior to analysis. Method performance for each pharmaceutical was determined for reagent water, groundwater, treated drinking water, surface water, treated wastewater effluent, and wastewater influent sample matrixes that this method will likely be applied to. Each matrix was evaluated in order of increasing complexity to demonstrate (1) the sensitivity of the method in different water matrixes and (2) the effect of sample matrix, particularly matrix enhancement or suppression of the precursor ion signal, on the quantitative determination of pharmaceutical concentrations. Recovery of water samples spiked (fortified) with the suite of pharmaceuticals determined by this method typically was greater than 90 percent in reagent water, groundwater, drinking water, and surface water. Correction for ambient environmental concentrations of pharmaceuticals hampered the determination of absolute recoveries and method sensitivity of some compounds in some water types, particularly for wastewater effluent and influent samples. The method detection limit of each pharmaceutical was determined from analysis of pharmaceuticals fortified at multiple concentrations in reagent water. The calibration range for each compound typically spanned three orders of magnitude of concentration. Absolute sensitivity for some compounds, using isotope-dilution quantitation, ranged from 0.45 to 94.1 nanograms per liter, primarily as a result of the inherent ionization efficiency of each pharmaceutical in the electrospray ionization process. Holding-time studies indicate that acceptable recoveries of pharmaceuticals can be obtained from filtered water samples held at 4 °C for as long as 9 days after sample collection. Freezing samples to provide for storage for longer periods currently (2014) is under evaluation by the National Water Quality Laboratory.

Real Time Monitoring of Containerless Microreactions in Acoustically Levitated Droplets via Ambient Ionization Mass Spectrometry.

PubMed

Crawford, Elizabeth A; Esen, Cemal; Volmer, Dietrich A

2016-09-06

Direct in-droplet (in stillo) microreaction monitoring using acoustically levitated micro droplets has been achieved by combining acoustic (ultrasonic) levitation for the first time with real time ambient tandem mass spectrometry (MS/MS). The acoustic levitation and inherent mixing of microliter volumes of reactants (3 μL droplets), yielding total reaction volumes of 6 μL, supported monitoring the acid-catalyzed degradation reaction of erythromycin A. This reaction was chosen to demonstrate the proof-of-principle of directly monitoring in stillo microreactions via hyphenated acoustic levitation and ambient ionization mass spectrometry. The microreactions took place completely in stillo over 30, 60, and 120 s within the containerless stable central pressure node of an acoustic levitator, thus readily promoting reaction miniaturization. For the evaluation of the miniaturized in stillo reactions, the degradation reactions were also carried out in vials (in vitro) with a total reaction volume of 400 μL. The reacted in vitro mixtures (6 μL total) were similarly introduced into the acoustic levitator prior to ambient ionization MS/MS analysis. The in stillo miniaturized reactions provided immediate real-time snap-shots of the degradation process for more accurate reaction monitoring and used a fraction of the reactants, while the larger scale in vitro reactions only yielded general reaction information.

Holographic interferometry imaging monitoring of photodynamic (PDT) reactions in gelatin biophantom

NASA Astrophysics Data System (ADS)

Davidenko, N.; Mahdi, H.; Zheng, X.; Davidenko, I.; Pavlov, V.; Kuranda, N.; Chuprina, N.; Studzinsky, S.; Pandya, A.; Karia, H.; Tajouri, S.; Dervenis, M.; Gergely, C.; Douplik, A.

2018-01-01

Heat and photochemical reactions with human hemoglobin and photosensitizer were monitored by holography interference method in gelatin phantom. The method has successfully facilitated monitoring the reactions as a highresolution refraction index mapping in real time video regime. Methylene Blue was exploited as a photosensitizer.

Real time monitoring of accelerated chemical reactions by ultrasonication-assisted spray ionization mass spectrometry.

PubMed

Lin, Shu-Hsuan; Lo, Ta-Ju; Kuo, Fang-Yin; Chen, Yu-Chie

2014-01-01

Ultrasonication has been used to accelerate chemical reactions. It would be ideal if ultrasonication-assisted chemical reactions could be monitored by suitable detection tools such as mass spectrometry in real time. It would be helpful to clarify reaction intermediates/products and to have a better understanding of reaction mechanism. In this work, we developed a system for ultrasonication-assisted spray ionization mass spectrometry (UASI-MS) with an ~1.7 MHz ultrasonic transducer to monitor chemical reactions in real time. We demonstrated that simply depositing a sample solution on the MHz-based ultrasonic transducer, which was placed in front of the orifice of a mass spectrometer, the analyte signals can be readily detected by the mass spectrometer. Singly and multiply charged ions from small and large molecules, respectively, can be observed in the UASI mass spectra. Furthermore, the ultrasonic transducer used in the UASI setup accelerates the chemical reactions while being monitored via UASI-MS. The feasibility of using this approach for real-time acceleration/monitoring of chemical reactions was demonstrated. The reactions of Girard T reagent and hydroxylamine with steroids were used as the model reactions. Upon the deposition of reactant solutions on the ultrasonic transducer, the intermediate/product ions are readily generated and instantaneously monitored using MS within 1 s. Additionally, we also showed the possibility of using this reactive UASI-MS approach to assist the confirmation of trace steroids from complex urine samples by monitoring the generation of the product ions. Copyright © 2014 John Wiley & Sons, Ltd.

Coupled charge migration and fluid mixing in reactive fronts

NASA Astrophysics Data System (ADS)

Ghosh, Uddipta; Bandopadhyay, Aditya; Jougnot, Damien; Le Borgne, Tanguy; Meheust, Yves

2017-04-01

Quantifying fluid mixing in subsurface environments and its consequence on biogeochemical reactions is of paramount importance owing to its role in processes such as contaminant migration, aquifer remediation, CO2 sequestration or clogging processes, to name a few (Dentz et al. 2011). The presence of strong velocity gradients in porous media is expected to lead to enhanced diffusive mixing and augmented reaction rates (Le Borgne et al. 2014). Accurate in situ imaging of subsurface reactive solute transport and mixing remains to date a challenging proposition: the opacity of the medium prevents optical imaging and field methods based on tracer tests do not provide spatial information. Recently developed geophysical methods based on the temporal monitoring of electrical conductivity and polarization have shown promises for mapping and monitoring biogeochemical reactions in the subsurface although it remains challenging to decipher the multiple sources of electrical signals (e.g. Knight et al. 2010). In this work, we explore the coupling between fluid mixing, reaction and charge migration in porous media to evaluate the potential of mapping reaction rates from electrical measurements. To this end, we develop a new theoretical framework based on a lamellar mixing model (Le Borgne et al. 2013) to quantify changes in electrical mobility induced by chemical reactions across mixing fronts. Electrical conductivity and induced polarization are strongly dependent on the concentration of ionic species, which in turn depend on the local reaction rates. Hence, our results suggest that variation in real and complex electrical conductivity may be quantitatively related to the mixing and reaction dynamics. Thus, the presented theory provides a novel upscaling framework for quantifying the coupling between mixing, reaction and charge migration in heterogeneous porous media flows. References: Dentz. et al., Mixing, spreading and reaction in heterogeneous media: A brief review J. Contam. Hydrol. 120-121, 1 (2011). Le Borgne et al. Impact of Fluid Deformation on Mixing-Induced Chemical Reactions in heterogeneous Flows, Geophys. Res. Lett. 41, 7898 (2014). Knight, et al., Geophysics at the interface: Response of geophysical properties to solid-fluid, fluid-fluid, and solid-solid interfaces. Rev. Geophys. 48, (2010). Le Borgne et al. (2013) Stretching, coalescence and mixing in porous media, Phys. Rev. Lett., 110, 204501

Appropriateness of Plasma Transfusion: A College of American Pathologists Q-Probes Study of Guidelines, Waste, and Serious Adverse Events.

PubMed

Alcorn, Kirsten; Ramsey, Glenn; Souers, Rhona; Lehman, Christopher M

2017-03-01

- Plasma transfusion guidelines support patient care and safety, management of product wastage, and compliance; yet, there is little information across multiple institutions about use of and adherence to plasma transfusion guidelines. - To survey multiple institutions regarding their plasma transfusion guidelines and compliance, plasma wastage rates, and incidence of transfusion reactions associated with plasma transfusion. - The College of American Pathologists Q-Probes model was used to collect data from 89 participating institutions. Each site was asked to provide data relevant to its most recent 40 adult patient plasma transfusion episodes, and complete a questionnaire regarding plasma transfusion guidelines, utilization and wastage of plasma, and transfusion reactions related to plasma transfusion. - The participating institutions reported a total of 3383 evaluable plasma transfusion episodes with transfusion of 9060 units of plasma. Compliance with institution-specific guidelines was seen in 3018 events (89%). Pretransfusion and posttransfusion coagulation testing was done in 3281 (97%) and 3043 (90%) of these episodes, respectively. Inappropriate criteria were noted for more than 100 transfusion episodes. Thirty-two plasma transfusion episodes (1%) were associated with a transfusion reaction. Serious and fatal reactions were reported. Median plasma wastage rate for the year preceding the study was 4.5%. - Most participating institutions are compliant with plasma transfusion guidelines based on published references, supported by appropriate testing. With transfusions for indications that lack evidence of efficacy and incidence of transfusion reactions, there is an ongoing role for transfusion service leaders to continue to update and monitor plasma transfusion practices.

The current status of clinical proteomics and the use of MRM and MRM(3) for biomarker validation.

PubMed

Lemoine, Jérôme; Fortin, Tanguy; Salvador, Arnaud; Jaffuel, Aurore; Charrier, Jean-Philippe; Choquet-Kastylevsky, Geneviève

2012-05-01

The transfer of biomarkers from the discovery field to clinical use is still, despite progress, on a road filled with pitfalls. Since the emergence of proteomics, thousands of putative biomarkers have been published, often with overlapping diagnostic capacities. The strengthening of the robustness of discovery technologies, particularly in mass spectrometry, has been followed by intense discussions on establishing well-defined evaluation procedures for the identified targets to ultimately allow the clinical validation and then the clinical use of some of these biomarkers. Some of the obstacles to the evaluation process have been the lack of the availability of quick and easy-to-develop, easy-to-use, robust, specific and sensitive alternative quantitative methods when immunoaffinity-based tests are unavailable. Multiple reaction monitoring (MRM; also called selected reaction monitoring) is currently proving its capabilities as a complementary or alternative technique to ELISA for large biomarker panel evaluation. Here, we present how MRM(3) can overcome the lack of specificity and sensitivity often encountered by MRM when tracking minor proteins diluted by complex biological matrices.

Biotransformation and induction: implications for toxicity, bioaccumulation and monitoring of environmental xenobiotics in fish.

PubMed Central

Kleinow, K M; Melancon, M J; Lech, J J

1987-01-01

Biotransformation of xenobiotics in fish occurs by many of the same reactions as in mammals. These reactions have been shown to affect the bioaccumulation, persistence, residue dynamics, and toxicity of select chemicals in fish. P-450-dependent monooxygenase activity of fish can be induced by polycyclic aromatic hydrocarbons, but phenobarbital-type agents induce poorly, if at all. Fish monooxygenase activity exhibits ideal temperature compensation and sex-related variation. Induction of monooxygenase activity by polycyclic aromatic hydrocarbons can result in qualitative as well as quantitative changes in the metabolic profile of a chemical. Induction can also alter toxicity. In addition, multiple P-450 isozymes have been described for several fish species. The biotransformation products of certain chemicals have been related to specific P-450 isozymes, and the formation of these products can be influenced by induction. Exposure of fish to low levels of certain environmental contaminants has resulted in induction of specific monooxygenase activities and monitoring of such activities has been suggested as a means of identifying areas of pollutant exposure in the wild. PMID:3297653

Targeted proteomics coming of age - SRM, PRM and DIA performance evaluated from a core facility perspective.

PubMed

Kockmann, Tobias; Trachsel, Christian; Panse, Christian; Wahlander, Asa; Selevsek, Nathalie; Grossmann, Jonas; Wolski, Witold E; Schlapbach, Ralph

2016-08-01

Quantitative mass spectrometry is a rapidly evolving methodology applied in a large number of omics-type research projects. During the past years, new designs of mass spectrometers have been developed and launched as commercial systems while in parallel new data acquisition schemes and data analysis paradigms have been introduced. Core facilities provide access to such technologies, but also actively support the researchers in finding and applying the best-suited analytical approach. In order to implement a solid fundament for this decision making process, core facilities need to constantly compare and benchmark the various approaches. In this article we compare the quantitative accuracy and precision of current state of the art targeted proteomics approaches single reaction monitoring (SRM), parallel reaction monitoring (PRM) and data independent acquisition (DIA) across multiple liquid chromatography mass spectrometry (LC-MS) platforms, using a readily available commercial standard sample. All workflows are able to reproducibly generate accurate quantitative data. However, SRM and PRM workflows show higher accuracy and precision compared to DIA approaches, especially when analyzing low concentrated analytes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ketamine-Based Anesthetic Protocols and Evoked Potential Monitoring: A Risk/Benefit Overview

PubMed Central

Stoicea, Nicoleta; Versteeg, Gregory; Florescu, Diana; Joseph, Nicholas; Fiorda-Diaz, Juan; Navarrete, Víctor; Bergese, Sergio D.

2016-01-01

Since its discovery, ketamine, a non-competitive N-methyl D-aspartate (NMDA) receptor antagonist related to phencyclidine, has been linked to multiple adverse reactions sometimes described as “out of body” and “near death experiences,” including emergence phenomena, delusions, hallucinations, delirium, and confusion. Due to these effects, ketamine has been withdrawn from mainstream anesthetic use in adult patients. Evoked potentials (EPs) are utilized to monitor neural pathways during surgery, detect intraoperative stress or damage, detect and define the level of neural lesions, and define abnormalities. Unfortunately, many of the volatile anesthetics commonly used during spinal and neurologic procedures suppress EP amplitude and monitoring. Ketamine has been found in several preclinical and clinical studies to actually increase EP amplitude and thus has been used as an analgesic adjunct in procedures where EP monitoring is critical. Once the gap in our knowledge of ketamine's risks has been sufficiently addressed in animal models, informed clinical trials should be conducted in order to properly incorporate ketamine-based anesthetic regimens during EP-monitored neurosurgeries. PMID:26909017

Quantitation of low molecular weight sugars by chemical derivatization-liquid chromatography/multiple reaction monitoring/mass spectrometry.

PubMed

Han, Jun; Lin, Karen; Sequria, Carita; Yang, Juncong; Borchers, Christoph H

2016-07-01

A new method for the separation and quantitation of 13 mono- and disaccharides has been developed by chemical derivatization/ultra-HPLC/negative-ion ESI-multiple-reaction monitoring MS. 3-Nitrophenylhydrazine (at 50°C for 60 min) was shown to be able to quantitatively derivatize low-molecular weight (LMW) reducing sugars. The nonreducing sugar, sucrose, was not derivatized. A pentafluorophenyl-bonded phase column was used for the chromatographic separation of the derivatized sugars. This method exhibits femtomole-level sensitivity, high precision (CVs of ≤ 4.6%) and high accuracy for the quantitation of LMW sugars in wine. Excellent linearity (R(2) ≥ 0.9993) and linear ranges of ∼500-fold for disaccharides and ∼1000-4000-fold for monosaccharides were achieved. With internal calibration ((13) C-labeled internal standards), recoveries were between 93.6% ± 1.6% (xylose) and 104.8% ± 5.2% (glucose). With external calibration, recoveries ranged from 82.5% ± 0.8% (ribulose) to 105.2% ± 2.1% (xylulose). Quantitation of sugars in two red wines and two white wines was performed using this method; quantitation of the central carbon metabolism-related carboxylic acids and tartaric acid was carried out using a previously established derivatization procedure with 3-nitrophenylhydrazine as well. The results showed that these two classes of compounds-both of which have important organoleptic properties-had different compositions in red and white wines. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitation of permethylated N-glycans through multiple-reaction monitoring (MRM) LC-MS/MS.

PubMed

Zhou, Shiyue; Hu, Yunli; DeSantos-Garcia, Janie L; Mechref, Yehia

2015-04-01

The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple-reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan was determined to be 30%, whereas it was found to be 35% for either fucosylated or sialylated N-glycans. The optimum CE for mannose and complex type N-glycan was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan compositions in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these glycans was achieved when the equivalence of 0.005 μL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitude. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples.

Hyperplex-MRM: a hybrid multiple reaction monitoring method using mTRAQ/iTRAQ labeling for multiplex absolute quantification of human colorectal cancer biomarker.

PubMed

Yin, Hong-Rui; Zhang, Lei; Xie, Li-Qi; Huang, Li-Yong; Xu, Ye; Cai, San-Jun; Yang, Peng-Yuan; Lu, Hao-Jie

2013-09-06

Novel biomarker verification assays are urgently required to improve the efficiency of biomarker development. Benefitting from lower development costs, multiple reaction monitoring (MRM) has been used for biomarker verification as an alternative to immunoassay. However, in general MRM analysis, only one sample can be quantified in a single experiment, which restricts its application. Here, a Hyperplex-MRM quantification approach, which combined mTRAQ for absolute quantification and iTRAQ for relative quantification, was developed to increase the throughput of biomarker verification. In this strategy, equal amounts of internal standard peptides were labeled with mTRAQ reagents Δ0 and Δ8, respectively, as double references, while 4-plex iTRAQ reagents were used to label four different samples as an alternative to mTRAQ Δ4. From the MRM trace and MS/MS spectrum, total amounts and relative ratios of target proteins/peptides of four samples could be acquired simultaneously. Accordingly, absolute amounts of target proteins/peptides in four different samples could be achieved in a single run. In addition, double references were used to increase the reliability of the quantification results. Using this approach, three biomarker candidates, ademosylhomocysteinase (AHCY), cathepsin D (CTSD), and lysozyme C (LYZ), were successfully quantified in colorectal cancer (CRC) tissue specimens of different stages with high accuracy, sensitivity, and reproducibility. To summarize, we demonstrated a promising quantification method for high-throughput verification of biomarker candidates.

Multiple reaction monitoring and multiple reaction monitoring cubed based assays for the quantitation of apolipoprotein F.

PubMed

Kumar, Abhinav; Gangadharan, Bevin; Zitzmann, Nicole

2016-10-15

Apolipoprotein F (APO-F) is a novel low abundance liver fibrosis biomarker and its concentration decreases in human serum and plasma across liver fibrosis stages. Current antibody based assays for APO-F suffer from limitations such as unspecific binding, antibody availability and undetectable target if the protein is degraded; and so an antibody-free assay has the potential to be a valuable diagnostic tool. We report an antibody-free, rapid, sensitive, selective and robust LC-MS/MS (MRM and MRM(3)) method for the detection and quantitation of APO-F in healthy human plasma. With further analysis of clinical samples, this LC-MS based method could be established as the first ever antibody-free biomarker assay for liver fibrosis. We explain the use of Skyline software for peptide selection and the creation of a reference library to aid in true peak identification of endogenous APO-F peptides in digests of human plasma without protein or peptide enrichment. Detection of a glycopeptide using MRM-EPI mode and reduction of interferences using MRM3 are explained. The amount of APO-F in human plasma from a healthy volunteer was determined to be 445.2ng/mL, the coefficient of variation (CV) of precision for 20 injections was <12% and the percentage error of each point along the calibration curve was calculated to be <8%, which is in line with the assay requirements for clinical samples. Copyright © 2016 Elsevier B.V. All rights reserved.

The application of multiple reaction monitoring and multi-analyte profiling to HDL proteins

PubMed Central

2014-01-01

Background HDL carries a rich protein cargo and examining HDL protein composition promises to improve our understanding of its functions. Conventional mass spectrometry methods can be lengthy and difficult to extend to large populations. In addition, without prior enrichment of the sample, the ability of these methods to detect low abundance proteins is limited. Our objective was to develop a high-throughput approach to examine HDL protein composition applicable to diabetes and cardiovascular disease (CVD). Methods We optimized two multiplexed assays to examine HDL proteins using a quantitative immunoassay (Multi-Analyte Profiling- MAP) and mass spectrometric-based quantitative proteomics (Multiple Reaction Monitoring-MRM). We screened HDL proteins using human xMAP (90 protein panel) and MRM (56 protein panel). We extended the application of these two methods to HDL isolated from a group of participants with diabetes and prior cardiovascular events and a group of non-diabetic controls. Results We were able to quantitate 69 HDL proteins using MAP and 32 proteins using MRM. For several common proteins, the use of MRM and MAP was highly correlated (p < 0.01). Using MAP, several low abundance proteins implicated in atherosclerosis and inflammation were found on HDL. On the other hand, MRM allowed the examination of several HDL proteins not available by MAP. Conclusions MAP and MRM offer a sensitive and high-throughput approach to examine changes in HDL proteins in diabetes and CVD. This approach can be used to measure the presented HDL proteins in large clinical studies. PMID:24397693

Quantitation of Permethylated N-Glycans through Multiple-Reaction Monitoring (MRM) LC-MS/MS

NASA Astrophysics Data System (ADS)

Zhou, Shiyue; Hu, Yunli; DeSantos-Garcia, Janie L.; Mechref, Yehia

2015-04-01

The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple-reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan was determined to be 30%, whereas it was found to be 35% for either fucosylated or sialylated N-glycans. The optimum CE for mannose and complex type N-glycan was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan compositions in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these glycans was achieved when the equivalence of 0.005 μL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitude. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples.

Detection and Site Localization of Phosphorylcholine-Modified Peptides by NanoLC-ESI-MS/MS Using Precursor Ion Scanning and Multiple Reaction Monitoring Experiments

NASA Astrophysics Data System (ADS)

Timm, Thomas; Lenz, Christof; Merkel, Dietrich; Sadiffo, Christian; Grabitzki, Julia; Klein, Jochen; Lochnit, Guenter

2015-03-01

Phosphorylcholine (PC)-modified biomolecules like lipopolysaccharides, glycosphingolipids, and (glyco)proteins are widespread, highly relevant antigens of parasites, since this small hapten shows potent immunomodulatory capacity, which allows the establishment of long-lasting infections of the host. Especially for PC-modified proteins, structural data is rare because of the zwitterionic nature of the PC substituent, resulting in low sensitivities and unusual but characteristic fragmentation patterns. We have developed a targeted mass spectrometric approach using hybrid triple quadrupole/linear ion trap (QTRAP) mass spectrometry coupled to nanoflow chromatography for the sensitive detection of PC-modified peptides from complex proteolytic digests, and the localization of the PC-modification within the peptide backbone. In a first step, proteolytic digests are screened using precursor ion scanning for the marker ions of choline ( m/z 104.1) and phosphorylcholine ( m/z 184.1) to establish the presence of PC-modified peptides. Potential PC-modified precursors are then subjected to a second analysis using multiple reaction monitoring (MRM)-triggered product ion spectra for the identification and site localization of the modified peptides. The approach was first established using synthetic PC-modified synthetic peptides and PC-modified model digests. Following the optimization of key parameters, we then successfully applied the method to the detection of PC-peptides in the background of a proteolytic digest of a whole proteome. This methodological invention will greatly facilitate the detection of PC-substituted biomolecules and their structural analysis.

Quantification of Inflammasome Adaptor Protein ASC in Biological Samples by Multiple-Reaction Monitoring Mass Spectrometry.

PubMed

Ulke-Lemée, Annegret; Lau, Arthur; Nelson, Michelle C; James, Matthew T; Muruve, Daniel A; MacDonald, Justin A

2018-06-09

Inflammation is an integral component of many diseases, including chronic kidney disease (CKD). ASC (apoptosis-associated speck-like protein containing CARD, also PYCARD) is the key inflammasome adaptor protein in the innate immune response. Since ASC specks, a macromolecular condensate of ASC protein, can be released by inflammasome-activated cells into the extracellular space to amplify inflammatory responses, the ASC protein could be an important biomarker in diagnostic applications. Herein, we describe the development and validation of a multiple reaction monitoring mass spectrometry (MRM-MS) assay for the accurate quantification of ASC in human biospecimens. Limits of detection and quantification for the signature DLLLQALR peptide (used as surrogate for the target ASC protein) were determined by the method of standard addition using synthetic isotope-labeled internal standard (SIS) peptide and urine matrix from a healthy donor (LOQ was 8.25 pM, with a ~ 1000-fold linear range). We further quantified ASC in the urine of CKD patients (8.4 ± 1.3 ng ASC/ml urine, n = 13). ASC was positively correlated with proteinuria and urinary IL-18 in CKD samples but not with urinary creatinine. Unfortunately, the ASC protein is susceptible to degradation, and patient urine that was thawed and refrozen lost 85% of the ASC signal. In summary, the MRM-MS assay provides a robust means to quantify ASC in biological samples, including clinical biospecimens; however, sample collection and storage conditions will have a critical impact on assay reliability.

Multiple reaction monitoring-ion pair finder: a systematic approach to transform nontargeted mode to pseudotargeted mode for metabolomics study based on liquid chromatography-mass spectrometry.

PubMed

Luo, Ping; Dai, Weidong; Yin, Peiyuan; Zeng, Zhongda; Kong, Hongwei; Zhou, Lina; Wang, Xiaolin; Chen, Shili; Lu, Xin; Xu, Guowang

2015-01-01

Pseudotargeted metabolic profiling is a novel strategy combining the advantages of both targeted and untargeted methods. The strategy obtains metabolites and their product ions from quadrupole time-of-flight (Q-TOF) MS by information-dependent acquisition (IDA) and then picks targeted ion pairs and measures them on a triple-quadrupole MS by multiple reaction monitoring (MRM). The picking of ion pairs from thousands of candidates is the most time-consuming step of the pseudotargeted strategy. Herein, a systematic and automated approach and software (MRM-Ion Pair Finder) were developed to acquire characteristic MRM ion pairs by precursor ions alignment, MS(2) spectrum extraction and reduction, characteristic product ion selection, and ion fusion. To test the reliability of the approach, a mixture of 15 metabolite standards was first analyzed; the representative ion pairs were correctly picked out. Then, pooled serum samples were further studied, and the results were confirmed by the manual selection. Finally, a comparison with a commercial peak alignment software was performed, and a good characteristic ion coverage of metabolites was obtained. As a proof of concept, the proposed approach was applied to a metabolomics study of liver cancer; 854 metabolite ion pairs were defined in the positive ion mode from serum. Our approach provides a high throughput method which is reliable to acquire MRM ion pairs for pseudotargeted metabolomics with improved metabolite coverage and facilitate more reliable biomarkers discoveries.

Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer

NASA Astrophysics Data System (ADS)

Wang, Evelyn H.; Combe, Peter C.; Schug, Kevin A.

2016-05-01

Methods that can efficiently and effectively quantify proteins are needed to support increasing demand in many bioanalytical fields. Triple quadrupole mass spectrometry (QQQ-MS) is sensitive and specific, and it is routinely used to quantify small molecules. However, low resolution fragmentation-dependent MS detection can pose inherent difficulties for intact proteins. In this research, we investigated variables that affect protein and fragment ion signals to enable protein quantitation using QQQ-MS. Collision induced dissociation gas pressure and collision energy were found to be the most crucial variables for optimization. Multiple reaction monitoring (MRM) transitions for seven standard proteins, including lysozyme, ubiquitin, cytochrome c from both equine and bovine, lactalbumin, myoglobin, and prostate-specific antigen (PSA) were determined. Assuming the eventual goal of applying such methodology is to analyze protein in biological fluids, a liquid chromatography method was developed. Calibration curves of six standard proteins (excluding PSA) were obtained to show the feasibility of intact protein quantification using QQQ-MS. Linearity (2-3 orders), limits of detection (0.5-50 μg/mL), accuracy (<5% error), and precision (1%-12% CV) were determined for each model protein. Sensitivities for different proteins varied considerably. Biological fluids, including human urine, equine plasma, and bovine plasma were used to demonstrate the specificity of the approach. The purpose of this model study was to identify, study, and demonstrate the advantages and challenges for QQQ-MS-based intact protein quantitation, a largely underutilized approach to date.

In house validation of a high resolution mass spectrometry Orbitrap-based method for multiple allergen detection in a processed model food.

PubMed

Pilolli, Rosa; De Angelis, Elisabetta; Monaci, Linda

2018-02-13

In recent years, mass spectrometry (MS) has been establishing its role in the development of analytical methods for multiple allergen detection, but most analyses are being carried out on low-resolution mass spectrometers such as triple quadrupole or ion traps. In this investigation, performance provided by a high resolution (HR) hybrid quadrupole-Orbitrap™ MS platform for the multiple allergens detection in processed food matrix is presented. In particular, three different acquisition modes were compared: full-MS, targeted-selected ion monitoring with data-dependent fragmentation (t-SIM/dd2), and parallel reaction monitoring. In order to challenge the HR-MS platform, the sample preparation was kept as simple as possible, limited to a 30-min ultrasound-aided protein extraction followed by clean-up with disposable size exclusion cartridges. Selected peptide markers tracing for five allergenic ingredients namely skim milk, whole egg, soy flour, ground hazelnut, and ground peanut were monitored in home-made cookies chosen as model processed matrix. Timed t-SIM/dd2 was found the best choice as a good compromise between sensitivity and accuracy, accomplishing the detection of 17 peptides originating from the five allergens in the same run. The optimized method was validated in-house through the evaluation of matrix and processing effects, recoveries, and precision. The selected quantitative markers for each allergenic ingredient provided quantification of 60-100 μg ingred /g allergenic ingredient/matrix in incurred cookies.

Substrate-Coated Illumination Droplet Spray Ionization: Real-Time Monitoring of Photocatalytic Reactions

NASA Astrophysics Data System (ADS)

Zhang, Hong; Li, Na; Zhao, Dandan; Jiang, Jie; You, Hong

2017-09-01

Real-time monitoring of photocatalytic reactions facilitates the elucidation of the mechanisms of the reactions. However, suitable tools for real-time monitoring are lacking. Herein, a novel method based on droplet spray ionization named substrate-coated illumination droplet spray ionization (SCI-DSI) for direct analysis of photocatalytic reaction solution is reported. SCI-DSI addresses many of the analytical limitations of electrospray ionization (ESI) for analysis of photocatalytic-reaction intermediates, and has potential for both in situ analysis and real-time monitoring of photocatalytic reactions. In SCI-DSI-mass spectrometry (MS), a photocatalytic reaction occurs by loading sample solutions onto the substrate-coated cover slip and by applying UV light above the modified slip; one corner of this slip adjacent to the inlet of a mass spectrometer is the high-electric-field location for launching a charged-droplet spray. After both testing and optimizing the performance of SCI-DSI, the value of this method for in situ analysis and real-time monitoring of photocatalytic reactions was demonstrated by the removal of cyclophosphamide (CP) in TiO2/UV. Reaction times ranged from seconds to minutes, and the proposed reaction intermediates were captured and identified by tandem mass spectrometry. Moreover, the free hydroxyl radical (·OH) was identified as the main radicals for CP removal. These results show that SCI-DSI is suitable for in situ analysis and real-time monitoring of CP removal under TiO2-based photocatalytic reactions. SCI-DSI is also a potential tool for in situ analysis and real-time assessment of the roles of radicals during CP removal under TiO2-based photocatalytic reactions. Graphical Abstract[Figure not available: see fulltext.

Design, implementation and multisite evaluation of a system suitability protocol for the quantitative assessment of instrument performance in liquid chromatography-multiple reaction monitoring-MS (LC-MRM-MS).

PubMed

Abbatiello, Susan E; Mani, D R; Schilling, Birgit; Maclean, Brendan; Zimmerman, Lisa J; Feng, Xingdong; Cusack, Michael P; Sedransk, Nell; Hall, Steven C; Addona, Terri; Allen, Simon; Dodder, Nathan G; Ghosh, Mousumi; Held, Jason M; Hedrick, Victoria; Inerowicz, H Dorota; Jackson, Angela; Keshishian, Hasmik; Kim, Jong Won; Lyssand, John S; Riley, C Paige; Rudnick, Paul; Sadowski, Pawel; Shaddox, Kent; Smith, Derek; Tomazela, Daniela; Wahlander, Asa; Waldemarson, Sofia; Whitwell, Corbin A; You, Jinsam; Zhang, Shucha; Kinsinger, Christopher R; Mesri, Mehdi; Rodriguez, Henry; Borchers, Christoph H; Buck, Charles; Fisher, Susan J; Gibson, Bradford W; Liebler, Daniel; Maccoss, Michael; Neubert, Thomas A; Paulovich, Amanda; Regnier, Fred; Skates, Steven J; Tempst, Paul; Wang, Mu; Carr, Steven A

2013-09-01

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities.

Design, Implementation and Multisite Evaluation of a System Suitability Protocol for the Quantitative Assessment of Instrument Performance in Liquid Chromatography-Multiple Reaction Monitoring-MS (LC-MRM-MS)*

PubMed Central

Abbatiello, Susan E.; Mani, D. R.; Schilling, Birgit; MacLean, Brendan; Zimmerman, Lisa J.; Feng, Xingdong; Cusack, Michael P.; Sedransk, Nell; Hall, Steven C.; Addona, Terri; Allen, Simon; Dodder, Nathan G.; Ghosh, Mousumi; Held, Jason M.; Hedrick, Victoria; Inerowicz, H. Dorota; Jackson, Angela; Keshishian, Hasmik; Kim, Jong Won; Lyssand, John S.; Riley, C. Paige; Rudnick, Paul; Sadowski, Pawel; Shaddox, Kent; Smith, Derek; Tomazela, Daniela; Wahlander, Asa; Waldemarson, Sofia; Whitwell, Corbin A.; You, Jinsam; Zhang, Shucha; Kinsinger, Christopher R.; Mesri, Mehdi; Rodriguez, Henry; Borchers, Christoph H.; Buck, Charles; Fisher, Susan J.; Gibson, Bradford W.; Liebler, Daniel; MacCoss, Michael; Neubert, Thomas A.; Paulovich, Amanda; Regnier, Fred; Skates, Steven J.; Tempst, Paul; Wang, Mu; Carr, Steven A.

2013-01-01

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities. PMID:23689285

Neurological adverse events associated with vaccination.

PubMed

Piyasirisilp, Sucheep; Hemachudha, Thiravat

2002-06-01

Public tolerance to adverse reactions is minimal. Several reporting systems have been established to monitor adverse events following immunization. The present review summarizes data on neurologic complications following vaccination, and provides evidence that indicates whether they were directly associated with the vaccines. These complications include autism (measles vaccine), multiple sclerosis (hepatitis B vaccine), meningoencephalitis (Japanese encephalitis vaccine), Guillain-Barré syndrome and giant cell arteritis (influenza vaccine), and reactions after exposure to animal rabies vaccine. Seizures and hypotonic/hyporesponsive episodes following pertussis vaccination and potential risks associated with varicella vaccination, as well as vaccine-associated paralytic poliomyelitis following oral poliovirus vaccination, are also described. In addition, claims that complications are caused by adjuvants, preservatives and contaminants [i.e. macrophagic myofasciitis (aluminium), neurotoxicity (thimerosal), and new variant Creutzfeldt-Jakob disease (bovine-derived materials)] are discussed.

Unravelling the mysteries of sub-second biochemical processes using time-resolved mass spectrometry.

PubMed

Lento, Cristina; Wilson, Derek J

2017-05-21

Many important chemical and biochemical phenomena proceed on sub-second time scales before entering equilibrium. In this mini-review, we explore the history and recent advancements of time-resolved mass spectrometry (TRMS) for the characterization of millisecond time-scale chemical reactions and biochemical processes. TRMS allows for the simultaneous tracking of multiple reactants, intermediates and products with no chromophoric species required, high sensitivity and temporal resolution. The method has most recently been used for the characterization of several short-lived reaction intermediates in rapid chemical reactions. Most of the reactions that occur in living organisms are accelerated by enzymes, with pre-steady state kinetics only attainable using time-resolved methods. TRMS has been increasingly used to monitor the conversion of substrates to products and the resulting changes to the enzyme during catalytic turnover. Early events in protein folding systems have also been elucidated, along with the characterization of dynamics and transient secondary structures in intrinsically disordered proteins. In this review, we will highlight representative examples where TRMS has been applied to study these phenomena.

Short term safety assessment of cilazapril.

PubMed

Coulter, D M

1993-11-24

To undertake an event monitoring study of cilazapril in general practice during the early marketing period, to provide some comparisons with other angiotensin converting enzyme inhibitors and to assess the monitoring method. The monitoring was undertaken in the Intensive Medicines Monitoring Programme. Cilazapril was prescribed for mild to moderate hypertension in 996 patients at a recommended dose of 2.5-5.0 mg daily. The monitoring period was six months and practitioners were asked to report all adverse events. A reaction profile was prepared and compared with profiles for lisinopril, enalapril and captopril. The chi-square test was applied to differences in proportions. There were 84 (8.4%) reports describing 133 adverse events; 124 (93%) were assessed as reactions. Withdrawals totalled 53 (5.3%). The most common reactions were cough (2.9%), nausea and vomiting (1.3%) and lethargy (1.1%). Cilazapril had a higher proportion of neurological reactions (p < 0.001) (mainly headache) but a lower proportion of skin reactions (p = 0.001) than the other ACE inhibitors. It also had relatively less diarrhoea and there were differences in the patterns of psychiatric reactions. Cilazapril has a similar reaction profile to other ACE inhibitors but this paper shows differences, some not previously reported, that may assist selection when prescribing. Although there was a high rate of reporting of known adverse reactions, other events were reported at a very low rate and spontaneous reporting is thus confirmed as an unreliable method of monitoring for unexpected adverse reactions.

Stratified premedication strategy for the prevention of contrast media hypersensitivity in high-risk patients.

PubMed

Lee, Suh-Young; Yang, Min Suk; Choi, Young-Hoon; Park, Chang Min; Park, Heung-Woo; Cho, Sang Heon; Kang, Hye-Ryun

2017-03-01

Although the severity of hypersensitivity reactions to iodinated contrast media varies, it is well correlated with the severity of recurrent reactions; however, prophylaxis protocols are not severity-stratified. To assess the outcomes of tailored prophylaxis according to the severity of hypersensitivity reactions to iodinated contrast media. Our premedication protocols were stratified based on the severity of previous reactions: (1) 4 mg of chlorpheniramine for mild reactions, (2) adding 40 mg of methylprednisolone for moderate reactions, and (3) adding multiple doses of 40 mg of methylprednisolone for severe index reactions. Cases of reexposure in patients with a history of hypersensitivity reactions were routinely monitored and mandatorily recorded. Among a total of 850 patients who underwent enhanced computed tomography after severity-tailored prophylaxis, breakthrough reactions occurred in 17.1%, but most breakthrough reactions (89.0%) were mild and did not require medical treatment. Additional corticosteroid use did not reduce the breakthrough reaction rate in cases with a mild index reaction (16.8% vs 17.2%, P = .70). However, underpremedication with a single dose of corticosteroid revealed significantly higher rates of breakthrough reaction than did double doses of corticosteroid in cases with a severe index reaction (55.6% vs 17.4%, P = .02). Changing the iodinated contrast media resulted in an additional reduction of the breakthrough reaction rate overall (14.9% vs 32.1%, P = .001). In a total severity-based stratified prophylaxis regimens and changing iodinated contrast media can be considered in patients with a history of previous hypersensitivity reaction to iodinated contrast media to reduce the risk of breakthrough reactions. Copyright © 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Effects of alcohol on complex performance.

DOT National Transportation Integrated Search

1969-08-01

Nine subjects were tested on a battery of tasks involving monitoring (simple reaction time, choice reaction time, and meter monitoring), two-dimensional compensatory tracking, and mental arithmetic. Three workloads were presented--monitoring plus tra...

In situ imaging of the soldering reactions in nanoscale Cu/Sn/Cu and Sn/Cu/Sn diffusion couples

NASA Astrophysics Data System (ADS)

Yin, Qiyue; Gao, Fan; Gu, Zhiyong; Wang, Jirui; Stach, Eric A.; Zhou, Guangwen

2018-01-01

The soldering reactions of three-segmented Sn/Cu/Sn and Cu/Sn/Cu diffusion couples are monitored by in-situ transmission electron microscopy to reveal the metallurgical reaction mechanism and the associated phase transformation pathway. For Sn/Cu/Sn diffusion couples, there is no ɛ-Cu3Sn formation due to the relatively insufficient Cu as compared to Sn. Kirkendall voids form initially in the Cu segment and then disappear due to the volume expansion associated with the continued intermetallic compound (IMC) formation as the reaction progresses. The incoming Sn atoms react with Cu to form η-Cu6Sn5, and the continuous reaction then transforms the entire nanowire to η-Cu6Sn5 grains with remaining Sn. With continued heating slightly above the melting point of Sn, an Sn-rich liquid phase forms between η-Cu6Sn5 grains. By contrast, the reaction in the Cu/Sn/Cu diffusion couples results in the intermetallic phases of both Cu3Sn and Cu6Sn5 and the development of Cu6Sn5 bulges on Cu3Sn grains. Kirkendall voids form in the two Cu segments, which grow and eventually break the nanowire into multiple segments.

A new parameter to simultaneously assess antioxidant activity for multiple phenolic compounds present in food products.

PubMed

Yang, Hong; Xue, Xuejia; Li, Huan; Tay-Chan, Su Chin; Ong, Seng Poon; Tian, Edmund Feng

2017-08-15

In this work, we established a new methodology to simultaneously assess the relative reaction rates of multiple antioxidant compounds in one experimental set-up. This new methodology hypothesizes that the competition among antioxidant compounds towards limiting amount of free radical (in this article, DPPH) would reflect their relative reaction rates. In contrast with the conventional detection of DPPH decrease at 515nm on a spectrophotometer, depletion of antioxidant compounds treated by a series of DPPH concentrations was monitored instead using liquid chromatography coupled with quadrupole time-of-flight (LC-QTOF). A new parameter, namely relative antioxidant activity (RAA), has been proposed to rank these antioxidants according to their reaction rate constants. We have investigated the applicability of RAA using pre-mixed standard phenolic compounds, and also extended this application to two food products, i.e. red wine and green tea. It has been found that RAA correlates well with the reported k values. This new parameter, RAA, provides a new perspective in evaluating antioxidant compounds present in food and herbal matrices. It not only realistically reflects the antioxidant activity of compounds when co-existing with competitive constituents; and it could also quicken up the discovery process in the search for potent yet rare antioxidants from many herbs of food/medicinal origins. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Computational Tool to Detect and Avoid Redundancy in Selected Reaction Monitoring

PubMed Central

Röst, Hannes; Malmström, Lars; Aebersold, Ruedi

2012-01-01

Selected reaction monitoring (SRM), also called multiple reaction monitoring, has become an invaluable tool for targeted quantitative proteomic analyses, but its application can be compromised by nonoptimal selection of transitions. In particular, complex backgrounds may cause ambiguities in SRM measurement results because peptides with interfering transitions similar to those of the target peptide may be present in the sample. Here, we developed a computer program, the SRMCollider, that calculates nonredundant theoretical SRM assays, also known as unique ion signatures (UIS), for a given proteomic background. We show theoretically that UIS of three transitions suffice to conclusively identify 90% of all yeast peptides and 85% of all human peptides. Using predicted retention times, the SRMCollider also simulates time-scheduled SRM acquisition, which reduces the number of interferences to consider and leads to fewer transitions necessary to construct an assay. By integrating experimental fragment ion intensities from large scale proteome synthesis efforts (SRMAtlas) with the information content-based UIS, we combine two orthogonal approaches to create high quality SRM assays ready to be deployed. We provide a user friendly, open source implementation of an algorithm to calculate UIS of any order that can be accessed online at http://www.srmcollider.org to find interfering transitions. Finally, our tool can also simulate the specificity of novel data-independent MS acquisition methods in Q1–Q3 space. This allows us to predict parameters for these methods that deliver a specificity comparable with that of SRM. Using SRM interference information in addition to other sources of information can increase the confidence in an SRM measurement. We expect that the consideration of information content will become a standard step in SRM assay design and analysis, facilitated by the SRMCollider. PMID:22535207

MIR-ATR sensor for process monitoring

NASA Astrophysics Data System (ADS)

Geörg, Daniel; Schalk, Robert; Methner, Frank-Jürgen; Beuermann, Thomas

2015-06-01

A mid-infrared attenuated total reflectance (MIR-ATR) sensor has been developed for chemical reaction monitoring. The optical setup of the compact and low-priced sensor consists of an IR emitter as light source, a zinc selenide (ZnSe) ATR prism as boundary to the process, and four thermopile detectors, each equipped with an optical bandpass filter. The practical applicability was tested during esterification of ethanol and formic acid to ethyl formate and water as a model reaction with subsequent distillation. For reference analysis, a Fourier transform mid-infrared (FT-MIR) spectrometer with diamond ATR module was applied. On-line measurements using the MIR-ATR sensor and the FT-MIR spectrometer were performed in a bypass loop. The sensor was calibrated by multiple linear regression in order to link the measured absorbance in the four optical channels to the analyte concentrations. The analytical potential of the MIR-ATR sensor was demonstrated by simultaneous real-time monitoring of all four chemical substances involved in the esterification and distillation process. The temporal courses of the sensor signals are in accordance with the concentration values achieved by the commercial FT-MIR spectrometer. The standard error of prediction for ethanol, formic acid, ethyl formate, and water were 0.38 mol L  -  1, 0.48 mol L  -  1, 0.38 mol L  -  1, and 1.12 mol L  -  1, respectively. A procedure based on MIR spectra is presented to simulate the response characteristics of the sensor if the transmission ranges of the filters are varied. Using this tool analyte specific bandpass filters for a particular chemical reaction can be identified. By exchanging the optical filters, the sensor can be adapted to a wide range of processes in the chemical, pharmaceutical, and beverage industries.

Development of methods to monitor ionization modification from dosing vehicles and phospholipids in study samples.

PubMed

Chang, Min; Li, Yongchao; Angeles, Reginald; Khan, Samina; Chen, Lian; Kaplan, Julia; Yang, Liyu

2011-08-01

Two approaches to monitor the matrix effect on ionization in study samples were described. One approach is the addition of multiple reaction monitoring transitions to the bioanalytical methods to monitor the presence of known ionization modification-causing components of the matrix, for example, m/z 184→125 (or m/z 184→184) and m/z 133→89 may be used for phospholipids and polyethylene oxide containing surfactants, respectively. This approach requires no additional equipment and can be readily adapted for most method. The approach detects only the intended interfering compounds and provides little quantitative indication if the matrix effect is within the tolerable range (±15%). The other approach requires the addition of an infusion pump and identifies an appropriate surrogate of the analyte to be infused for the determination of modification on the ionization of the analyte. The second approach detects interferences in the sample regardless of the sources (i.e., dosing vehicle components, co-administrated drugs, their metabolites, phospholipids, plasticizers and endogenous components introduced due to disease stage).

Multiple Reaction Monitoring Enables Precise Quantification of 97 Proteins in Dried Blood Spots*

PubMed Central

Chambers, Andrew G.; Percy, Andrew J.; Yang, Juncong; Borchers, Christoph H.

2015-01-01

The dried blood spot (DBS) methodology provides a minimally invasive approach to sample collection and enables room-temperature storage for most analytes. DBS samples have successfully been analyzed by liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM-MS) to quantify a large range of small molecule biomarkers and drugs; however, this strategy has only recently been explored for MS-based proteomics applications. Here we report the development of a highly multiplexed MRM assay to quantify endogenous proteins in human DBS samples. This assay uses matching stable isotope-labeled standard peptides for precise, relative quantification, and standard curves to characterize the analytical performance. A total of 169 peptides, corresponding to 97 proteins, were quantified in the final assay with an average linear dynamic range of 207-fold and an average R2 value of 0.987. The total range of this assay spanned almost 5 orders of magnitude from serum albumin (P02768) at 18.0 mg/ml down to cholinesterase (P06276) at 190 ng/ml. The average intra-assay and inter-assay precision for 6 biological samples ranged from 6.1–7.5% CV and 9.5–11.0% CV, respectively. The majority of peptide targets were stable after 154 days at storage temperatures from −20 °C to 37 °C. Furthermore, protein concentration ratios between matching DBS and whole blood samples were largely constant (<20% CV) across six biological samples. This assay represents the highest multiplexing yet achieved for targeted protein quantification in DBS samples and is suitable for biomedical research applications. PMID:26342038

Quantitation of Permethylated N-Glycans through Multiple-Reaction Monitoring (MRM) LC-MS/MS

PubMed Central

Zhou, Shiyue; Hu, Yunli; DeSantos-Garcia, Janie L.; Mechref, Yehia

2015-01-01

The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan structures was determined to be 30% while it was found to be 35% for either fucosylated or sialylated structures The optimum CE for mannose and complex type N-glycan structures was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan structures in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these structures was achieved when the equivalence of 0.005 μL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitudes. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan structures enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples. PMID:25698222

Multiple Hotspot Mutations Scanning by Single Droplet Digital PCR.

PubMed

Decraene, Charles; Silveira, Amanda B; Bidard, François-Clément; Vallée, Audrey; Michel, Marc; Melaabi, Samia; Vincent-Salomon, Anne; Saliou, Adrien; Houy, Alexandre; Milder, Maud; Lantz, Olivier; Ychou, Marc; Denis, Marc G; Pierga, Jean-Yves; Stern, Marc-Henri; Proudhon, Charlotte

2018-02-01

Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan ® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. The KRAS and EGFR assays were highly specific and both reached a limit of detection of <0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different KRAS or EGFR mutations. This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy. © 2017 American Association for Clinical Chemistry.

[Response Mechanism of Trace Metals in the Bishuiyan Subterranean River to the Rainfall and Their Source Analysis].

PubMed

Zou, Yan-e; Jiang, Ping-ping; Zhang, Qiang; Tang, Qing-jia; Kang, Zhi-qiang; Gong, Xiao- ping; Chen, Chang-jie; Yu, Jian-guo

2015-12-01

High-frequency sampling was conducted at the outlet of Guangxi Bishuiyan karst subterranean river using an automatic sampler during the rainfall events. The hydrochemical drymanic variation characteristics of trace metals (Cu, Pb, Zn, Cd) at the outlet of Guangxi Bishuiyan karst subterranean river were analyzed, and the sources of the trace metals in the subterranean river as well as their response to rainfall were explored. The results showed that the rainfall provoked a sharp decrease in the major elements (Ca²⁺, Mg²⁺, HCO₃⁻, etc.) due to dilution and precipitation, while it also caused an increase in the concentrations of dissolved metals including Al, Mn, Cu, Zn and Cd, due to water-rock reaction, sediment remobilization, and soil erosion. The water-rock reaction was more sensitive to rainfall than the others, while the sediment remobilization and soil erosion took the main responsibility for the chemical change of the heavy metals. The curves of the heavy metal concentrations presented multiple peaks, of which the maximum was reached at 9 hours later after the largest precipitation. Different metal sources and the double-inlet structure of the subterranean river were supposed to be the reasons for the formation of multiple peaks. During the monitoring period, the average speed of the solute in the river reached about 0.47 km · h⁻¹, indicating fast migration of the pollutants. Therefore, monitoring the chemical dynamics of the karst subterranean river, mastering the sources and migration characteristics of trace metal components have great significance for the subterranean river environment pollution treatment.

Air and Surface Sampling Method for Assessing Exposures to Quaternary Ammonium Compounds Using Liquid Chromatography Tandem Mass Spectrometry.

PubMed

LeBouf, Ryan F; Virji, Mohammed Abbas; Ranpara, Anand; Stefaniak, Aleksandr B

2017-07-01

This method was designed for sampling select quaternary ammonium (quat) compounds in air or on surfaces followed by analysis using ultraperformance liquid chromatography tandem mass spectrometry. Target quats were benzethonium chloride, didecyldimethylammonium bromide, benzyldimethyldodecylammonium chloride, benzyldimethyltetradecylammonium chloride, and benzyldimethylhexadecylammonium chloride. For air sampling, polytetrafluoroethylene (PTFE) filters are recommended for 15-min to 24-hour sampling. For surface sampling, Pro-wipe® 880 (PW) media was chosen. Samples were extracted in 60:40 acetonitrile:0.1% formic acid for 1 hour on an orbital shaker. Method detection limits range from 0.3 to 2 ng/ml depending on media and analyte. Matrix effects of media are minimized through the use of multiple reaction monitoring versus selected ion recording. Upper confidence limits on accuracy meet the National Institute for Occupational Safety and Health 25% criterion for PTFE and PW media for all analytes. Using PTFE and PW analyzed with multiple reaction monitoring, the method quantifies levels among the different quats compounds with high precision (<10% relative standard deviation) and low bias (<11%). The method is sensitive enough with very low method detection limits to capture quats on air sampling filters with only a 15-min sample duration with a maximum assessed storage time of 103 days before sample extraction. This method will support future exposure assessment and quantitative epidemiologic studies to explore exposure-response relationships and establish levels of quats exposures associated with adverse health effects. © The Author 2017. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.

Geoelectrical signatures of reactive mixing

NASA Astrophysics Data System (ADS)

Ghosh, U.; Bandopadhyay, A.; Jougnot, D.; Le Borgne, T.; Meheust, Y.

2017-12-01

Characterizing the effects of fluid mixing on geochemical reactions in the subsurface is of paramount importance owing to their pivotal role in processes such as contaminant migration or aquifer remediation, to name a few [1]. Large velocity gradients in the porous media are expected to lead to enhanced diffusive mixing accompanied by augmented reaction rates [2]. Despite its importance, accurate monitoring of such processes still remains an open challenge, mainly due to the opacity of the medium and to the lack of access to it. However, in recent years, geophysical methods based on electrical conductivity and polarization have come up as a promising tool for mapping and monitoring such reactions in the subsurface. In this regard, one of the main challenges is to properly characterize the multiple sources of electrical signals and in particular isolate the influence of reactive mixing on the electrical conductivity from those of other sources [3]. In this work, we explore the coupling between fluid mixing, reaction and charge migration in porous media to evaluate the potential of obtaining a spatially-resolved measurement of local reaction rates in the subsurface from electrical measurements. To this end, we employ a lamellar description of the mixing interface [4] with novel semi-analytical upscaling techniques to quantify changes in electrical conductivity induced by chemical reactions across mixing fronts. The changes in electrical conductivity are strongly dependent on the concentration of ionic species as well as on the polarization of the pore (water) solution around the grains, which in turn are controlled by local reaction rates and, consequently, by the local velocity gradients. Hence, our results essentially suggest that local variations in the electrical conductivity may be quantitatively related to the mixing and reaction dynamics, and thus be used as a measurement tool to characterize these dynamics. References 1. M. Dentz, T. Le Borgne, A. Englert, and B. Bijeljic, J. Cont. Hyd., 120, 1-17, 2011. 2. T. Le Borgne, T. R. Ginn, and M. Dentz, Geophys. Res. Lett., 41(22), 7898-7906, 2014. 3. R Knight et al. Reviews of Geophysics, 48(4), 2010. 4. T. Le Borgne, M. Dentz, and E. Villermaux, J. Fluid Mech., 770, 458-498, 2015.

High mass accuracy assay for trimethylamine N-oxide using stable-isotope dilution with liquid chromatography coupled to orthogonal acceleration time of flight mass spectrometry with multiple reaction monitoring.

PubMed

Heaney, Liam M; Jones, Donald J L; Mbasu, Richard J; Ng, Leong L; Suzuki, Toru

2016-01-01

Trimethylamine N-oxide (TMAO) has attracted interest as circulating levels have reported prognostic value in patients with cardiovascular conditions, such as heart failure. With continual advances in accurate mass measurements, robust methods that can employ the capabilities of time of flight mass spectrometers would offer additional utility in the analysis of complex clinical samples. A Waters Acquity UPLC was coupled to a Waters Synapt G2-S high-resolution mass spectrometer. TMAO was measured in plasma by stable-isotope dilution-hydrophilic interaction liquid chromatography-time of flight mass spectrometry with multiple reaction monitoring (LC-ToF-MRM). Two transitions were monitored: m/z 76.1 to 58.066/59.073 and m/z 85.1 to 66.116/68.130. The method was assessed for linearity, lower limits of detection and quantitation, and reproducibility. A selected cohort of patients with systolic heart failure (SHF; n = 43) and healthy controls (n = 42) were measured to verify the assay is suitable for the analysis of clinical samples. Quantitative analysis of TMAO using LC-ToF-MRM enabled linearity to be established between 0.1 and 75 μmol/L, with a lower limit of detection of 0.05 μmol/L. Relative standard deviations reported an inter-day variation of ≤20.8% and an intra-day variation of ≤11.4% with an intra-study quality control variation of 2.7%. Run times were 2.5 min. Clinical application of the method reported that TMAO in SHF was elevated compared to that of healthy controls (p < 0.0005). LC-ToF-MRM offers a highly selective method for accurate mass measurement of TMAO with rapid and reproducible results. Applicability of the method was shown in a selected cohort of patient samples.

Characterization and application of droplet spray ionization for real-time reaction monitoring.

PubMed

Zhang, Hong; Li, Na; Li, Xiao-di; Jiang, Jie; Zhao, Dan-Dan; You, Hong

2016-08-01

The ionization source for real-time reaction monitoring has attracted tremendous interest in recent years. We have previously reported a reliable approach in which droplet spray ionization (DSI) was used for monitoring chemical reactions in real-time. Herein, we systematically investigated the characterization and application of DSI for real-time reaction monitoring. Analyte ions are generated by loading a sample solution onto a corner of a microscope cover glass positioned in front of the MS inlet and applying a high voltage to the sample. The tolerance to positioning, solvent effect, spray angle and spray time were investigated. Extension to real-time monitoring of macromolecule reactions was also demonstrated by the charge state change of cytochrome c in the presence of acetic acid. The corner could be positioned within an area of approximately 10 × 6 × 5 mm (x, y, z) in front of the MS inlet. The broad polarities of solvents from methanol to PhF were suitable for DSI. It featured monitoring real-time changes in reactions on the time scale of seconds to minutes. A real-time charge state change of cytochrome c was captured. DSI-MS features ease of use, durability of the spray platform and reusability of the ion source. Eliminating the need for a sample transport capillary, DSI opens a new avenue for the in situ analysis and real-time monitoring of short-lived key reaction intermediates even at subsecond dead times. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Monitoring chemical reactions by low-field benchtop NMR at 45 MHz: pros and cons.

PubMed

Silva Elipe, Maria Victoria; Milburn, Robert R

2016-06-01

Monitoring chemical reactions is the key to controlling chemical processes where NMR can provide support. High-field NMR gives detailed structural information on chemical compounds and reactions; however, it is expensive and complex to operate. Conversely, low-field NMR instruments are simple and relatively inexpensive alternatives. While low-field NMR does not provide the detailed information as the high-field instruments as a result of their smaller chemical shift dispersion and the complex secondary coupling, it remains of practical value as a process analytical technology (PAT) tool and is complimentary to other established methods, such as ReactIR and Raman spectroscopy. We have tested a picoSpin-45 (currently under ThermoFisher Scientific) benchtop NMR instrument to monitor three types of reactions by 1D (1) H NMR: a Fischer esterification, a Suzuki cross-coupling, and the formation of an oxime. The Fischer esterification is a relatively simple reaction run at high concentration and served as proof of concept. The Suzuki coupling is an example of a more complex, commonly used reaction involving overlapping signals. Finally, the oxime formation involved a reaction in two phases that cannot be monitored by other PAT tools. Here, we discuss the pros and cons of monitoring these reactions at a low-field of 45 MHz by 1D (1) H NMR. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Hair-based rapid analyses for multiple drugs in forensics and doping: application of dynamic multiple reaction monitoring with LC-MS/MS.

PubMed

Shah, Iltaf; Petroczi, Andrea; Uvacsek, Martina; Ránky, Márta; Naughton, Declan P

2014-01-01

Considerable efforts are being extended to develop more effective methods to detect drugs in forensic science for applications such as preventing doping in sport. The aim of this study was to develop a sensitive and accurate method for analytes of forensic and toxicological nature in human hair at sub-pg levels. The hair test covers a range of different classes of drugs and metabolites of forensic and toxicological nature including selected anabolic steroids, cocaine, amphetamines, cannabinoids, opiates, bronchodilators, phencyclidine and ketamine. For extraction purposes, the hair samples were decontaminated using dichloromethane, ground and treated with 1 M sodium hydroxide and neutralised with hydrochloric acid and phosphate buffer and the homogenate was later extracted with hexane using liquid-liquid extraction (LLE). Following extraction from hair samples, drug-screening employed liquid chromatography coupled to tandem mass spectrometric (LC-MS/MS) analysis using dynamic multiple reaction monitoring (DYN-MRM) method using proprietary software. The screening method (for > 200 drugs/metabolites) was calibrated with a tailored drug mixture and was validated for 20 selected drugs for this study. Using standard additions to hair sample extracts, validation was in line with FDA guidance. A Zorbax Eclipse plus C18 (2.1 mm internal diameter × 100 mm length × 1.8 μm particle size) column was used for analysis. Total instrument run time was 8 minutes with no noted matrix interferences. The LOD of compounds ranged between 0.05-0.5 pg/mg of hair. 233 human hair samples were screened using this new method and samples were confirmed positive for 20 different drugs, mainly steroids and drugs of abuse. This is the first report of the application of this proprietary system to investigate the presence of drugs in human hair samples. The method is selective, sensitive and robust for the screening and confirmation of multiple drugs in a single analysis and has potential as a very useful tool for the analysis of large array of controlled substances and drugs of abuse.

Multiple reaction monitoring (MRM)-profiling for biomarker discovery applied to human polycystic ovarian syndrome.

PubMed

Cordeiro, Fernanda B; Ferreira, Christina R; Sobreira, Tiago Jose P; Yannell, Karen E; Jarmusch, Alan K; Cedenho, Agnaldo P; Lo Turco, Edson G; Cooks, R Graham

2017-09-15

We describe multiple reaction monitoring (MRM)-profiling, which provides accelerated discovery of discriminating molecular features, and its application to human polycystic ovary syndrome (PCOS) diagnosis. The discovery phase of the MRM-profiling seeks molecular features based on some prior knowledge of the chemical functional groups likely to be present in the sample. It does this through use of a limited number of pre-chosen and chemically specific neutral loss and/or precursor ion MS/MS scans. The output of the discovery phase is a set of precursor/product transitions. In the screening phase these MRM transitions are used to interrogate multiple samples (hence the name MRM-profiling). MRM-profiling was applied to follicular fluid samples of 22 controls and 29 clinically diagnosed PCOS patients. Representative samples were delivered by flow injection to a triple quadrupole mass spectrometer set to perform a number of pre-chosen and chemically specific neutral loss and/or precursor ion MS/MS scans. The output of this discovery phase was a set of 1012 precursor/product transitions. In the screening phase each individual sample was interrogated for these MRM transitions. Principal component analysis (PCA) and receiver operating characteristic (ROC) curves were used for statistical analysis. To evaluate the method's performance, half the samples were used to build a classification model (testing set) and half were blinded (validation set). Twenty transitions were used for the classification of the blind samples, most of them (N = 19) showed lower abundances in the PCOS group and corresponded to phosphatidylethanolamine (PE) and phosphatidylserine (PS) lipids. Agreement of 73% with clinical diagnosis was found when classifying the 26 blind samples. MRM-profiling is a supervised method characterized by its simplicity, speed and the absence of chromatographic separation. It can be used to rapidly isolate discriminating molecules in healthy/disease conditions by tailored screening of signals associated with hundreds of molecules in complex samples. Copyright © 2017 John Wiley & Sons, Ltd.

MRMaid, the web-based tool for designing multiple reaction monitoring (MRM) transitions.

PubMed

Mead, Jennifer A; Bianco, Luca; Ottone, Vanessa; Barton, Chris; Kay, Richard G; Lilley, Kathryn S; Bond, Nicholas J; Bessant, Conrad

2009-04-01

Multiple reaction monitoring (MRM) of peptides uses tandem mass spectrometry to quantify selected proteins of interest, such as those previously identified in differential studies. Using this technique, the specificity of precursor to product transitions is harnessed for quantitative analysis of multiple proteins in a single sample. The design of transitions is critical for the success of MRM experiments, but predicting signal intensity of peptides and fragmentation patterns ab initio is challenging given existing methods. The tool presented here, MRMaid (pronounced "mermaid") offers a novel alternative for rapid design of MRM transitions for the proteomics researcher. The program uses a combination of knowledge of the properties of optimal MRM transitions taken from expert practitioners and literature with MS/MS evidence derived from interrogation of a database of peptide identifications and their associated mass spectra. The tool also predicts retention time using a published model, allowing ordering of transition candidates. By exploiting available knowledge and resources to generate the most reliable transitions, this approach negates the need for theoretical prediction of fragmentation and the need to undertake prior "discovery" MS studies. MRMaid is a modular tool built around the Genome Annotating Proteomic Pipeline framework, providing a web-based solution with both descriptive and graphical visualizations of transitions. Predicted transition candidates are ranked based on a novel transition scoring system, and users may filter the results by selecting optional stringency criteria, such as omitting frequently modified residues, constraining the length of peptides, or omitting missed cleavages. Comparison with published transitions showed that MRMaid successfully predicted the peptide and product ion pairs in the majority of cases with appropriate retention time estimates. As the data content of the Genome Annotating Proteomic Pipeline repository increases, the coverage and reliability of MRMaid are set to increase further. MRMaid is freely available over the internet as an executable web-based service at www.mrmaid.info.

MRMaid, the Web-based Tool for Designing Multiple Reaction Monitoring (MRM) Transitions*

PubMed Central

Mead, Jennifer A.; Bianco, Luca; Ottone, Vanessa; Barton, Chris; Kay, Richard G.; Lilley, Kathryn S.; Bond, Nicholas J.; Bessant, Conrad

2009-01-01

Multiple reaction monitoring (MRM) of peptides uses tandem mass spectrometry to quantify selected proteins of interest, such as those previously identified in differential studies. Using this technique, the specificity of precursor to product transitions is harnessed for quantitative analysis of multiple proteins in a single sample. The design of transitions is critical for the success of MRM experiments, but predicting signal intensity of peptides and fragmentation patterns ab initio is challenging given existing methods. The tool presented here, MRMaid (pronounced “mermaid”) offers a novel alternative for rapid design of MRM transitions for the proteomics researcher. The program uses a combination of knowledge of the properties of optimal MRM transitions taken from expert practitioners and literature with MS/MS evidence derived from interrogation of a database of peptide identifications and their associated mass spectra. The tool also predicts retention time using a published model, allowing ordering of transition candidates. By exploiting available knowledge and resources to generate the most reliable transitions, this approach negates the need for theoretical prediction of fragmentation and the need to undertake prior “discovery” MS studies. MRMaid is a modular tool built around the Genome Annotating Proteomic Pipeline framework, providing a web-based solution with both descriptive and graphical visualizations of transitions. Predicted transition candidates are ranked based on a novel transition scoring system, and users may filter the results by selecting optional stringency criteria, such as omitting frequently modified residues, constraining the length of peptides, or omitting missed cleavages. Comparison with published transitions showed that MRMaid successfully predicted the peptide and product ion pairs in the majority of cases with appropriate retention time estimates. As the data content of the Genome Annotating Proteomic Pipeline repository increases, the coverage and reliability of MRMaid are set to increase further. MRMaid is freely available over the internet as an executable web-based service at www.mrmaid.info. PMID:19011259

A simple and selective method for the measurement of azadirachtin and related azadirachtoid levels in fruits and vegetables using liquid chromatography electrospray ionization tandem mass spectrometry.

PubMed

Sarais, Giorgia; Caboni, Pierluigi; Sarritzu, Erika; Russo, Mariateresa; Cabras, Paolo

2008-05-14

Neem-based insecticides containing azadirachtin and related azadirachtoids are widely used in agriculture. Here, we report an analytical method for the rapid and accurate quantification of the insecticide azadirachtin A and B and other azadirachtoids such as salannin, nimbin, and their deacetylated analogues on tomatoes and peaches. Azadirachtoids were extracted from fruits and vegetables with acetonitrile. Using high-performance liquid chromatography/electrospray ionization tandem mass spectrometer, azadirachtoids were selectively detected monitoring the multiple reaction transitions of sodium adduct precursor ions. For azadirachtin A, calibration was linear over a working range of 1-1000 microg/L with r > 0.996. The limit of detection and limit of quantification for azadirachtin A were 0.4 and 0.8 microg/kg, respectively. The presence of interfering compounds in the peach and tomato extracts was evaluated and found to be minimal. Because of the linear behavior, it was concluded that the multiple reaction transitions of sodium adduct ions can be used for analytical purposes, that is, for the identification and quantification of azadirachtin A and B and related azadirachtoids in fruit and vegetable extracts at trace levels.

Simultaneous determination of three anticonvulsants using hydrophilic interaction LC-MS.

PubMed

Oertel, Reinhard; Arenz, Norman; Pietsch, Jörg; Kirch, Wilhelm

2009-01-01

A specific and automated method was developed to quantify the anticonvulsants gabapentin, pregabalin and vigabatrin simultaneously in human serum. Samples were prepared with a protein precipitation. The hydrophilic interaction chromatography (HILIC) with a mobile phase gradient was used to divide off ions of the matrix and for separation of the analytes. Four different HILIC-columns and two different column temperatures were tested. The Tosoh-Amid column gave the best results: single small peaks. The anticonvulsants were detected in the multiple reaction monitoring mode (MRM) with ESI-MS-MS. Using a volume of 100 microL biological sample the lowest point of the standard curve, i.e. the lower LOQs were 312 ng/mL. The described HILIC-MS-MS method is suitable for therapeutic drug monitoring and for clinical and pharmcokinetical investigations of the anticonvulsives.

Lysine-Derived Protein-Bound Heyns Compounds in Bakery Products.

PubMed

Treibmann, Stephanie; Hellwig, Anne; Hellwig, Michael; Henle, Thomas

2017-12-06

Fructose and dicarbonyl compounds resulting from fructose in heated foods have been linked to pathophysiological pathways of several metabolic disorders. Up to now, very little has been known about the Maillard reaction of fructose in food. Heyns rearrangement compounds (HRCs), the first stable intermediates of the Maillard reaction between amino components and fructose, have not yet been quantitated as protein-bound products in food. Therefore, the HRCs glucosyllysine and mannosyllysine were synthesized and characterized by NMR. Protein-bound HRCs in cookies containing various sugars and in commercial bakery products were quantitated after enzymatic hydrolysis by RP-HPLC-ESI-MS/MS in the multiple reaction monitoring mode through application of the standard addition method. Protein-bound HRCs were quantitated for the first time in model cookies and in commercial bakery products containing honey, banana, and invert sugar syrup. Concentrations of HRCs from 19 to 287 mg/kg were found, which were similar to or exceeded the content of other frequently analyzed Maillard reaction products, such as N-ε-carboxymethyllysine (10-76 mg/kg), N-ε-carboxyethyllysine (2.5-53 mg/kg), and methylglyoxal-derived hydroimidazolone 1 (10-218 mg/kg) in the analyzed cookies. These results show that substantial amounts of HRCs form during food processing. Analysis of protein-bound HRCs in cookies is therefore useful to evaluate the Maillard reaction of fructose.

40 CFR 257.24 - Detection monitoring program.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Disposal Units Ground-Water Monitoring and Corrective Action § 257.24 Detection monitoring program. (a... unit; (ii) The mobility, stability, and persistence of waste constituents or their reaction products in... constituents, and reaction products in the ground water; and (iv) The concentration or values and coefficients...

Yeast Metabolites of Glycated Amino Acids in Beer.

PubMed

Hellwig, Michael; Beer, Falco; Witte, Sophia; Henle, Thomas

2018-06-01

Glycation reactions (Maillard reactions) during the malting and brewing processes are important for the development of the characteristic color and flavor of beer. Recently, free and protein-bound Maillard reaction products (MRPs) such as pyrraline, formyline, and maltosine were found in beer. Furthermore, these amino acid derivatives are metabolized by Saccharomyces cerevisiae via the Ehrlich pathway. In this study, a method was developed for quantitation of individual Ehrlich intermediates derived from pyrraline, formyline, and maltosine. Following synthesis of the corresponding reference material, the MRP-derived new Ehrlich alcohols pyrralinol (up to 207 μg/L), formylinol (up to 50 μg/L), and maltosinol (up to 6.9 μg/L) were quantitated for the first time in commercial beer samples by reverse phase high performance liquid chromatography tandem mass spectrometry in the multiple reaction monitoring mode. This is equivalent to ca. 20-40% of the concentrations of the parent glycated amino acids. The metabolites were almost absent from alcohol-free beers and malt-based beverages. Two previously unknown valine-derived pyrrole derivatives were characterized and qualitatively identified in beer. The metabolites investigated represent new process-induced alkaloids that may influence brewing yeast performance due to structural similarities to quorum sensing and metal-binding molecules.

In Situ Monitoring of Chemical Reactions at a Solid-Water Interface by Femtosecond Acoustics.

PubMed

Shen, Chih-Chiang; Weng, Meng-Yu; Sheu, Jinn-Kong; Yao, Yi-Ting; Sun, Chi-Kuang

2017-11-02

Chemical reactions at a solid-liquid interface are of fundamental importance. Interfacial chemical reactions occur not only at the very interface but also in the subsurface area, while existing monitoring techniques either provide limited spatial resolution or are applicable only for the outmost atomic layer. Here, with the aid of the time-domain analysis with femtosecond acoustics, we demonstrate a subatomic-level-resolution technique to longitudinally monitor chemical reactions at solid-water interfaces, capable of in situ monitoring even the subsurface area under atmospheric conditions. Our work was proven by monitoring the already-known anode oxidation process occurring during photoelectrochemical water splitting. Furthermore, whenever the oxide layer thickness equals an integer  number of the effective atomic layer thickness, the measured acoustic echo will show higher signal-to-noise ratios with reduced speckle noise, indicating the quantum-like behavior of this coherent-phonon-based technique.

[Analysis of saponins from panax notoginseng using pressurized solvent extraction coupled with liquid chromatography-electrospray mass spectrum].

PubMed

Wan, Jianbo; Li, Changming; Li, Shaopin; Kong, Lingyi; Wang, Yitao

2005-10-01

To establish a method for qualitative analysis of saponins from Panax notoginseng using pressurized solvent extraction coupled with LC-ESI-MS. The PSE technology was applied to the process of extraction for Panax notoginseng, and the negative ion detection and multiple reaction monitoring model were used. The saponins were investigated based on total ion chromatogram (TIC) and MRM chromatogram. According to the fragment character of saponins, the molecular weight and their structures could be identified. The method can be used for qualitative analysis of saponins from Panax notoginseng.

Rapid LC-MS/MS profiling of protein amino acids and metabolically related compounds for large-scale assessment of metabolic phenotypes.

PubMed

Gu, Liping; Jones, A Daniel; Last, Robert L

2012-01-01

Amino acids extracted from a biological matrix can be resolved and measured using a 6-min per sample method through high-performance liquid chromatography with a short C18 column and rapid gradient using the ion-pairing reagent perfluoroheptanoic acid. LC-tandem mass spectrometry with multiple reaction monitoring (MRM) transitions selective for each compound allows simultaneous quantification of the 20 proteinogenic amino acids and 5 metabolically related compounds. Distinct MRM transitions were also established for selective detection of the isomers leucine/isoleucine and threonine/homoserine.

Contour temperature programmed desorption for monitoring multiple chemical reaction products

NASA Astrophysics Data System (ADS)

Chusuei, C. C.; de la Peña, J. V.; Schreifels, J. A.

1999-09-01

A simple method for obtaining a comprehensive overview of major compounds desorbing from the surface during temperature programmed desorption (TPD) experiments is outlined. Standard commercially available equipment is used to perform the experiment. The method is particularly valuable when high molecular mass compounds are being studied. The acquisition of contour temperature programmed desorption (CTPD) spectra, sampling 50-dalton mass ranges at a time in the thermal desorption experiments, is described and demonstrated for the interaction of benzotriazole adsorbed on a Ni(111) surface. Conventional two-dimensional TPD spectra can be extracted from the CTPD by taking vertical slices of the contour.

Optimal Design and Operation of In-Situ Chemical Oxidation Using Stochastic Cost Optimization Toolkit

NASA Astrophysics Data System (ADS)

Kim, U.; Parker, J.; Borden, R. C.

2014-12-01

In-situ chemical oxidation (ISCO) has been applied at many dense non-aqueous phase liquid (DNAPL) contaminated sites. A stirred reactor-type model was developed that considers DNAPL dissolution using a field-scale mass transfer function, instantaneous reaction of oxidant with aqueous and adsorbed contaminant and with readily oxidizable natural oxygen demand ("fast NOD"), and second-order kinetic reactions with "slow NOD." DNAPL dissolution enhancement as a function of oxidant concentration and inhibition due to manganese dioxide precipitation during permanganate injection are included in the model. The DNAPL source area is divided into multiple treatment zones with different areas, depths, and contaminant masses based on site characterization data. The performance model is coupled with a cost module that involves a set of unit costs representing specific fixed and operating costs. Monitoring of groundwater and/or soil concentrations in each treatment zone is employed to assess ISCO performance and make real-time decisions on oxidant reinjection or ISCO termination. Key ISCO design variables include the oxidant concentration to be injected, time to begin performance monitoring, groundwater and/or soil contaminant concentrations to trigger reinjection or terminate ISCO, number of monitoring wells or geoprobe locations per treatment zone, number of samples per sampling event and location, and monitoring frequency. Design variables for each treatment zone may be optimized to minimize expected cost over a set of Monte Carlo simulations that consider uncertainty in site parameters. The model is incorporated in the Stochastic Cost Optimization Toolkit (SCOToolkit) program, which couples the ISCO model with a dissolved plume transport model and with modules for other remediation strategies. An example problem is presented that illustrates design tradeoffs required to deal with characterization and monitoring uncertainty. Monitoring soil concentration changes during ISCO was found to be important to avoid decision errors associated with slow rebound of groundwater concentrations.

A Targeted LC-MS/MS Method for the Simultaneous Detection and Quantitation of Egg, Milk, and Peanut Allergens in Sugar Cookies.

PubMed

Boo, Chelsea C; Parker, Christine H; Jackson, Lauren S

2018-01-01

Food allergy is a growing public health concern, with many individuals reporting allergies to multiple food sources. Compliance with food labeling regulations and prevention of inadvertent cross-contact in manufacturing requires the use of reliable methods for the detection and quantitation of allergens in processed foods. In this work, a novel liquid chromatography-tandem mass spectrometry multiple-reaction monitoring method for multiallergen detection and quantitation of egg, milk, and peanut was developed and evaluated in an allergen-incurred baked sugar cookie matrix. A systematic evaluation of method parameters, including sample extraction, concentration, and digestion, were optimized for candidate allergen peptide markers. The optimized method enabled the reliable detection and quantitation of egg, milk, and peanut allergens in sugar cookies, with allergen concentrations as low as 5 ppm allergen-incurred ingredient.

Electrochemical Detection of Multiple Bioprocess Analytes

NASA Technical Reports Server (NTRS)

Rauh, R. David

2010-01-01

An apparatus that includes highly miniaturized thin-film electrochemical sensor array has been demonstrated as a prototype of instruments for simultaneous detection of multiple substances of interest (analytes) and measurement of acidity or alkalinity in bioprocess streams. Measurements of pH and of concentrations of nutrients and wastes in cell-culture media, made by use of these instruments, are to be used as feedback for optimizing the growth of cells or the production of desired substances by the cultured cells. The apparatus is designed to utilize samples of minimal volume so as to minimize any perturbation of monitored processes. The apparatus can function in a potentiometric mode (for measuring pH), an amperometric mode (detecting analytes via oxidation/reduction reactions), or both. The sensor array is planar and includes multiple thin-film microelectrodes covered with hydrous iridium oxide. The oxide layer on each electrode serves as both a protective and electrochemical transducing layer. In its transducing role, the oxide provides electrical conductivity for amperometric measurement or pH response for potentiometric measurement. The oxide on an electrode can also serve as a matrix for one or more enzymes that render the electrode sensitive to a specific analyte. In addition to transducing electrodes, the array includes electrodes for potential control. The array can be fabricated by techniques familiar to the microelectronics industry. The sensor array is housed in a thin-film liquid-flow cell that has a total volume of about 100 mL. The flow cell is connected to a computer-controlled subsystem that periodically draws samples from the bioprocess stream to be monitored. Before entering the cell, each 100-mL sample is subjected to tangential-flow filtration to remove particles. In the present version of the apparatus, the electrodes are operated under control by a potentiostat and are used to simultaneously measure the pH and the concentration of glucose. It is anticipated that development of procedures for trapping more enzymes into hydrous iridium oxide (and possibly into other electroactive metal oxides) and of means for imparting long-term stability to the transducer layers should make it possible to monitor concentrations of products of many enzyme reactions for example, such key bioprocess analytes as amino acids, vitamins, lactose, and acetate.

Determination of gamma-aminobutyric acid in food matrices by isotope dilution hydrophilic interaction chromatography coupled to mass spectrometry.

PubMed

Zazzeroni, Raniero; Homan, Andrew; Thain, Emma

2009-08-01

The estimation of the dietary intake of gamma-aminobutyric acid (GABA) is dependent upon the knowledge of its concentration values in food matrices. To this end, an isotope dilution liquid chromatography-mass spectrometry method has been developed employing the hydrophilic interaction chromatography technique for analyte separation. This approach enabled accurate quantification of GABA in apple, potato, soybeans, and orange juice without the need of a pre- or post-column derivatization reaction. A selective and precise analytical measurement has been obtained with a triple quadrupole mass spectrometer operating in multiple reaction monitoring using the method of standard additions and GABA-d(6) as an internal standard. The concentrations of GABA found in the matrices tested are 7 microg/g of apple, 342 microg/g of potatoes, 211 microg/g of soybeans, and 344 microg/mL of orange juice.

Reaction Buildup of PBX Explosives JOB-9003 under Different Initiation Pressures

NASA Astrophysics Data System (ADS)

Zhang, Xu; Wang, Yan-fei; Hung, Wen-bin; Gu, Yan; Zhao, Feng; Wu, Qiang; Yu, Xin; Yu, Heng

2017-04-01

Aluminum-based embedded multiple electromagnetic particle velocity gauge technique has been developed in order to measure the shock initiation behavior of JOB-9003 explosives. In addition, another gauge element called a shock tracker has been used to monitor the progress of the shock front as a function of time, thus providing a position-time trajectory of the wave front as it moves through the explosive sample. The data are used to determine the position and time for shock to detonation transition. All the experimental results show that: the rising-up time of Al-based electromagnetic particle velocity gauge was very fast and less than 20 ns; the reaction buildup velocity profiles and the position-time for shock to detonation transition of HMX-based PBX explosive JOB-9003 with 1-8 mm depth from the origin of impact plane under different initiation pressures are obtained with high accuracy.

Structure determination of 3-O-caffeoyl-epi-gamma-quinide, an orphan bitter lactone in roasted coffee.

PubMed

Frank, Oliver; Blumberg, Simone; Krümpel, Gudrun; Hofmann, Thomas

2008-10-22

Recent investigations on the bitterness of coffee as well as 5- O-caffeoyl quinic acid roasting mixtures indicated the existence of another, yet unknown, bitter lactone besides the previously identified bitter compounds 5- O-caffeoyl- muco-gamma-quinide, 3- O-caffeoyl-gamma-quinide, 4- O-caffeoyl- muco-gamma-quinide, 5- O-caffeoyl- epi-delta-quinide, and 4- O-caffeoyl-gamma-quinide. In the present study, this orphan bitter lactone was isolated from the reaction products generated by dry heating of 5- O-caffeoylquinic acid model, and its structure was determined as the previously unreported 3- O-caffeoyl- epi-gamma-quinide by means of liquid chromatography-mass spectrometry (LC-MS) and one-/two-dimensional NMR experiments. The occurrence of this bitter lactone, exhibiting a low bitter recognition threshold of 58 micromol/L, in coffee beverages could be confirmed by LC-MS/MS (negative electrospray ionization) operating in the multiple reaction monitoring mode.

Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

PubMed Central

Ullrich, Thomas; Ermantraut, Eugen; Schulz, Torsten; Steinmetzer, Katrin

2012-01-01

Background State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests. PMID:22539973

Rapid and sensitive method for simultaneous determination of first-line anti-tuberculosis drugs in human plasma by HPLC-MS/MS: Application to therapeutic drug monitoring.

PubMed

Gao, Shouhong; Wang, Zhipeng; Xie, Xinfang; You, Chunhua; Yang, Yang; Xi, Yanhai; Chen, Wansheng

2018-03-01

First-line anti-tuberculosis drugs are playing vital roles for curbing rapid spread of tuberculosis. Multidrug therapies are commonly applied in clinical to achieve better treatment outcomes. However, drug resistance and adverse reactions come along with this therapies and therapeutic drug monitoring is a feasible way to precaution them. For this reasons, a simple and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and single protein precipitation was developed and validated for simultaneously quantifying of pyrazinamide, isoniazid, ethambutol, streptomycin and rifampicin in human plasma. Optimized chromatographic separation was achieved on a ZORBAX SB-C 18 column with heptafluorobutyric acid, an ion-pair reagent, in the mobile phase at a flow rate of 0.3 mL/min. The mass detection was achieved using electrospray ionization in the positive ion mode with a multiple reaction monitoring mode. The lower limit of quantification (LLOQ) and dynamic range of pyrazinamide, isoniazid, ethambutol, streptomycin and rifampicin were 200-4000 ng/mL, 80-2000 ng/mL, 0.2-1000 ng/mL, 2000-200000 ng/mL and 200-4000 ng/mL, respectively. The Inter-day and intra-day accuracy and precision were within ±15.0% and less than 15%. The method had been successfully applied to simultaneous determination of four first-line Anti-tuberculosis drugs in plasma from tuberculosis patients. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of MRM-based assays for the absolute quantitation of plasma proteins.

PubMed

Kuzyk, Michael A; Parker, Carol E; Domanski, Dominik; Borchers, Christoph H

2013-01-01

Multiple reaction monitoring (MRM), sometimes called selected reaction monitoring (SRM), is a directed tandem mass spectrometric technique performed on to triple quadrupole mass spectrometers. MRM assays can be used to sensitively and specifically quantify proteins based on peptides that are specific to the target protein. Stable-isotope-labeled standard peptide analogues (SIS peptides) of target peptides are added to enzymatic digests of samples, and quantified along with the native peptides during MRM analysis. Monitoring of the intact peptide and a collision-induced fragment of this peptide (an ion pair) can be used to provide information on the absolute peptide concentration of the peptide in the sample and, by inference, the concentration of the intact protein. This technique provides high specificity by selecting for biophysical parameters that are unique to the target peptides: (1) the molecular weight of the peptide, (2) the generation of a specific fragment from the peptide, and (3) the HPLC retention time during LC/MRM-MS analysis. MRM is a highly sensitive technique that has been shown to be capable of detecting attomole levels of target peptides in complex samples such as tryptic digests of human plasma. This chapter provides a detailed description of how to develop and use an MRM protein assay. It includes sections on the critical "first step" of selecting the target peptides, as well as optimization of MRM acquisition parameters for maximum sensitivity of the ion pairs that will be used in the final method, and characterization of the final MRM assay.

Detection of Onchocerca volvulus in Skin Snips by Microscopy and Real-Time Polymerase Chain Reaction: Implications for Monitoring and Evaluation Activities.

PubMed

Thiele, Elizabeth A; Cama, Vitaliano A; Lakwo, Thomson; Mekasha, Sindeaw; Abanyie, Francisca; Sleshi, Markos; Kebede, Amha; Cantey, Paul T

2016-04-01

Microscopic evaluation of skin biopsies is the monitoring and evaluation (M and E) method currently used by multiple onchocerciasis elimination programs in Africa. However, as repeated mass drug administration suppresses microfilarial loads, the sensitivity and programmatic utility of skin snip microscopy is expected to decrease. Using a pan-filarial real-time polymerase chain reaction with melt curve analysis (qPCR-MCA), we evaluated 1) the use of a single-step molecular assay for detecting and identifying Onchocerca volvulus microfilariae in residual skin snips and 2) the sensitivity of skin snip microscopy relative to qPCR-MCA. Skin snips were collected and examined with routine microscopy in hyperendemic regions of Uganda and Ethiopia (N= 500 each) and "residual" skin snips (tissue remaining after induced microfilarial emergence) were tested with qPCR-MCA. qPCR-MCA detected Onchocerca DNA in 223 residual snips: 139 of 147 microscopy(+) and 84 among microscopy(-) snips, suggesting overall sensitivity of microscopy was 62.3% (139/223) relative to qPCR-MCA (75.6% in Uganda and 28.6% in Ethiopia). These findings demonstrate the insufficient sensitivity of skin snip microscopy for reliable programmatic monitoring. Molecular tools such as qPCR-MCA can augment sensitivity and provide diagnostic confirmation of skin biopsies and will be useful for evaluation or validation of new onchocerciasis M and E tools. © The American Society of Tropical Medicine and Hygiene.

Chemo-selective high yield microwave assisted reaction turns cellulose to green chemicals.

PubMed

Hassanzadeh, Salman; Aminlashgari, Nina; Hakkarainen, Minna

2014-11-04

Exceptionally high cellulose liquefaction yields, up to 87% as calculated from the amount of solid residue, were obtained under mild conditions by utilizing the synergistic effect of microwave radiation and acid catalysis. The effect of processing conditions on degradation products was fingerprinted by rapid laser desorption ionization-mass spectrometry (LDI-MS) method. The reaction was chemo-tunable, enabling production of glucose (Glc) or levulinic acid (LeA) at significantly high selectivity and yields, the relative molar yields being up to 50 and 69%, respectively. A turning point from pure depolymerization to glucose to further degradation to levulinic acid and formic acid was observed at approximately 50% liquefaction or above 140 °C. This was accompanied by the formation of small amounts of solid spherical carbonized residues. The reaction was monitored by multiple analytical techniques. The high yields were connected to the ability of the process to break the strong secondary interactions in cellulose. The developed method has great potential for future production of green platform chemicals. Copyright © 2014 Elsevier Ltd. All rights reserved.

Quantitative determination of free D-Asp, L-Asp and N-methyl-D-aspartate in mouse brain tissues by chiral separation and Multiple Reaction Monitoring tandem mass spectrometry.

PubMed

Fontanarosa, Carolina; Pane, Francesca; Sepe, Nunzio; Pinto, Gabriella; Trifuoggi, Marco; Squillace, Marta; Errico, Francesco; Usiello, Alessandro; Pucci, Piero; Amoresano, Angela

2017-01-01

Several studies have suggested that free d-Asp has a crucial role in N-methyl d-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free d-Asp, l-Asp and N-methyl d-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing l-Asp, d-Asp and N-methyl d-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75-110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for d-Asp, 0.46 pg/μl for l-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for d-Asp, 1.41 pg/μl for l-Asp and 1.64 pg/μl for NMDA, respectively. Different concentration levels were used for constructing the calibration curves which showed good linearity. The validated method was then successfully applied to the simultaneous detection of d-Asp, l-Asp and NMDA in mouse brain tissues. The concurrent, sensitive, fast, and reproducible measurement of these metabolites in brain tissues will be useful to correlate the amount of free d-Asp with relevant neurological processes, making the LC-MS/MS MRM method well suited, not only for research work but also for clinical analyses.

Pulsed Multiple Reaction Monitoring Approach to Enhancing Sensitivity of a Tandem Quadrupole Mass Spectrometer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Belov, Mikhail E.; Prasad, Satendra; Prior, David C.

2011-02-23

Liquid chromatography (LC)-triple quadrupole mass spectrometers operating in a Multiple Reaction Monitoring (MRM) mode are increasingly used for quantitative analysis of low abundance analytes in highly complex biochemical matrices. After development and selection of optimum MRM transition, sensitivity and data quality limitations are largely related to mass spectral peak interferences from sample or matrix constituents and statistical limitations at low number of ions reaching the detector. Herein, we report a new approach to enhancing MRM sensitivity by converting the continuous stream of ions from the ion source into a pulsed ion beam through the use of an Ion Funnel Trapmore » (IFT). Evaluation of the pulsed MRM approach was performed with a tryptic digest of Shewanella oneidensis strain MR-1 spiked with several reference peptides. The sensitivity improvement observed with the IFT coupled to the triple quadrupole instrument is based on several unique features. First, ion accumulation in the radio frequency (RF) ion trap facilitates improved droplet desolvation, which is manifested in the reduced background ion noise at the detector. Second, signal amplitude for a given transition is enhanced because of an order-of-magnitude increase in the ion charge density per unit time compared to a continuous mode of operation. Third, signal detection at the full duty cycle is obtained, as the trap use eliminates dead times between transitions, which are inevitable with continuous ion streams. In comparison with the conventional approach, the pulsed MRM signals showed up to 5-fold enhanced peak amplitude and 2-3 fold reduced chemical background, resulting in an improvement in the limit of detection (LOD) by a factor of ~ 4 to ~ 8.« less

Precision of Multiple Reaction Monitoring Mass Spectrometry Analysis of Formalin-Fixed, Paraffin-Embedded Tissue

PubMed Central

2012-01-01

We compared the reproducibility of multiple reaction monitoring (MRM) mass spectrometry-based peptide quantitation in tryptic digests from formalin-fixed, paraffin-embedded (FFPE) and frozen clear cell renal cell carcinoma tissues. The analyses targeted a candidate set of 114 peptides previously identified in shotgun proteomic analyses, of which 104 were detectable in FFPE and frozen tissue. Although signal intensities for MRM of peptides from FFPE tissue were on average 66% of those in frozen tissue, median coefficients of variation (CV) for measurements in FFPE and frozen tissues were nearly identical (18–20%). Measurements of lysine C-terminal peptides and arginine C-terminal peptides from FFPE tissue were similarly reproducible (19.5% and 18.3% median CV, respectively). We further evaluated the precision of MRM-based quantitation by analysis of peptides from the Her2 receptor in FFPE and frozen tissues from a Her2 overexpressing mouse xenograft model of breast cancer and in human FFPE breast cancer specimens. We obtained equivalent MRM measurements of HER2 receptor levels in FFPE and frozen mouse xenografts derived from HER2-overexpressing BT474 cells and HER2-negative Sum159 cells. MRM analyses of 5 HER2-positive and 5 HER-negative human FFPE breast tumors confirmed the results of immunohistochemical analyses, thus demonstrating the feasibility of HER2 protein quantification in FFPE tissue specimens. The data demonstrate that MRM analyses can be performed with equal precision on FFPE and frozen tissues and that lysine-containing peptides can be selected for quantitative comparisons, despite the greater impact of formalin fixation on lysine residues. The data further illustrate the feasibility of applying MRM to quantify clinically important tissue biomarkers in FFPE specimens. PMID:22530795

Quantitative liquid chromatography-mass spectrometry-multiple reaction monitoring (LC-MS-MRM) analysis of site-specific glycoforms of haptoglobin in liver disease.

PubMed

Sanda, Miloslav; Pompach, Petr; Brnakova, Zuzana; Wu, Jing; Makambi, Kepher; Goldman, Radoslav

2013-05-01

Development of liver disease is associated with the appearance of multiply fucosylated glycoforms of haptoglobin. To analyze the disease-related haptoglobin glycoforms in liver cirrhosis and hepatocellular carcinoma, we have optimized an LC-MS-multiple reaction monitoring (MRM) workflow for glycopeptide quantification. The final quantitative analysis included 24 site-specific glycoforms generated by treatment of a tryptic digest of haptoglobin with α(2-3,6,8)-neuraminidase and β(1-4)-galactosidase. The combination of LC-MS-MRM with exoglycosidase digests allowed resolution of isobaric glycoforms of the haptoglobin-T3 glycopeptide for quantification of the multiply fucosylated Lewis Y-containing glycoforms we have identified in the context of liver disease. Fourteen multiply fucosylated glycoforms of the 20 examined increased significantly in the liver disease group compared with healthy controls with an average 5-fold increase in intensity (p < 0.05). At the same time, two tri-antennary glycoforms without fucoses did not increase in the liver disease group, and two tetra-antennary glycoforms without fucoses showed a marginal increase (at most 40%) in intensity. Our analysis of 30 individual patient samples (10 healthy controls, 10 cirrhosis patients, and 10 hepatocellular carcinoma patients) showed that these glycoforms were substantially increased in a small subgroup of liver disease patients but did not significantly differ between the groups of hepatocellular carcinoma and cirrhosis patients. The tri- and tetra-antennary singly fucosylated glycoforms are associated with a MELD score and low platelet counts (p < 0.05). The exoglycosidase-assisted LC-MS-MRM workflow, optimized for the quantification of fucosylated glycoforms of haptoglobin, can be used for quantification of these glycoforms on other glycopeptides with appropriate analytical behavior.

Prediction of Response to Sorafenib in Hepatocellular Carcinoma: A Putative Marker Panel by Multiple Reaction Monitoring-Mass Spectrometry (MRM-MS).

PubMed

Kim, Hyunsoo; Yu, Su Jong; Yeo, Injun; Cho, Young Youn; Lee, Dong Hyeon; Cho, Yuri; Cho, Eun Ju; Lee, Jeong-Hoon; Kim, Yoon Jun; Lee, Sungyoung; Jun, Jongsoo; Park, Taesung; Yoon, Jung-Hwan; Kim, Youngsoo

2017-07-01

Sorafenib is the only standard treatment for unresectable hepatocellular carcinoma (HCC), but it provides modest survival benefits over placebo, necessitating predictive biomarkers of the response to sorafenib. Serum samples were obtained from 115 consecutive patients with HCC before sorafenib treatment and analyzed by multiple reaction monitoring-mass spectrometry (MRM-MS) and ELISA to quantify candidate biomarkers. We verified a triple-marker panel to be predictive of the response to sorafenib by MRM-MS, comprising CD5 antigen-like (CD5L), immunoglobulin J (IGJ), and galectin-3-binding protein (LGALS3BP), in HCC patients. This panel was a significant predictor (AUROC > 0.950) of the response to sorafenib treatment, having the best cut-off value (0.4) by multivariate analysis. In the training set, patients who exceeded this cut-off value had significantly better overall survival (median, 21.4 months) than those with lower values (median, 8.6 months; p = 0.001). Further, a value that was lower than this cutoff was an independent predictor of poor overall survival [hazard ratio (HR), 2.728; 95% confidence interval (CI), 1.312-5.672; p = 0.007] and remained an independent predictive factor of rapid progression (HR, 2.631; 95% CI, 1.448-4.780; p = 0.002). When applied to the independent validation set, levels of the cut-off value for triple-marker panel maintained their prognostic value for poor clinical outcomes. On the contrast, the triple-marker panel was not a prognostic factor for patients who were treated with transarterial chemoembolization (TACE). The discriminatory signature of a triple-marker panel provides new insights into targeted proteomic biomarkers for individualized sorafenib therapy. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Quantitation of 47 human tear proteins using high resolution multiple reaction monitoring (HR-MRM) based-mass spectrometry.

PubMed

Tong, Louis; Zhou, Xi Yuan; Jylha, Antti; Aapola, Ulla; Liu, Dan Ning; Koh, Siew Kwan; Tian, Dechao; Quah, Joanne; Uusitalo, Hannu; Beuerman, Roger W; Zhou, Lei

2015-02-06

Tear proteins are intimately related to the pathophysiology of the ocular surface. Many recent studies have demonstrated that the tear is an accessible fluid for studying eye diseases and biomarker discovery. This study describes a high resolution multiple reaction monitoring (HR-MRM) approach for developing assays for quantification of biologically important tear proteins. Human tear samples were collected from 1000 subjects with no eye complaints (411 male, 589 female, average age: 55.5±14.5years) after obtaining informed consent. Tear samples were collected using Schirmer's strips and pooled into a single global control sample. Quantification of proteins was carried out by selecting "signature" peptides derived by trypsin digestion. A 1-h nanoLC-MS/MS run was used to quantify the tear proteins in HR-MRM mode. Good reproducibility of signal intensity (using peak areas) was demonstrated for all 47 HR-MRM assays with an average coefficient of variation (CV%) of 4.82% (range: 1.52-10.30%). All assays showed consistent retention time with a CV of less than 0.80% (average: 0.57%). HR-MRM absolute quantitation of eight tear proteins was demonstrated using stable isotope-labeled peptides. In this study, we demonstrated for the first time the technique to quantify 47 human tear proteins in HR-MRM mode using approximately 1μl of human tear sample. These multiplexed HR-MRM-based assays show great promise of further development for biomarker validation in human tear samples. Both discovery-based and targeted quantitative proteomics can be achieved in a single quadrupole time-of-flight mass spectrometer platform (TripleTOF 5600 system). Copyright © 2015 Elsevier B.V. All rights reserved.

Application of adenosine triphosphate affinity probe and scheduled multiple-reaction monitoring analysis for profiling global kinome in human cells in response to arsenite treatment.

PubMed

Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

2014-11-04

Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli.

Development of Biomarkers for Screening Hepatocellular Carcinoma Using Global Data Mining and Multiple Reaction Monitoring

PubMed Central

Yu, Su Jong; Jang, Eun Sun; Yu, Jiyoung; Cho, Geunhee; Yoon, Jung-Hwan; Kim, Youngsoo

2013-01-01

Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers and is associated with a poor survival rate. Clinically, the level of alpha-fetoprotein (AFP) has been used as a biomarker for the diagnosis of HCC. The discovery of useful biomarkers for HCC, focused solely on the proteome, has been difficult; thus, wide-ranging global data mining of genomic and proteomic databases from previous reports would be valuable in screening biomarker candidates. Further, multiple reaction monitoring (MRM), based on triple quadrupole mass spectrometry, has been effective with regard to high-throughput verification, complementing antibody-based verification pipelines. In this study, global data mining was performed using 5 types of HCC data to screen for candidate biomarker proteins: cDNA microarray, copy number variation, somatic mutation, epigenetic, and quantitative proteomics data. Next, we applied MRM to verify HCC candidate biomarkers in individual serum samples from 3 groups: a healthy control group, patients who have been diagnosed with HCC (Before HCC treatment group), and HCC patients who underwent locoregional therapy (After HCC treatment group). After determining the relative quantities of the candidate proteins by MRM, we compared their expression levels between the 3 groups, identifying 4 potential biomarkers: the actin-binding protein anillin (ANLN), filamin-B (FLNB), complementary C4-A (C4A), and AFP. The combination of 2 markers (ANLN, FLNB) improved the discrimination of the before HCC treatment group from the healthy control group compared with AFP. We conclude that the combination of global data mining and MRM verification enhances the screening and verification of potential HCC biomarkers. This efficacious integrative strategy is applicable to the development of markers for cancer and other diseases. PMID:23717429

Application of Adenosine Triphosphate Affinity Probe and Scheduled Multiple-Reaction Monitoring Analysis for Profiling Global Kinome in Human Cells in Response to Arsenite Treatment

PubMed Central

2015-01-01

Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli. PMID:25301106

Quantitative Liquid Chromatography-Mass Spectrometry-Multiple Reaction Monitoring (LC-MS-MRM) Analysis of Site-specific Glycoforms of Haptoglobin in Liver Disease*

PubMed Central

Sanda, Miloslav; Pompach, Petr; Brnakova, Zuzana; Wu, Jing; Makambi, Kepher; Goldman, Radoslav

2013-01-01

Development of liver disease is associated with the appearance of multiply fucosylated glycoforms of haptoglobin. To analyze the disease-related haptoglobin glycoforms in liver cirrhosis and hepatocellular carcinoma, we have optimized an LC-MS-multiple reaction monitoring (MRM) workflow for glycopeptide quantification. The final quantitative analysis included 24 site-specific glycoforms generated by treatment of a tryptic digest of haptoglobin with α(2–3,6,8)-neuraminidase and β(1–4)-galactosidase. The combination of LC-MS-MRM with exoglycosidase digests allowed resolution of isobaric glycoforms of the haptoglobin-T3 glycopeptide for quantification of the multiply fucosylated Lewis Y-containing glycoforms we have identified in the context of liver disease. Fourteen multiply fucosylated glycoforms of the 20 examined increased significantly in the liver disease group compared with healthy controls with an average 5-fold increase in intensity (p < 0.05). At the same time, two tri-antennary glycoforms without fucoses did not increase in the liver disease group, and two tetra-antennary glycoforms without fucoses showed a marginal increase (at most 40%) in intensity. Our analysis of 30 individual patient samples (10 healthy controls, 10 cirrhosis patients, and 10 hepatocellular carcinoma patients) showed that these glycoforms were substantially increased in a small subgroup of liver disease patients but did not significantly differ between the groups of hepatocellular carcinoma and cirrhosis patients. The tri- and tetra-antennary singly fucosylated glycoforms are associated with a MELD score and low platelet counts (p < 0.05). The exoglycosidase-assisted LC-MS-MRM workflow, optimized for the quantification of fucosylated glycoforms of haptoglobin, can be used for quantification of these glycoforms on other glycopeptides with appropriate analytical behavior. PMID:23389048

Scheduled multiple reaction monitoring algorithm as a way to analyse new designer drugs combined with synthetic cannabinoids in human serum with liquid chromatography-tandem mass spectrometry.

PubMed

Dziadosz, Marek; Weller, Jens-Peter; Klintschar, Michael; Teske, Jörg

2013-06-15

Here, we describe the development and application of a liquid chromatography-tandem mass spectrometry method with positive electrospray ionisation and scheduled multiple reaction monitoring algorithm (s-MRM) to analyse synthetic cannabinoids (SC) combined with new designer drugs (NDD) in human serum. A Luna 5μm C18 (2) 100A, 150mm×2mm analytical column and a mobile phase consisted of A (H2O/methanol=95/5, v/v) and B (H2O/methanol=3/97, v/v) - both with 10mM ammonium acetate and 0.1% acetic acid (pH=3.2), were used for the separation. A binary flow pumping mode with a total flow rate of 0.400mL/min was used. A single sample extraction with 1-chlorobutane for both substance groups was performed. Acceptable linearity in the validated calibration ranges of 0.05-1ng/mL for SC and 1-50ng/mL for NDD was achieved. The limit of detection was not greater than 0.02/0.40ng/mL and the limit of quantification not greater than 0.05/0.50ng/mL for SC/NDD respectively. The presented study revealed that this method is a very effective way for sensitive SC and NDD identification in human serum and has useful application in hospitals, therapy centres and forensic psychiatric centres. S-MRM ensures a method upgrade with a smaller loss of sensitivity, precision and accuracy in comparison to traditional MRM methods. Also addition of new SC and NDD can be performed in the future. Copyright © 2013 Elsevier B.V. All rights reserved.

Proteomic analysis of serum biomarkers for prediabetes using the Long-Evans Agouti rat, a spontaneous animal model of type 2 diabetes mellitus.

PubMed

Takahashi, Eri; Unoki-Kubota, Hiroyuki; Shimizu, Yukiko; Okamura, Tadashi; Iwata, Wakiko; Kajio, Hiroshi; Yamamoto-Honda, Ritsuko; Shiga, Tomoko; Yamashita, Shigeo; Tobe, Kazuyuki; Okumura, Akinori; Matsumoto, Michihiro; Yasuda, Kazuki; Noda, Mitsuhiko; Kaburagi, Yasushi

2017-09-01

To identify candidate serum molecules associated with the progression of type 2 diabetes mellitus, differential serum proteomic analysis was carried out on a spontaneous animal model of type 2 diabetes mellitus without obesity, the Long-Evans Agouti (LEA) rat. We carried out quantitative proteomic analysis using serum samples from 8- and 16-week-old LEA and control Brown Norway (BN) rats (n = 4/group). Differentially expressed proteins were validated by multiple reaction monitoring analysis using the sera collected from 8-, 16-, and 24-week-old LEA (n = 4/each group) and BN rats (n = 5/each group). Among the validated proteins, we also examined the possible relevance of the human homolog of serine protease inhibitor A3 (SERPINA3) to type 2 diabetes mellitus. The use of 2-D fluorescence difference gel electrophoresis analysis and the following liquid chromatography-multiple reaction monitoring analysis showed that the serum levels of five proteins were differentially changed between LEA rats and BN rats at all three time-points examined. Among the five proteins, SERPINA3N was increased significantly in the sera of LEA rats compared with age-matched BN rats. The serum level of SERPINA3 was also found to be significantly higher in type 2 diabetes mellitus patients than in healthy control participants. Furthermore, glycated hemoglobin, fasting insulin and estimated glomerular filtration rate were independently associated with the SERPINA3 levels. These findings suggest a possible role for SERPINA3 in the development of the early stages of type 2 diabetes mellitus, although further replication studies and functional investigations regarding their role are required. © 2017 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

Development of biomarkers for screening hepatocellular carcinoma using global data mining and multiple reaction monitoring.

PubMed

Kim, Hyunsoo; Kim, Kyunggon; Yu, Su Jong; Jang, Eun Sun; Yu, Jiyoung; Cho, Geunhee; Yoon, Jung-Hwan; Kim, Youngsoo

2013-01-01

Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers and is associated with a poor survival rate. Clinically, the level of alpha-fetoprotein (AFP) has been used as a biomarker for the diagnosis of HCC. The discovery of useful biomarkers for HCC, focused solely on the proteome, has been difficult; thus, wide-ranging global data mining of genomic and proteomic databases from previous reports would be valuable in screening biomarker candidates. Further, multiple reaction monitoring (MRM), based on triple quadrupole mass spectrometry, has been effective with regard to high-throughput verification, complementing antibody-based verification pipelines. In this study, global data mining was performed using 5 types of HCC data to screen for candidate biomarker proteins: cDNA microarray, copy number variation, somatic mutation, epigenetic, and quantitative proteomics data. Next, we applied MRM to verify HCC candidate biomarkers in individual serum samples from 3 groups: a healthy control group, patients who have been diagnosed with HCC (Before HCC treatment group), and HCC patients who underwent locoregional therapy (After HCC treatment group). After determining the relative quantities of the candidate proteins by MRM, we compared their expression levels between the 3 groups, identifying 4 potential biomarkers: the actin-binding protein anillin (ANLN), filamin-B (FLNB), complementary C4-A (C4A), and AFP. The combination of 2 markers (ANLN, FLNB) improved the discrimination of the before HCC treatment group from the healthy control group compared with AFP. We conclude that the combination of global data mining and MRM verification enhances the screening and verification of potential HCC biomarkers. This efficacious integrative strategy is applicable to the development of markers for cancer and other diseases.

When goals collide: monitoring the goals of multiple characters.

PubMed

Magliano, Joseph P; Taylor, Holly A; Kim, Hyun-Jeong Joyce

2005-12-01

Most story plots contain multiple characters who are independent, interact, and often have conflicting goals. One would expect that narrative understanding would require monitoring of the goals, concerns, and situations of multiple agents. There is considerable evidence that understanders monitor the primary protagonist's goal plans (e.g., Suh & Trabasso, 1993). However, there is relatively little research on the extent to which understanders monitor the goals of multiple agents. We investigated the impact of characters' roles and prominence on the extent to which understanders monitor the goal plans of multiple characters in a feature length film. In Experiment 1, participants made situation change judgments, and in Experiment 2, they verbally described scenes. Both types of judgments indicated that viewers monitor the goals and plans of multiple agents but do so to a greater extent for characters more prominent to the plotline.

Ambient Mechanochemical Solid-State Reactions of Carbon Nanotubes and Their Reactions via Covalent Coordinate Bond in Solution

NASA Astrophysics Data System (ADS)

Kabbani, Mohamad A.

In its first part, this thesis deals with ambient mechanochemical solid-state reactions of differently functionalized multiple walled carbon nanotubes (MWCNTs) while in its second part it investigates the cross-linking reactions of CNTs in solution via covalent coordinate bonds with transitions metals and carboxylate groups decorating their surfaces. In the first part a series of mechanochemical reactions involving different reactive functionalities on the CNTs such as COOH/OH, COOH/NH2 and COCl/OH were performed. The solid-state unzipping of CNTs leading to graphene formation was confirmed using spectroscopic, thermal and electron microscopy techniques. The non-grapheme products were established using in-situ quadruple mass spectroscopy. The experimental results were confirmed by theoretical simulation calculations using the 'hot spots' protocol. The kinetics of the reaction between MWCNT-COOH and MWCNT-OH was monitored using variable temperature Raman spectroscopy. The low activation energy was discussed in terms of hydrogen bond mediated proton transfer mechanism. The second part involves the reaction of MWCNTII COOH with Zn (II) and Cu (II) to form CNT metal-organic frame (MOFs) products that were tested for their effective use as counter-electrodes in dyes sensitized solar cells (DSSC). The thesis concludes by the study of the room temperature reaction between the functionalized graphenes, GOH and G'-COOH followed by the application of compressive loads. The 3D solid graphene pellet product ( 0.6gm/cc) is conductive and reflective with a 35MPa ultimate strength as compared to 10MPa strength of graphite electrode ( 2.2gm/cc).

Detection of 22 antiepileptic drugs by ultra-performance liquid chromatography coupled with tandem mass spectrometry applicable to routine therapeutic drug monitoring.

PubMed

Shibata, Mai; Hashi, Sachiyo; Nakanishi, Haruka; Masuda, Satohiro; Katsura, Toshiya; Yano, Ikuko

2012-12-01

The purpose of this study was to develop an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method of 22 antiepileptics for routine therapeutic monitoring. The antiepileptics used in the analyses were carbamazepine, carbamazepine-10,11-epoxide, clobazam, N-desmethylclobazam, clonazepam, diazepam, N-desmethyldiazepam, ethosuximide, felbamate, gabapentin, lamotrigine, levetiracetam, N-desmethylmesuximide, nitrazepam, phenobarbital, phenytoin, primidone, tiagabine, topiramate, valproic acid, vigabatrin and zonisamide. After protein precipitation of 50 μL plasma with methanol, the supernatant was diluted with water or was evaporated to dryness and reconstituted with mobile phase in the case of benzodiazepines. Separation was achieved on an Acquity UPLC BEH C₁₈ column with a gradient mobile phase of 10 mm ammonium acetate containing 0.1% formic acid and methanol at a flow rate of 0.4 mL/min. An Acquity TQD instrument in multiple reaction monitoring mode with ion mode switching was used for detection. All antiepileptics were detected and quantified within 10 min, with no endogenous interference. All the calibration curves showed good linearity in the therapeutic range (r²  < 0.99). The precision and accuracy values for intra- and inter-assays were within ±15% except for phenobarbital and tiagabine. A good correlation was observed between the concentration of clinical samples measured by the new method described here and the conventional methods. The values of carbamazepine and phenytoin by UPLC-MS/MS were lower than those detected by the immunoassays, which might be caused by the cross-reaction of antibodies with their metabolites. In conclusion, we developed a simple and selective UPLC-MS/MS method suitable for routine therapeutic monitoring of antiepileptics. Copyright © 2012 John Wiley & Sons, Ltd.

Real-time pH monitoring of industrially relevant enzymatic reactions in a microfluidic side-entry reactor (μSER) shows potential for pH control.

PubMed

Gruber, Pia; Marques, Marco P C; Sulzer, Philipp; Wohlgemuth, Roland; Mayr, Torsten; Baganz, Frank; Szita, Nicolas

2017-06-01

Monitoring and control of pH is essential for the control of reaction conditions and reaction progress for any biocatalytic or biotechnological process. Microfluidic enzymatic reactors are increasingly proposed for process development, however typically lack instrumentation, such as pH monitoring. We present a microfluidic side-entry reactor (μSER) and demonstrate for the first time real-time pH monitoring of the progression of an enzymatic reaction in a microfluidic reactor as a first step towards achieving pH control. Two different types of optical pH sensors were integrated at several positions in the reactor channel which enabled pH monitoring between pH 3.5 and pH 8.5, thus a broader range than typically reported. The sensors withstood the thermal bonding temperatures typical of microfluidic device fabrication. Additionally, fluidic inputs along the reaction channel were implemented to adjust the pH of the reaction. Time-course profiles of pH were recorded for a transketolase and a penicillin G acylase catalyzed reaction. Without pH adjustment, the former showed a pH increase of 1 pH unit and the latter a pH decrease of about 2.5 pH units. With pH adjustment, the pH drop of the penicillin G acylase catalyzed reaction was significantly attenuated, the reaction condition kept at a pH suitable for the operation of the enzyme, and the product yield increased. This contribution represents a further step towards fully instrumented and controlled microfluidic reactors for biocatalytic process development. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Monitoring technique for multiple power splitter-passive optical networks using a tunable OTDR and FBGs

NASA Astrophysics Data System (ADS)

Hann, Swook; Kim, Dong-Hwan; Park, Chang-Soo

2006-04-01

A monitoring technique for multiple power splitter-passive optical networks (PS-PON) is presented. The technique is based on the remote sensing of fiber Bragg grating (FBG) using a tunable OTDR. To monitor the multiple PS-PON, the FBG can be used for a wavelength dependent reflective reference on each branch end of the PS. The FBG helps discern an individual event of the multiple PS-PON for the monitoring in collaborate with information of Rayleigh backscattered power. The multiple PS-PON can be analyzed by the monitoring method at the central office under 10-Gbit/s in-service.

Quantification of the Triazole Antifungal Compounds Voriconazole and Posaconazole in Human Serum or Plasma Using Liquid Chromatography Electrospray Tandem Mass Spectrometry (HPLC-ESI-MS/MS).

PubMed

Molinelli, Alejandro R; Rose, Charles H

2016-01-01

Voriconazole and posaconazole are triazole antifungal compounds used in the treatment of fungal infections. Therapeutic drug monitoring of both compounds is recommended in order to guide drug dosing to achieve optimal blood concentrations. In this chapter we describe an HPLC-ESI-MS/MS method for the quantification of both compounds in human plasma or serum following a simple specimen preparation procedure. Specimen preparation consists of protein precipitation using methanol and acetonitrile followed by a cleanup step that involves filtration through a cellulose acetate membrane. The specimen is then injected into an HPLC-ESI-MS/MS equipped with a C18 column and separated over an acetonitrile gradient. Quantification of the drugs in the specimen is achieved by comparing the response of the unknown specimen to that of the calibrators in the standard curve using multiple reaction monitoring.

Multiplexed LC-MS/MS analysis of horse plasma proteins to study doping in sport.

PubMed

Barton, Chris; Beck, Paul; Kay, Richard; Teale, Phil; Roberts, Jane

2009-06-01

The development of protein biomarkers for the indirect detection of doping in horse is a potential solution to doping threats such as gene and protein doping. A method for biomarker candidate discovery in horse plasma is presented using targeted analysis of proteotypic peptides from horse proteins. These peptides were first identified in a novel list of the abundant proteins in horse plasma. To monitor these peptides, an LC-MS/MS method using multiple reaction monitoring was developed to study the quantity of 49 proteins in horse plasma in a single run. The method was optimised and validated, and then applied to a population of race-horses to study protein variance within a population. The method was finally applied to longitudinal time courses of horse plasma collected after administration of an anabolic steroid to demonstrate utility for hypothesis-driven discovery of doping biomarker candidates.

Effect of vehicular size on chain-reaction crash

NASA Astrophysics Data System (ADS)

Nagatani, Takashi

2015-11-01

We present the dynamic model of the chain-reaction crash to take account of the vehicular size. Drivers brake according to taillights of the forward vehicle. We investigate the effect of the vehicular size on the chain-reaction crash (multiple-vehicle collision) in the traffic flow controlled by taillights. In the multiple-vehicle collision, the first crash induces more collisions. We investigate how the first collision induces the chain-reaction crash numerically. We derive, analytically, the transition points and the region maps for the chain-reaction crash in the traffic flow of vehicles with finite sizes. We clarify the effect of the vehicular size on the multiple-vehicle collision.

Human placenta-derived cells (PDA-001) for the treatment of adults with multiple sclerosis: a randomized, placebo-controlled, multiple-dose study.

PubMed

Lublin, Fred D; Bowen, James D; Huddlestone, John; Kremenchutzky, Marcelo; Carpenter, Adam; Corboy, John R; Freedman, Mark S; Krupp, Lauren; Paulo, Corri; Hariri, Robert J; Fischkoff, Steven A

2014-11-01

Infusion of PDA-001, a preparation of mesenchymal-like cells derived from full-term human placenta, is a new approach in the treatment of patients with multiple sclerosis. This safety study aimed to rule out the possibility of paradoxical exacerbation of disease activity by PDA-001 in patients with multiple sclerosis. This was a phase 1b, multicenter, randomized, double-blind, placebo-controlled, 2-dose ranging study including patients with relapsing-remitting multiple sclerosis or secondary progressive multiple sclerosis. The study was conducted at 6 sites in the United States and 2 sites in Canada. Patients were randomized 3:1 to receive 2 low-dose infusions of PDA-001 (150×10(6) cells) or placebo, given 1 week apart. After completing this cohort, subsequent patients received high-dose PDA-001 (600×10(6) cells) or placebo. Monthly brain magnetic resonance imaging scans were performed. The primary end point was ruling out the possibility of paradoxical worsening of MS disease activity. This was monitored using Cutter׳s rule (≥5 new gadolinium lesions on 2 consecutive scans) by brain magnetic resonance imaging on a monthly basis for six months and also the frequency of multiple sclerosis relapse. Ten patients with relapsing-remitting multiple sclerosis and 6 with secondary progressive multiple sclerosis were randomly assigned to treatment: 6 to low-dose PDA-001, 6 to high-dose PDA-001, and 4 to placebo. No patient met Cutter׳s rule. One patient receiving high-dose PDA-001 had an increase in T2 and gadolinium lesions and in Expanded Disability Status Scale score during a multiple sclerosis flare 5 months after receiving PDA-001. No other patient had an increase in Expanded Disability Status Scale score>0.5, and most had stable or decreasing Expanded Disability Status Scale scores. With high-dose PDA-001, 1 patient experienced a grade 1 anaphylactoid reaction and 1 had grade 2 superficial thrombophlebitis. Other adverse events were mild to moderate and included headache, fatigue, infusion site reactions, and urinary tract infection. PDA-001 infusions were safe and well tolerated in relapsing-remitting multiple sclerosis and secondary progressive multiple sclerosis patients. No paradoxical worsening of lesion counts was noted with either dose. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Multiplexed detection of anthrax-related toxin genes.

PubMed

Moser, Michael J; Christensen, Deanna R; Norwood, David; Prudent, James R

2006-02-01

Simultaneous analysis of three targets in three colors on any real-time polymerase chain reaction (PCR) instrument would increase the flexibility of real-time PCR. For the detection of Bacillus strains that can cause inhalation anthrax-related illness, this ability would be valuable because two plasmids confer virulence, and internal positive controls are needed to monitor the testing in cases lacking target-specific signals. Using a real-time PCR platform called MultiCode-RTx, multiple assays were developed that specifically monitor the presence of Bacillus anthracis-specific virulence plasmid-associated genes. In particular for use on LightCycler-1, two triplex RTx systems demonstrated high sensitivity with limits of detection nearing single-copy levels for both plasmids. Specificity was established using a combination of Ct values and correct amplicon melting temperatures. All reactions were further verified by detection of an internal positive control. For these two triplex RTx assays, the analytical detection limit was one to nine plasmid copy equivalents, 100% analytical specificity with a 95% confidence interval (CI) of 9%, and 100% analytical sensitivity with a CI of 2%. Although further testing using clinical or environmental samples will be required to assess diagnostic sensitivity and specificity, the RTx platform achieves similar results to those of probe-based real-time systems.

Post-translational quantitation by SRM/MRM: applications in cardiology.

PubMed

Gianazza, Erica; Banfi, Cristina

2018-06-04

Post-translational modifications (PTMs) have an important role in the regulation of protein function, localization and interaction with other molecules. PTMs apply a dynamic control of proteins both in physiological and pathological conditions. The study of disease-specific PTMs allows identifying potential biomarkers and developing effective drugs. Enrichment techniques combined with high-resolution MS/MS analysis provide attractive results on PTMs characterization. Selected reaction monitoring/multiple reaction monitoring (SRM/MRM) is a powerful targeted assay for the quantitation and validation of PTMs in complex biological samples. Areas covered: The most frequent PTMs are described in terms of biological role and analytical methods commonly used to detect them. The applications of SRM/MRM for the absolute quantitation of PTMs are reported and a specific section is focused on PTMs detection in proteins that are involved in cardiovascular system and heart diseases. Expert commentary: PTMs characterization in relation to disease pathology is still in progress, but targeted proteomics by LC-MS/MS has significantly upgraded the knowledge in the last years. Advances in enrichment strategies and software tools will facilitate the interpretation of high PTMs complexity. Promising studies confirm the great potentiality of SRM/MRM to study PTMs in cardiovascular field and PTMomics could be very useful in a clinical perspective.

Compatibility studies of acyclovir and lactose in physical mixtures and commercial tablets.

PubMed

Monajjemzadeh, Farnaz; Hassanzadeh, Davoud; Valizadeh, Hadi; Siahi-Shadbad, Mohammad R; Mojarrad, Javid Shahbazi; Robertson, Thomas A; Roberts, Michael S

2009-11-01

This study documents drug-excipient incompatibility studies of acyclovir in physical mixtures with lactose and in different tablet brands. Differential scanning calorimetry (DSC) was initially used to assess compatibility of mixtures. The Fourier-transform infrared (FTIR) spectrum was also compared with the spectra of pure drug and excipient. Although DSC results indicated incompatibility with lactose, FTIR spectra were mostly unmodified due to overlapping peaks. Samples of isothermally stressed physical mixture were stored at 95 degrees C for 24 h. The residual drug was monitored using a validated high-performance liquid chromatography (HPLC) assay and data fitting to solid-state kinetic models was performed. The drug loss kinetics followed a diffusion model. The aqueous mixture of drug and excipient was heated in order to prepare an adduct mixture. HPLC analysis revealed one extra peak that was fractionated and subsequently injected into the liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) system. The MRM (Multiple Reaction Monitoring) chromatograms characterized the peak with molecular mass corresponding to an acyclovir-lactose Maillard reaction product. The presence of lactose in commercial tablets was checked using a new TLC method. Overall, the incompatibility of acyclovir with lactose was successfully evaluated using a combination of thermal methods and LC-MS/MS.

Monitoring biodiesel reactions of soybean oil and sunflower oil using ultrasonic parameters

NASA Astrophysics Data System (ADS)

Figueiredo, M. K. K.; Silva, C. E. R.; Alvarenga, A. V.; Costa-Félix, R. P. B.

2015-01-01

Biodiesel is an innovation that attempts to substitute diesel oil with biomass. The aim of this paper is to show the development of a real-time method to monitor transesterification reactions by using low-power ultrasound and pulse/echo techniques. The results showed that it is possible to identify different events during the transesterification process by using the proposed parameters, showing that the proposed method is a feasible way to monitor the reactions of biodiesel during its fabrication, in real time, and with relatively low- cost equipment.

HPLC method for rapidly following biodiesel fuel transesterification reaction progress using a core-shell column.

PubMed

Allen, Samuel J; Ott, Lisa S

2012-07-01

There are a wide and growing variety of feedstocks for biodiesel fuel. Most commonly, these feedstocks contain triglycerides which are transesterified into the fatty acid alkyl esters (FAAEs) which comprise biodiesel fuel. While the tranesterification reaction itself is simple, monitoring the reaction progress and reaction products is not. Gas chromatography-mass spectrometry is useful for assessing the FAAE products, but does not directly address either the tri-, di-, or monoglycerides present from incomplete transesterification or the free fatty acids which may also be present. Analysis of the biodiesel reaction mixture is complicated by the solubility and physical property differences among the components of the tranesterification reaction mixture. In this contribution, we present a simple, rapid HPLC method which allows for monitoring all of the main components in a biodiesel fuel transesterification reaction, with specific emphasis on the ability to monitor the reaction as a function of time. The utilization of a relatively new, core-shell stationary phase for the HPLC column allows for efficient separation of peaks with short elution times, saving both time and solvent.

Esterification Reaction Utilizing Sense of Smell and Eyesight for Conversion and Catalyst Recovery Monitoring

ERIC Educational Resources Information Center

Janssens, Nikki; Wee, Lik H.; Martens, Johan A.

2014-01-01

The esterification reaction of salicylic acid with ethanol is performed in presence of dissolved 12-tungstophosphoric Brønsted-Lowry acid catalyst, a Keggin-type polyoxometalate (POM). The monitoring of the reaction with smell and the recovery of the catalyst with sight is presented. Formation of the sweet-scented ester is apparent from the smell.…

[Comparative study on four kinds of assessment methods of post-marketing safety of Danhong injection].

PubMed

Li, Xuelin; Tang, Jinfa; Meng, Fei; Li, Chunxiao; Xie, Yanming

2011-10-01

To study the adverse reaction of Danhong injection with four kinds of methods, central monitoring method, chart review method, literature study method and spontaneous reporting method, and to compare the differences between them, explore an appropriate method to carry out post-marketing safety evaluation of traditional Chinese medicine injection. Set down the adverse reactions' questionnaire of four kinds of methods, central monitoring method, chart review method, literature study method and collect the information on adverse reactions in a certain period. Danhong injection adverse reaction information from Henan Province spontaneous reporting system was collected with spontaneous reporting method. Carry on data summary and descriptive analysis. Study the adverse reaction of Danhong injection with four methods of central monitoring method, chart review method, literature study method and spontaneous reporting method, the rates of adverse events were 0.993%, 0.336%, 0.515%, 0.067%, respectively. Cyanosis, arrhythmia, hypotension, sweating, erythema, hemorrhage dermatitis, rash, irritability, bleeding gums, toothache, tinnitus, asthma, elevated aminotransferases, constipation, pain are new discovered adverse reactions. The central monitoring method is the appropriate method to carry out post-marketing safety evaluation of traditional Chinese medicine injection, which could objectively reflect the real world of clinical usage.

Expertise and responsibility effects on pilots' reactions to flight deck alerts in a simulator.

PubMed

Zheng, Yiyuan; Lu, Yanyu; Yang, Zheng; Fu, Shan

2014-11-01

Flight deck alerts provide system malfunction information designed to lead corresponding pilot reactions aimed at guaranteeing flight safety. This study examined the roles of expertise and flight responsibility and their relationship to pilots' reactions to flight deck alerts. There were 17 pilots composing 12 flight crews that were assigned into pairs according to flight hours and responsibilities. The experiment included 9 flight scenarios and was carried out in a CRJ-200 flight simulator. Pilot performance was recorded by a wide angle video camera, and four kinds of reactions to alerts were defined for analysis. Pilots tended to have immediate reactions to uninterrupted cautions, with a turning off rate as high as 75%. However, this rate decreased sharply when pilots encountered interrupted cautions and warnings; they also exhibited many wrong reactions to warnings. Pilots with more expertise had more reactions to uninterrupted cautions than those with less expertise, both as pilot flying and pilot monitoring. Meanwhile, the pilot monitoring, regardless of level of expertise, exhibited more reactions than the pilot flying. In addition, more experienced pilots were more likely to have wrong reactions to warnings while acting as the monitoring pilot. These results suggest that both expertise and flight responsibility influence pilots' reactions to alerts. Considering crew pairing strategy, when a pilot flying is a less experienced pilot, a more experience pilot is suggested to be the monitoring pilot. The results of this study have implications for understanding pilots' behaviors to flight deck alerts, calling for specialized training and design of approach alarms on the flight deck.

Rapid determination of quinolones in cosmetic products by ultra high performance liquid chromatography with tandem mass spectrometry.

PubMed

Liu, Shao-Ying; Huang, Xi-Hui; Wang, Xiao-Fang; Jin, Quan; Zhu, Guo-Nian

2014-05-01

This study developed an improved analytical method for the simultaneous quantification of 13 quinolones in cosmetics by ultra high performance liquid chromatography combined with ESI triple quadrupole MS/MS under the multiple reaction monitoring mode. The analytes were extracted and purified by using an SPE cartridge. The limits of quantification ranged from 0.03 to 3.02 μg/kg. The precision for determining the quinolones was <19.39%. The proposed method was successfully developed for the determination of quinolones in real cosmetic samples. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Assay of lovastatin containing dietary supplement by LC-MS/MS under MRM condition.

PubMed

Di Donna, Leonardo; Bartella, Lucia; Napoli, Anna; Sindona, Giovanni; Mazzotti, Fabio

2018-05-16

Monacolin K, the active ingredient present in dietary supplement, is a nutraceutical whose health benefits have been widely documented. A fast approach for the assay of lovastatin in both form, lactone and acid, by mass spectrometry is presented. The quantitative assay is carried out by HPLC-MS/MS using the multiple reaction monitoring (MRM) mode and simvastatin and pravastatin as internal standards. The accuracy values ranged from 97 to 101%; the analytical parameters values of LOQ, LOD, recovery and reproducibility, were calculated analyzing fortified samples, confirming the reliability of the proposed approach. This article is protected by copyright. All rights reserved.

Quartz crystal microbalance for the cardiac markers/antibodies binding kinetic measurements in the plasma samples

NASA Astrophysics Data System (ADS)

Agafonova, L. E.; Shumyantseva, V. V.; Archakov, A. I.

2014-06-01

The quartz crystal microbalance (QCM) was exploited for cardiac markers detection and kinetic studies of immunochemical reaction of cardiac troponin I (cTnI) and human heart fatty acid binding protein (H-FABP) with the corresponding monoclonal antibodies in undiluted plasma (serum) and standard solutions. The QCM technique allowed to dynamically monitor the kinetic differences in specific interactions and nonspecific sorption, without multiple labeling procedures and separation steps. The affinity binding process was characterized by the association (ka) and the dissociation (kd) kinetic constants and the equilibrium association (K) constant, all of which were obtained from experimental data.

In situ loading of well-dispersed silver nanoparticles on nanocrystalline magnesium oxide for real-time monitoring of catalytic reactions by surface enhanced Raman spectroscopy.

PubMed

Zhang, Kaige; Li, Gongke; Hu, Yuling

2015-10-28

The surface-enhanced Raman spectroscopy (SERS) technique is of great importance for insight into the transient reaction intermediates and mechanistic pathways involved in heterogeneously catalyzed chemical reactions under actual reaction conditions, especially in water. Herein, we demonstrate a facile method for in situ synthesis of nanocrystalline magnesium oxide-Ag(0) (nano MgO-Ag(0)) hybrid nanomaterials with dispersed Ag nanoparticles (Ag NPs) on the surface of nanocrystalline magnesium oxide (nano MgO) via Sn(2+) linkage and reduction. As a benefit from the synergy effect of nano MgO and Ag NPs, the nano MgO-Ag(0) exhibited both excellent SERS and catalytic activities for the reduction of 4-nitrothiophenol in the presence of NaBH4. The nano MgO-Ag(0) was used for real-time monitoring of the catalytic reaction process of 4-nitrothiophenol to 4-aminothiophenol in an aqueous medium by observing the SERS signals of the reactant, intermediate and final products. The intrinsic reaction kinetics and reaction mechanism of this reaction were also investigated. This SERS-based synergy technique provides a novel approach for quantitative in situ monitoring of catalytic chemical reaction processes.

UPLC-MS/MS method for therapeutic drug monitoring of 10 antibiotics used in intensive care units.

PubMed

El-Najjar, Nahed; Hösl, Julian; Holzmann, Thomas; Jantsch, Jonathan; Gessner, André

2018-03-01

A large variation in the levels of different ß-lactams and other antibiotics used in critically ill patients has been documented. The aim of this study is to establish and validate a fast, ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous analysis of ten antibiotics (Meropenem, Cefepime, Ceftazidime, Piperacillin, Benzylpenicillin, Ampicillin, Flucloxacillin, Linezolid, and Sulfamethoxazole/Trimethoprim) in human plasma according to European Medicines Agency (EMA) guidelines. Protein precipitation with ice-cold methanol containing 9 isotopically labeled internal standards was used for sample clean up. Antibiotics were detected, following a 4-minute gradient separation, in multiple reactions monitoring (MRM) using API 4000 instrument equipped with electrospray source operating in positive ion mode. The lower limit of quantification was 0.1 mg/L for Meropenem, Ceftazidime, Piperacillin, Ampicillin, Flucloxacillin, and Sulfamethoxazole; 0.05 mg/L for Cefepime, Benzylpenicillin, and Trimethoprim; and 0.02 mg/L for Linezolid. The method proved to be precise and accurate and applicable for therapeutic drug monitoring and other pharmacokinetic studies. Copyright © 2017 John Wiley & Sons, Ltd.

A rapid, automated approach to optimisation of multiple reaction monitoring conditions for quantitative bioanalytical mass spectrometry.

PubMed

Higton, D M

2001-01-01

An improvement to the procedure for the rapid optimisation of mass spectrometry (PROMS), for the development of multiple reaction methods (MRM) for quantitative bioanalytical liquid chromatography/tandem mass spectrometry (LC/MS/MS), is presented. PROMS is an automated protocol that uses flow-injection analysis (FIA) and AppleScripts to create methods and acquire the data for optimisation. The protocol determines the optimum orifice potential, the MRM conditions for each compound, and finally creates the MRM methods needed for sample analysis. The sensitivities of the MRM methods created by PROMS approach those created manually. MRM method development using PROMS currently takes less than three minutes per compound compared to at least fifteen minutes manually. To further enhance throughput, approaches to MRM optimisation using one injection per compound, two injections per pool of five compounds and one injection per pool of five compounds have been investigated. No significant difference in the optimised instrumental parameters for MRM methods were found between the original PROMS approach and these new methods, which are up to ten times faster. The time taken for an AppleScript to determine the optimum conditions and build the MRM methods is the same with all approaches. Copyright 2001 John Wiley & Sons, Ltd.

The Application of the Weighted k-Partite Graph Problem to the Multiple Alignment for Metabolic Pathways.

PubMed

Chen, Wenbin; Hendrix, William; Samatova, Nagiza F

2017-12-01

The problem of aligning multiple metabolic pathways is one of very challenging problems in computational biology. A metabolic pathway consists of three types of entities: reactions, compounds, and enzymes. Based on similarities between enzymes, Tohsato et al. gave an algorithm for aligning multiple metabolic pathways. However, the algorithm given by Tohsato et al. neglects the similarities among reactions, compounds, enzymes, and pathway topology. How to design algorithms for the alignment problem of multiple metabolic pathways based on the similarity of reactions, compounds, and enzymes? It is a difficult computational problem. In this article, we propose an algorithm for the problem of aligning multiple metabolic pathways based on the similarities among reactions, compounds, enzymes, and pathway topology. First, we compute a weight between each pair of like entities in different input pathways based on the entities' similarity score and topological structure using Ay et al.'s methods. We then construct a weighted k-partite graph for the reactions, compounds, and enzymes. We extract a mapping between these entities by solving the maximum-weighted k-partite matching problem by applying a novel heuristic algorithm. By analyzing the alignment results of multiple pathways in different organisms, we show that the alignments found by our algorithm correctly identify common subnetworks among multiple pathways.

Fission time scale from pre-scission neutron and α multiplicities in the 16O + 194Pt reaction

NASA Astrophysics Data System (ADS)

Kapoor, K.; Verma, S.; Sharma, P.; Mahajan, R.; Kaur, N.; Kaur, G.; Behera, B. R.; Singh, K. P.; Kumar, A.; Singh, H.; Dubey, R.; Saneesh, N.; Jhingan, A.; Sugathan, P.; Mohanto, G.; Nayak, B. K.; Saxena, A.; Sharma, H. P.; Chamoli, S. K.; Mukul, I.; Singh, V.

2017-11-01

Pre- and post-scission α -particle multiplicities have been measured for the reaction 16O+P194t at 98.4 MeV forming R210n compound nucleus. α particles were measured at various angles in coincidence with the fission fragments. Moving source technique was used to extract the pre- and post-scission contributions to the particle multiplicity. Study of the fission mechanism using the different probes are helpful in understanding the detailed reaction dynamics. The neutron multiplicities for this reaction have been reported earlier. The multiplicities of neutrons and α particles were reproduced using standard statistical model code joanne2 by varying the transient (τt r) and saddle to scission (τs s c) times. This code includes deformation dependent-particle transmission coefficients, binding energies and level densities. Fission time scales of the order of 50-65 ×10-21 s are required to reproduce the neutron and α -particle multiplicities.

A multimodal optical and electrochemical device for monitoring surface reactions: redox active surfaces in porous silicon Rugate filters.

PubMed

Ciampi, Simone; Guan, Bin; Darwish, Nadim A; Zhu, Ying; Reece, Peter J; Gooding, J Justin

2012-12-21

Herein, mesoporous silicon (PSi) is configured as a single sensing device that has dual readouts; as a photonic crystal sensor in a Rugate filter configuration, and as a high surface area porous electrode. The as-prepared PSi is chemically modified to provide it with stability in aqueous media and to allow for the subsequent coupling of chemical species, such as via Cu(I)-catalyzed cycloaddition reactions between 1-alkynes and azides ("click" reactions). The utility of the bimodal capabilities of the PSi sensor for monitoring surface coupling procedures is demonstrated by the covalent coupling of a ferrocene derivative, as well as by demonstrating ligand-exchange reactions (LER) at the PSi surface. Both types of reactions were monitored through optical reflectivity measurements, as well as electrochemically via the oxidation/reduction of the surface tethered redox species.

Multiple Reaction Monitoring Profiling to Assess Compliance with an Almond Consumption Intervention

PubMed Central

Mattes, Richard D

2017-01-01

Abstract Background: Almonds are extremely rich sources of lipids and flavonoids, and their consumption is associated with several health benefits. However, there are no analytical methods available to document compliance with prescribed or self-reported chronic almond consumption. Objective: The aim was to use an analytical approach that identifies metabolic profiles associated with long-term almond consumption to ascertain compliance with prescribed consumption. Methods: A multiple reaction monitoring (MRM)–profiling strategy was designed to isolate metabolic changes in erythrocytes after 12 wk of almond consumption. MRM-profiling data acquisition and analysis involve performing separate discovery and screening steps to detect molecular features related to metabolic changes between experimental groups. Samples used for this research were erythrocytes recovered at baseline, after 12 wk of almond consumption (W12-almond group), and after 12 wk of a nut-free diet (W12-control group). For the MRM-profiling discovery step, representative samples (pools) of erythrocytes from individuals of all groups were interrogated by precursor ion and neutral loss scan experiments on the basis of previous knowledge of chemical functional groups present in the samples. The outputs of the discovery phase were methods used for the MRM-profiling screening phase to interrogate individual samples on the basis of fast-MRM measurements. In addition, we screened the literature for flavonoids identified in almond skins and included them for individual sample screening. Results: Of the 254 m/z values monitored, 5 ratios and combinations of specific ions with receiver operating characteristic curve AUCs >0.89 provided a sensitivity of 74.2% and a specificity of 90% for blind samples presented in the model. Eight of the 31 participants (25.8%) in the W12-almond group and 3 of the 30 (10%) participants in the W12-control group were misclassified by all 5 ratios. Ratios and combinations of specific transitions were mainly related to membrane lipids. Conclusion: The misclassifications observed as a result of ratio performance evaluation may indicate noncompliance as supported by the dietary intake data. The parent trial was registered at www.clinicaltrials.gov as NCT02360787.

Comparison of Quantitative Mass Spectrometry Platforms for Monitoring Kinase ATP Probe Uptake in Lung Cancer.

PubMed

Hoffman, Melissa A; Fang, Bin; Haura, Eric B; Rix, Uwe; Koomen, John M

2018-01-05

Recent developments in instrumentation and bioinformatics have led to new quantitative mass spectrometry platforms including LC-MS/MS with data-independent acquisition (DIA) and targeted analysis using parallel reaction monitoring mass spectrometry (LC-PRM), which provide alternatives to well-established methods, such as LC-MS/MS with data-dependent acquisition (DDA) and targeted analysis using multiple reaction monitoring mass spectrometry (LC-MRM). These tools have been used to identify signaling perturbations in lung cancers and other malignancies, supporting the development of effective kinase inhibitors and, more recently, providing insights into therapeutic resistance mechanisms and drug repurposing opportunities. However, detection of kinases in biological matrices can be challenging; therefore, activity-based protein profiling enrichment of ATP-utilizing proteins was selected as a test case for exploring the limits of detection of low-abundance analytes in complex biological samples. To examine the impact of different MS acquisition platforms, quantification of kinase ATP uptake following kinase inhibitor treatment was analyzed by four different methods: LC-MS/MS with DDA and DIA, LC-MRM, and LC-PRM. For discovery data sets, DIA increased the number of identified kinases by 21% and reduced missingness when compared with DDA. In this context, MRM and PRM were most effective at identifying global kinome responses to inhibitor treatment, highlighting the value of a priori target identification and manual evaluation of quantitative proteomics data sets. We compare results for a selected set of desthiobiotinylated peptides from PRM, MRM, and DIA and identify considerations for selecting a quantification method and postprocessing steps that should be used for each data acquisition strategy.

Quantitative diagnosis and prognosis framework for concrete degradation due to alkali-silica reaction

NASA Astrophysics Data System (ADS)

Mahadevan, Sankaran; Neal, Kyle; Nath, Paromita; Bao, Yanqing; Cai, Guowei; Orme, Peter; Adams, Douglas; Agarwal, Vivek

2017-02-01

This research is seeking to develop a probabilistic framework for health diagnosis and prognosis of aging concrete structures in nuclear power plants that are subjected to physical, chemical, environment, and mechanical degradation. The proposed framework consists of four elements: monitoring, data analytics, uncertainty quantification, and prognosis. The current work focuses on degradation caused by ASR (alkali-silica reaction). Controlled concrete specimens with reactive aggregate are prepared to develop accelerated ASR degradation. Different monitoring techniques — infrared thermography, digital image correlation (DIC), mechanical deformation measurements, nonlinear impact resonance acoustic spectroscopy (NIRAS), and vibro-acoustic modulation (VAM) — are studied for ASR diagnosis of the specimens. Both DIC and mechanical measurements record the specimen deformation caused by ASR gel expansion. Thermography is used to compare the thermal response of pristine and damaged concrete specimens and generate a 2-D map of the damage (i.e., ASR gel and cracked area), thus facilitating localization and quantification of damage. NIRAS and VAM are two separate vibration-based techniques that detect nonlinear changes in dynamic properties caused by the damage. The diagnosis results from multiple techniques are then fused using a Bayesian network, which also helps to quantify the uncertainty in the diagnosis. Prognosis of ASR degradation is then performed based on the current state of degradation obtained from diagnosis, by using a coupled thermo-hydro-mechanical-chemical (THMC) model for ASR degradation. This comprehensive approach of monitoring, data analytics, and uncertainty-quantified diagnosis and prognosis will facilitate the development of a quantitative, risk informed framework that will support continuous assessment and risk management of structural health and performance.

Gene transcription in sea otters (Enhydra lutris); development of a diagnostic tool for sea otter and ecosystem health

USGS Publications Warehouse

Bowen, Lizabeth; Miles, A. Keith; Murray, Michael; Haulena, Martin; Tuttle, Judy; van Bonn, William; Adams, Lance; Bodkin, James L.; Ballachey, Brenda E.; Estes, James A.; Tinker, M. Tim; Keister, Robin; Stott, Jeffrey L.

2012-01-01

Gene transcription analysis for diagnosing or monitoring wildlife health requires the ability to distinguish pathophysiological change from natural variation. Herein, we describe methodology for the development of quantitative real-time polymerase chain reaction (qPCR) assays to measure differential transcript levels of multiple immune function genes in the sea otter (Enhydra lutris); sea otter-specific qPCR primer sequences for the genes of interest are defined. We establish a ‘reference’ range of transcripts for each gene in a group of clinically healthy captive and free-ranging sea otters. The 10 genes of interest represent multiple physiological systems that play a role in immuno-modulation, inflammation, cell protection, tumour suppression, cellular stress response, xenobiotic metabolizing enzymes, antioxidant enzymes and cell–cell adhesion. The cycle threshold (CT) measures for most genes were normally distributed; the complement cytolysis inhibitor was the exception. The relative enumeration of multiple gene transcripts in simple peripheral blood samples expands the diagnostic capability currently available to assess the health of sea otters in situ and provides a better understanding of the state of their environment.

Quantitative Proteomics via High Resolution MS Quantification: Capabilities and Limitations

PubMed Central

Higgs, Richard E.; Butler, Jon P.; Han, Bomie; Knierman, Michael D.

2013-01-01

Recent improvements in the mass accuracy and resolution of mass spectrometers have led to renewed interest in label-free quantification using data from the primary mass spectrum (MS1) acquired from data-dependent proteomics experiments. The capacity for higher specificity quantification of peptides from samples enriched for proteins of biological interest offers distinct advantages for hypothesis generating experiments relative to immunoassay detection methods or prespecified peptide ions measured by multiple reaction monitoring (MRM) approaches. Here we describe an evaluation of different methods to post-process peptide level quantification information to support protein level inference. We characterize the methods by examining their ability to recover a known dilution of a standard protein in background matrices of varying complexity. Additionally, the MS1 quantification results are compared to a standard, targeted, MRM approach on the same samples under equivalent instrument conditions. We show the existence of multiple peptides with MS1 quantification sensitivity similar to the best MRM peptides for each of the background matrices studied. Based on these results we provide recommendations on preferred approaches to leveraging quantitative measurements of multiple peptides to improve protein level inference. PMID:23710359

Chain-reaction crash on a highway in high visibility

NASA Astrophysics Data System (ADS)

Nagatani, Takashi

2016-05-01

We study the chain-reaction crash (multiple-vehicle collision) in high-visibility condition on a highway. In the traffic situation, drivers control their vehicles by both gear-changing and braking. Drivers change the gears according to the headway and brake according to taillights of the forward vehicle. We investigate whether or not the first collision induces the chain-reaction crash numerically. It is shown that dynamic transitions occur from no collisions, through a single collision, to multiple collisions with decreasing the headway. Also, we find that the dynamic transition occurs from the finite chain reaction to the infinite chain reaction when the headway is less than the critical value. We compare the multiple-vehicle collisions in high-visibility with that in low-visibility. We derive the transition points and the region maps for the chain-reaction crash in high visibility.

A lectin-coupled, targeted proteomic mass spectrometry (MRM MS) platform for identification of multiple liver cancer biomarkers in human plasma.

PubMed

Ahn, Yeong Hee; Shin, Park Min; Oh, Na Ree; Park, Gun Wook; Kim, Hoguen; Yoo, Jong Shin

2012-09-18

Aberrantly glycosylated proteins related to liver cancer progression were captured with specific lectin and identified from human plasma by multiple reaction monitoring (MRM) mass spectrometry as multiple biomarkers for hepatocellular carcinoma (HCC). The lectin fractionation for fucosylated protein glycoforms in human plasma was conducted with a fucose-specific aleuria aurantia lectin (AAL). Following tryptic digestion of the lectin-captured fraction, plasma samples from 30 control cases (including 10 healthy, 10 hepatitis B virus [HBV], and 10 cirrhosis cases) and 10 HCC cases were quantitatively analyzed by MRM to identify which glycoproteins are viable HCC biomarkers. A1AG1, AACT, A1AT, and CERU were found to be potent biomarkers to differentiate HCC plasma from control plasmas. The AUROC generated independently from these four biomarker candidates ranged from 0.73 to 0.92. However, the lectin-coupled MRM assay with multiple combinations of biomarker candidates is superior statistically to those generated from the individual candidates with AUROC more than 0.95, which can be an alternative to the immunoassay inevitably requiring tedious development of multiple antibodies against biomarker candidates to be verified. Eventually the lectin-coupled, targeted proteomic mass spectrometry (MRM MS) platform was found to be efficient to identify multiple biomarkers from human plasma according to cancer progression. Copyright © 2012 Elsevier B.V. All rights reserved.

In Site Analysis of a High Temperature Cure Reaction in Real Time Using Modulated Fiber-Optic FT-Raman Spectroscopy

NASA Technical Reports Server (NTRS)

Cooper, John; Aust, Jeffrey F.; Wise, Kent L.; Jensen, Brian J.

1999-01-01

The vibrational spectrum of a high temperature (330 C) polymerization reaction was successfully monitored in real time using a modulated fiber-optic FT-Raman spectrometer. A phenylethynyl terminated monomer was cured, and spectral evidence for two different reaction products was acquired. The products are a conjugated polyene chain and a cyclized trimer. This is the first report describing the use of FT-Raman spectroscopy to monitor a high temperature (greater than 250 C) reaction in real time.

In Situ Analysis of a High-Temperature Cure Reaction in Real Time Using Modulated Fiber-Optic FT-Raman Spectroscopy

NASA Technical Reports Server (NTRS)

Aust, Jeffrey F.; Cooper, John B.; Wise, Kent L.; Jensen, Brian J.

1999-01-01

The vibrational spectrum of a high-temperature (330 C) polymerization reaction was successfully monitored in real time with the use of a modulated fiber-optic Fourier transform (FT)-Raman spectrometer. A phenylethynyl-terminated monomer was cured, and spectral evidence for two different reaction products was acquired. The products are a conjugated polyene chain and a cyclized trimer. This is the first report describing the use of FT-Raman spectroscopy to monitor a high temperature (greater than 250 C) reaction in real time.

40 CFR 258.54 - Detection monitoring program.

Code of Federal Regulations, 2010 CFR

2010-07-01

... FOR MUNICIPAL SOLID WASTE LANDFILLS Ground-Water Monitoring and Corrective Action § 258.54 Detection... reaction products in the unsaturated zone beneath the MSWLF unit; (iii) The detectability of indicator parameters, waste constituents, and reaction products in the ground water; and (iv) The concentration or...

Development of a new microtiter plate format for clinically relevant assays.

PubMed

Piletska, Elena V; Piletsky, Stanislav S; Whitcombe, Michael J; Chianella, Iva; Piletsky, Sergey A

2012-02-21

A new format for the microtiter plate-based assays was proposed. The novelty involves the use of disk-shaped inserts for immobilization of biological and chemical reagents. The internal opening of the disks allows measurements of the reactions by standard microtiter plate readers without any additional steps involving liquid handling. Ideally the plate end-users just have to add the sample and take the measurement without any need of multiple reagent additions or transfer of the liquid to a different plate. The novel assay format also allows handling of reagents which are not soluble in an aqueous environment. As a proof of concept we describe here several model reactions which are compatible with microtiter plate format, such as monitoring enzymatic reactions catalyzed by glucose oxidase (GOx) and urease, measurements of proteins by BCA assay, analysis of pH, and concentration of antioxidants. The "mix and match" approach in the disk-shape format allows multiplexing and could be particularly useful for high throughput screening. One of the potential application areas for this novel assay format could be in a multianalyte system for measurement of clinically relevant analytes in primary care.

Multi-template polymerase chain reaction.

PubMed

Kalle, Elena; Kubista, Mikael; Rensing, Christopher

2014-12-01

PCR is a formidable and potent technology that serves as an indispensable tool in a wide range of biological disciplines. However, due to the ease of use and often lack of rigorous standards many PCR applications can lead to highly variable, inaccurate, and ultimately meaningless results. Thus, rigorous method validation must precede its broad adoption to any new application. Multi-template samples possess particular features, which make their PCR analysis prone to artifacts and biases: multiple homologous templates present in copy numbers that vary within several orders of magnitude. Such conditions are a breeding ground for chimeras and heteroduplexes. Differences in template amplification efficiencies and template competition for reaction compounds undermine correct preservation of the original template ratio. In addition, the presence of inhibitors aggravates all of the above-mentioned problems. Inhibitors might also have ambivalent effects on the different templates within the same sample. Yet, no standard approaches exist for monitoring inhibitory effects in multitemplate PCR, which is crucial for establishing compatibility between samples.

Non-target screening of Allura Red AC photodegradation products in a beverage through ultra high performance liquid chromatography coupled with hybrid triple quadrupole/linear ion trap mass spectrometry.

PubMed

Gosetti, Fabio; Chiuminatto, Ugo; Mazzucco, Eleonora; Calabrese, Giorgio; Gennaro, Maria Carla; Marengo, Emilio

2013-01-15

The study deals with the identification of the degradation products formed by simulated sunlight photoirradiation in a commercial beverage that contains Allura Red AC dye. An UHPLC-MS/MS method, that makes use of hybrid triple quadrupole/linear ion trap, was developed. In the identification step the software tool information dependent acquisition (IDA) was used to automatically obtain information about the species present and to build a multiple reaction monitoring (MRM) method with the MS/MS fragmentation pattern of the species considered. The results indicate that the identified degradation products are formed from side-reactions and/or interactions among the dye and other ingredients present in the beverage (ascorbic acid, citric acid, sucrose, aromas, strawberry juice, and extract of chamomile flowers). The presence of aromatic amine or amide functionalities in the chemical structures proposed for the degradation products might suggest potential hazards to consumer health. Copyright © 2012 Elsevier Ltd. All rights reserved.

A model of human decision making in multiple process monitoring situations

NASA Technical Reports Server (NTRS)

Greenstein, J. S.; Rouse, W. B.

1982-01-01

Human decision making in multiple process monitoring situations is considered. It is proposed that human decision making in many multiple process monitoring situations can be modeled in terms of the human's detection of process related events and his allocation of attention among processes once he feels event have occurred. A mathematical model of human event detection and attention allocation performance in multiple process monitoring situations is developed. An assumption made in developing the model is that, in attempting to detect events, the human generates estimates of the probabilities that events have occurred. An elementary pattern recognition technique, discriminant analysis, is used to model the human's generation of these probability estimates. The performance of the model is compared to that of four subjects in a multiple process monitoring situation requiring allocation of attention among processes.

Field assessment and enhancement of cognitive performance: development of an ambulatory vigilance monitor.

PubMed

Lieberman, Harris R; Kramer, F Matthew; Montain, Scott J; Niro, Philip

2007-05-01

Limited opportunities to study human cognitive performance in non-laboratory, ambulatory situations exist. However, advances in technology make it possible to extend behavioral assessments to the field. One of the first devices to measure human behavior in the field was the wrist-worn actigraph. This device acquires minute-by-minute information on an individual's physical activity and can distinguish sleep from waking, the most basic aspect of behavior. Our laboratory developed a series of wrist-worn devices, not much larger than a watch, which assess reaction time, vigilance and memory. The devices concurrently assess motor activity with greater temporal resolution than standard actigraphs. They also continuously monitor multiple environmental variables including temperature, humidity, sound, and light. These monitors have been employed during training and simulated military operations to collect behavioral and environmental information that would typically be unavailable under such circumstances. Development of the vigilance monitor, and how each successive version extended capabilities of the device are described. Data from several studies are presented, including studies conducted in harsh field environments during a simulated infantry assault, an officer training course. The monitors simultaneously documented environmental conditions, patterns of sleep and activity and effects of nutritional manipulations on cognitive performance. They provide a new method to relate cognitive performance to real world environmental conditions and assess effects of various interventions on human behavior in the field. They can also monitor cognitive performance in real time, and if it is degraded, attempt to intervene to maintain

Online monitoring of chemical reactions by polarization-induced electrospray ionization.

PubMed

Meher, Anil Kumar; Chen, Yu-Chie

2016-09-21

Polarization-induced electrospray ionization (PI-ESI) is a simple technique for instant generation of gas-phase ions directly from a microliter-sized droplet for mass spectrometric analysis. A sample droplet was placed over a dielectric substrate and in proximity (2-3 mm) to the inlet of a mass spectrometer. Owing to the polarization effect induced by the high electric field provided by the mass spectrometer, the droplet was polarized and the electrospray was generated from the apex of the droplet. The polarization-induced electrospray could last for tens of seconds, which was sufficiently long to monitor fast reactions occurring within few seconds. Thus, we demonstrated the feasibility of using the droplet-based PI-ESI MS for the online monitoring of fast reactions by simply mixing two droplets (5-10 μL) containing reactants on a dielectric substrate placed in front of a mass spectrometer applied with a high voltage (-4500 V). Schiff base reactions and oxidation reactions that can generate intermediates/products within a few seconds were selected as the model reactions. The ionic reaction species generated from intermediates and products can be simultaneously monitored by PI-ESI MS in real time. We also used this approach to selectively detect acetone from a urine sample, in which acetone was derivatized in situ. In addition, the possibility of using this approach for quantitative analysis of acetone from urine samples was examined. Copyright © 2016 Elsevier B.V. All rights reserved.

Detection of Gelatin Adulteration in Traditional Chinese Medicine: Analysis of Deer-Horn Glue by Rapid-Resolution Liquid Chromatography-Triple Quadrupole Mass Spectrometry

PubMed Central

Chen, Jia; Cheng, Xian-Long; Wei, Feng; Zhang, Qian-Qian; Li, Ming-Hua; Ma, Shuang-Cheng

2015-01-01

Simultaneous identification of donkey-hide gelatin and bovine-hide gelatin in deer-horn glue was established by rapid-resolution liquid chromatography-triple quadrupole mass spectrometry. Water containing 1% NH4HCO3 was used for sample dissolution and trypsin was used for hydrolysis of the gelatins. After separation by a SB-C18 reversed-phase analytical column, collagen marker peptides were detected by mass spectrometry in positive electrospray ionization mode with multiple reaction monitoring. The method was specific, precise and reliable, and suitable for detection of adulterants derived from donkey-hide gelatin and bovine-hide gelatin in deer-horn glue. PMID:26504613

Phenolics from Castanea sativa leaves and their effects on UVB-induced damage.

PubMed

Cerulli, Antonietta; Masullo, Milena; Mari, Angela; Balato, Anna; Filosa, Rosanna; Lembo, Serena; Napolitano, Assunta; Piacente, Sonia

2018-05-01

The phytochemical investigation of the methanol extract of the leaves of Castanea sativa Mill., source of the Italian PGI (Protected Geographical Indication) product 'Marrone di Roccadaspide' (Campania region) afforded as main compounds crenatin (1), chestanin (2), gallic acid (3), cretanin (4), 5-O-p-coumaroylquinic acid (5), p-methylgallic acid (6) and quercetin-3-O-glucoside (7). To quantify the isolated compounds a LC-ESI(QqQ)MS method working with a very sensitive and selective mass tandem experiment called Multiple Reaction Monitoring (MRM) has been developed. Moreover the antioxidant capacity by TEAC assay and the ability of compounds 1-7 to protect HaCaT human keratinocytes from UVB-induced damage has been investigated.

Quantitation of Met tyrosine phosphorylation using MRM-MS.

PubMed

Meng, Zhaojing; Srivastava, Apurva K; Zhou, Ming; Veenstra, Timothy

2013-01-01

Phosphorylation has long been accepted as a key cellular regulator of cell signaling pathways. The recent development of multiple-reaction monitoring mass spectrometry (MRM-MS) provides a useful tool for measuring the absolute quantity of phosphorylation occupancy at pivotal sites within signaling proteins, even when the phosphorylation sites are in close proximity. Here, we described a targeted quantitation approach to measure the absolute phosphorylation occupancy at Y1234 and Y1235 of Met. The approach is utilized to obtain absolute occupancy of the two phosphorylation sites in the full-length recombinant Met. It is further applied to quantitate the phosphorylation state of these two sites in SNU-5 cells treated with a Met inhibitor.

Multiplexed, quantitative, and targeted metabolite profiling by LC-MS/MRM.

PubMed

Wei, Ru; Li, Guodong; Seymour, Albert B

2014-01-01

Targeted metabolomics, which focuses on a subset of known metabolites representative of biologically relevant metabolic pathways, is a valuable tool to discover biomarkers and link disease phenotypes to underlying mechanisms or therapeutic modes of action. A key advantage of targeted metabolomics, compared to discovery metabolomics, is its immediate readiness for extracting biological information derived from known metabolites and quantitative measurements. However, simultaneously analyzing hundreds of endogenous metabolites presents a challenge due to their diverse chemical structures and properties. Here we report a method which combines different chromatographic separation conditions, optimal ionization polarities, and the most sensitive triple-quadrupole MS-based data acquisition mode, multiple reaction monitoring (MRM), to quantitatively profile 205 endogenous metabolites in 10 min.

Determination of virginiamycin M1 residue in tissues of swine and chicken by ultra-performance liquid chromatography tandem mass spectrometry.

PubMed

Wang, Xiaoyang; Wang, Mi; Zhang, Keyu; Hou, Ting; Zhang, Lifang; Fei, Chenzong; Xue, Feiqun; Hang, Taijun

2018-06-01

A reliable UPLC-MS/MS method with high sensitivity was developed and validated for the determination of virginiamycin M1 in muscle, fat, liver, and kidney samples of chicken and swine. Analytes were extracted using acetonitrile and extracts were defatted by N-hexane. Chromatographic separation was performed on a BEH C18 liquid chromatography column. The analytes were then detected using triplequadrupole mass spectrometry in positive electrospray ionization and multiple reaction monitoring mode. Calibration plots were constructed using standard working solutions and showed good linearity. Limits of quantification ranged from 2 to 60 ng mL -1 . Copyright © 2018 Elsevier Ltd. All rights reserved.

RAPID MONITORING BY QUANTITATIVE POLYMERASE CHAIN REACTION FOR PATHOGENIC ASPERGILLUS DURING CARPET REMOVAL FROM A HOSPITAL

EPA Science Inventory

Monitoring for pathogenic Aspergillus species using a rapid, highly sensitive, quantitative polumerase chain reaction technique during carpet removal in a burn unit provided data which allowed the patients to be safely returned to the re-floored area sooner than if only conventio...

Action Monitoring and Perfectionism in Anorexia Nervosa

ERIC Educational Resources Information Center

Pieters, Guido L. M.; de Bruijn, Ellen R. A.; Maas, Yvonne; Hulstijn, Wouter; Vandereycken, Walter; Peuskens, Joseph; Sabbe, Bernard G.

2007-01-01

To study action monitoring in anorexia nervosa, behavioral and EEG measures were obtained in underweight anorexia nervosa patients (n=17) and matched healthy controls (n=19) while performing a speeded choice-reaction task. Our main measures of interest were questionnaire outcomes, reaction times, error rates, and the error-related negativity ERP…

Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes.

PubMed

Börner, Tim; Grey, Carl; Adlercreutz, Patrick

2016-08-01

Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein biomarker discovery and fast monitoring for the identification and detection of Anisakids by parallel reaction monitoring (PRM) mass spectrometry.

PubMed

Carrera, Mónica; Gallardo, José M; Pascual, Santiago; González, Ángel F; Medina, Isabel

2016-06-16

Anisakids are fish-borne parasites that are responsible for a large number of human infections and allergic reactions around the world. World health organizations and food safety authorities aim to control and prevent this emerging health problem. In the present work, a new method for the fast monitoring of these parasites is described. The strategy is divided in three steps: (i) purification of thermostable proteins from fish-borne parasites (Anisakids), (ii) in-solution HIFU trypsin digestion and (iii) monitoring of several peptide markers by parallel reaction monitoring (PRM) mass spectrometry. This methodology allows the fast detection of Anisakids in <2h. An affordable assay utilizing this methodology will facilitate testing for regulatory and safety applications. The work describes for the first time, the Protein Biomarker Discovery and the Fast Monitoring for the identification and detection of Anisakids in fishery products. The strategy is based on the purification of thermostable proteins, the use of accelerated in-solution trypsin digestions under an ultrasonic field provided by High-Intensity Focused Ultrasound (HIFU) and the monitoring of several peptide biomarkers by Parallel Reaction Monitoring (PRM) Mass Spectrometry in a linear ion trap mass spectrometer. The workflow allows the unequivocal detection of Anisakids, in <2h. The present strategy constitutes the fastest method for Anisakids detection, whose application in the food quality control area, could provide to the authorities an effective and rapid method to guarantee the safety to the consumers. Copyright © 2016 Elsevier B.V. All rights reserved.

Imaging thermal conductivity with nanoscale resolution using a scanning spin probe

DOE PAGES

Laraoui, Abdelghani; Aycock-Rizzo, Halley; Gao, Yang; ...

2015-11-20

The ability to probe nanoscale heat flow in a material is often limited by lack of spatial resolution. Here, we use a diamond-nanocrystal-hosted nitrogen-vacancy centre attached to the apex of a silicon thermal tip as a local temperature sensor. We apply an electrical current to heat up the tip and rely on the nitrogen vacancy to monitor the thermal changes the tip experiences as it is brought into contact with surfaces of varying thermal conductivity. By combining atomic force and confocal microscopy, we image phantom microstructures with nanoscale resolution, and attain excellent agreement between the thermal conductivity and topographic maps.more » The small mass and high thermal conductivity of the diamond host make the time response of our technique short, which we demonstrate by monitoring the tip temperature upon application of a heat pulse. Our approach promises multiple applications, from the investigation of phonon dynamics in nanostructures to the characterization of heterogeneous phase transitions and chemical reactions in various solid-state systems.« less

External cavity tunable quantum cascade lasers and their applications to trace gas monitoring.

PubMed

Rao, Gottipaty N; Karpf, Andreas

2011-02-01

Since the first quantum cascade laser (QCL) was demonstrated approximately 16 years ago, we have witnessed an explosion of interesting developments in QCL technology and QCL-based trace gas sensors. QCLs operate in the mid-IR region (3-24 μm) and can directly access the rotational vibrational bands of most molecular species and, therefore, are ideally suited for trace gas detection with high specificity and sensitivity. These sensors have applications in a wide range of fields, including environmental monitoring, atmospheric chemistry, medical diagnostics, homeland security, detection of explosive compounds, and industrial process control, to name a few. Tunable external cavity (EC)-QCLs in particular offer narrow linewidths, wide ranges of tunability, and stable power outputs, which open up new possibilities for sensor development. These features allow for the simultaneous detection of multiple species and the study of large molecules, free radicals, ions, and reaction kinetics. In this article, we review the current status of EC-QCLs and sensor developments based on them and speculate on possible future developments.

Simple and Fast Extraction-Coupled UPLC-MS/MS Method for the Determination of Mequindox and Its Major Metabolites in Food Animal Tissues.

PubMed

You, Yanli; Song, Liting; Li, Yanshen; Wu, Yongtao; Xin, Mao

2016-03-23

This research described a sensitive and rapid UPLC-MS/MS method for the determination of mequindox and its six major metabolites in chicken muscle, chicken liver, swine muscle, and swine liver. Among the metabolites, carbonyl reduction-1,4-bisdesoxy-mequindox is novel. Target analytes could be extracted by ethyl acetate without any acidolysis or enzymolysis steps. After purification by a Bond Elut C18 cartridge, analysis was carried out by UPLC-MS/MS using positive ion multiple reaction monitoring (MRM) mode. Validation was performed in spiked samples, and mean recoveries ranged from 64.3 to 114.4%, with intraday and interday variations of less than 14.7 and 19.2%, respectively. The limit of detection (LOD) was <1.0 μg kg(-1), whereas the limit of quantification (LOQ) was <4.0 μg kg(-1). This procedure will help monitor mequindox residues in animal-derived food, and it will also facilitate further pharmacokinetics of mequindox.

Monitoring the Reaction Process During the S2 → S3 Transition in Photosynthetic Water Oxidation Using Time-Resolved Infrared Spectroscopy.

PubMed

Sakamoto, Hiroki; Shimizu, Tatsuki; Nagao, Ryo; Noguchi, Takumi

2017-02-08

Photosynthetic water oxidation performed at the Mn 4 CaO 5 cluster in photosystem II plays a crucial role in energy production as electron and proton sources necessary for CO 2 fixation. Molecular oxygen, a byproduct, is a source of the oxygenic atmosphere that sustains life on earth. However, the molecular mechanism of water oxidation is not yet well-understood. In the reaction cycle of intermediates called S states, the S 2 → S 3 transition is particularly important; it consists of multiple processes of electron transfer, proton release, and water insertion, and generates an intermediate leading to O-O bond formation. In this study, we monitored the reaction process during the S 2 → S 3 transition using time-resolved infrared spectroscopy to clarify its molecular mechanism. A change in the hydrogen-bond interaction of the oxidized Y Z • radical, an immediate electron acceptor of the Mn 4 CaO 5 cluster, was clearly observed as a ∼100 μs phase before the electron-transfer phase with a time constant of ∼350 μs. This observation provides strong experimental evidence that rearrangement of the hydrogen-bond network around Y Z • , possibly due to the movement of a water molecule located near Y Z • to the Mn site, takes place before the electron transfer. The electron transfer was coupled with proton release, as revealed by a relatively high deuterium kinetic isotope effect of 1.9. This proton release, which decreases the redox potential of the Mn 4 CaO 5 cluster to facilitate electron transfer to Y Z • , was proposed to determine, as a rate-limiting step, the relatively slow electron-transfer rate of the S 2 → S 3 transition.

Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

PubMed

Fu, Glenn K; Wilhelmy, Julie; Stern, David; Fan, H Christina; Fodor, Stephen P A

2014-03-18

We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification.

Metabolomic Analysis of Key Central Carbon Metabolism Carboxylic Acids as Their 3-Nitrophenylhydrazones by UPLC/ESI-MS

PubMed Central

Han, Jun; Gagnon, Susannah; Eckle, Tobias; Borchers, Christoph H.

2014-01-01

Multiple hydroxy-, keto-, di-, and tri-carboxylic acids are among the cellular metabolites of central carbon metabolism (CCM). Sensitive and reliable analysis of these carboxylates is important for many biological and cell engineering studies. In this work, we examined 3-nitrophenylhydrazine as a derivatizing reagent and optimized the reaction conditions for the measurement of ten CCM related carboxylic compounds, including glycolate, lactate, malate, fumarate, succinate, citrate, isocitrate, pyruvate, oxaloacetate, and α-ketoglutarate as their 3-nitrophenylhydrazones using LC/MS with electrospray ionization. With the derivatization protocol which we have developed, and using negative-ion multiple reaction monitoring on a triple-quadrupole instrument, all of the carboxylates showed good linearity within a dynamic range of ca. 200 to more than 2000. The on-column limits of detection and quantitation were from high femtomoles to low picomoles. The analytical accuracies for eight of the ten analytes were determined to be between 89.5 to 114.8% (CV≤7.4%, n=6). Using a quadrupole time-of-flight instrument, the isotopic distribution patterns of these carboxylates, extracted from a 13C-labeled mouse heart, were successfully determined by UPLC/MS with full-mass detection, indicating the possible utility of this analytical method for metabolic flux analysis. In summary, this work demonstrates an efficient chemical derivatization LC/MS method for metabolomic analysis of these key CCM intermediates in a biological matrix. PMID:23580203

Towards rapid prototyped convective microfluidic DNA amplification platform

NASA Astrophysics Data System (ADS)

Ajit, Smrithi; Praveen, Hemanth Mithun; Puneeth, S. B.; Dave, Abhishek; Sesham, Bharat; Mohan, K. N.; Goel, Sanket

2017-02-01

Today, Polymerase Chain Reaction (PCR) based DNA amplification plays an indispensable role in the field of biomedical research. Its inherent ability to exponentially amplify sample DNA has proven useful for the identification of virulent pathogens like those causing Multiple Drug-Resistant Tuberculosis (MDR-TB). The intervention of Microfluidics technology has revolutionized the concept of PCR from being a laborious and time consuming process into one that is faster, easily portable and capable of being multifunctional. The Microfluidics based PCR outweighs its traditional counterpart in terms of flexibility of varying reaction rate, operation simplicity, need of a fraction of volume and capability of being integrated with other functional elements. The scope of the present work involves the development of a real-time continuous flow microfluidic device, fabricated by 3D printing-governed rapid prototyping method, eventually leading to an automated and robust platform to process multiple DNA samples for detection of MDRTB-associated mutations. The thermal gradient characteristic to the PCR process is produced using peltier units appropriate to the microfluidic environment fully monitored and controlled by a low cost controller driven by a Data Acquisition System. The process efficiency achieved in the microfluidic environment in terms of output per cycle is expected to be on par with the traditional PCR and capable of earning the additional advantages of being faster and minimizing the handling.

Determination of the molecular weight of poly(ethylene glycol) in biological samples by reversed-phase LC-MS with in-source fragmentation.

PubMed

Warrack, Bethanne M; Redding, Brian P; Chen, Guodong; Bolgar, Mark S

2013-05-01

PEGylation has been widely used to improve the biopharmaceutical properties of therapeutic proteins and peptides. Previous studies have used multiple analytical techniques to determine the fate of both the therapeutic molecule and unconjugated poly(ethylene glycol) (PEG) after drug administration. A straightforward strategy utilizing liquid chromatography-mass spectrometry (LC-MS) to characterize high-molecular weight PEG in biologic matrices without a need for complex sample preparation is presented. The method is capable of determining whether high-MW PEG is cleaved in vivo to lower-molecular weight PEG species. Reversed-phase chromatographic separation is used to take advantage of the retention principles of polymeric materials whereby elution order correlates with PEG molecular weight. In-source collision-induced dissociation (CID) combined with selected reaction monitoring (SRM) or selected ion monitoring (SIM) mass spectrometry (MS) is then used to monitor characteristic PEG fragment ions in biological samples. MS provides high sensitivity and specificity for PEG and the observed retention times in reversed-phase LC enable estimation of molecular weight. This method was successfully used to characterize PEG molecular weight in mouse serum samples. No change in molecular weight was observed for 48 h after dosing.

A sensitive LC-MS/MS method for the simultaneous determination of amoxicillin and ambroxol in human plasma with segmental monitoring.

PubMed

Dong, Xin; Ding, Li; Cao, Xiaomei; Jiang, Liyuan; Zhong, Shuisheng

2013-04-01

Amoxicillin (AMO) degrades in plasma at room temperature and readily undergoes hydrolysis by the plasma amidase. In this paper, a novel, rapid and sensitive LC-MS/MS method operated in segmental and multiple reaction monitoring has been developed for the simultaneous determination of amoxicillin and ambroxol in human plasma. The degradation of amoxicillin in plasma was well prevented by immediate addition of 20 μL glacial acetic acid to 200 μL aliquot of freshly collected plasma samples before storage at -80°C. The sensitivity of the method was improved with segmental monitoring of the analytes, and lower limits of quantitation of 0.5 ng/mL for ambroxol and 5 ng/mL for amoxicillin were obtained. The sensitivity of our method was five times better than those of the existing methods. Furthermore, the mass response saturation problem with amoxicillin was avoided by diluting the deproteinized plasma samples with water before injection into the LC-MS/MS system. The method was successfully employed in a pharmacokinetic study of the compound amoxicillin and ambroxol hydrochloride tablets. Copyright © 2012 John Wiley & Sons, Ltd.

Employing Magnetic Levitation to Monitor Reaction Kinetics and Measure Activation Energy

ERIC Educational Resources Information Center

Benz, Lauren; Cesafsky, Karen E.; Le, Tran; Park, Aileen; Malicky, David

2012-01-01

This article describes a simple and inexpensive undergraduate-level kinetics experiment that uses magnetic levitation to monitor the progress and determine the activation energy of a condensation reaction on a polymeric solid support. The method employs a cuvette filled with a paramagnetic solution positioned between two strong magnets. The…

Simultaneous quantification of multiple components in rat plasma by UPLC-MS/MS and pharmacokinetic study after oral administration of Huangqi decoction.

PubMed

Zeng, Jia-Kai; Li, Yuan-Yuan; Wang, Tian-Ming; Zhong, Jie; Wu, Jia-Sheng; Liu, Ping; Zhang, Hua; Ma, Yue-Ming

2018-05-01

A rapid, sensitive and accurate UPLC-MS/MS method was developed for the simultaneous quantification of components of Huangqi decoction (HQD), such as calycosin-7-O-β-d-glucoside, calycosin-glucuronide, liquiritin, formononetin-glucuronide, isoliquiritin, liquiritigenin, ononin, calycosin, isoliquiritigenin, formononetin, glycyrrhizic acid, astragaloside IV, cycloastragenol, and glycyrrhetinic acid, in rat plasma. After plasma samples were extracted by protein precipitation, chromatographic separation was performed with a C 18 column, using a gradient of methanol and 0.05% acetic acid containing 4mm ammonium acetate as the mobile phase. Multiple reaction monitoring scanning was performed to quantify the analytes, and the electrospray ion source polarity was switched between positive and negative modes in a single run of 10 min. Method validation showed that specificity, linearity, accuracy, precision, extraction recovery, matrix effect and stability for 14 components met the requirements for their quantitation in biological samples. The established method was successfully applied to the pharmacokinetic study of multiple components in rats after intragastric administration of HQD. The results clarified the pharmacokinetic characteristics of multiple components found in HQD. This research provides useful information for understanding the relation between the chemical components of HQD and their therapeutic effects. Copyright © 2017 John Wiley & Sons, Ltd.

[High-sensitive detection of multiple allergenic proteins in infant food with high-resolution mass spectrometry].

PubMed

Wu, Ci; Chen, Xi; Liu, Jianhui; Zhang, Xiaolin; Xue, Weifeng; Liang, Zhen; Liu, Mengyao; Cui, Yan; Huang, Daliang; Zhang, Lihua

2017-10-08

A novel method of the simultaneous detection of multiple kinds of allergenic proteins in infant food with parallel reaction monitoring (PRM) mode using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established. In this method, unique peptides with good stability and high sensibility were used to quantify the corresponding allergenic proteins. Furthermore, multiple kinds of allergenic proteins are inspected simultaneously with high sensitivity. In addition, such method was successfully used for the detection of multiple allergenic proteins in infant food. As for the sample preparation for infant food, compared with the traditional acetone precipitation strategy, the protein extraction efficiency and capacity of resisting disturbance are both higher with in-situ filter-aided sample pretreatment (i-FASP) method. All allergenic proteins gave a good linear response with the correlation coefficients ( R 2 ) ≥ 0.99, and the largest concentration range of the allergenic proteins could be four orders of magnitude, and the lowest detection limit was 0.028 mg/L, which was better than that reported in references. Finally, the method was conveniently used to detect the allergens from four imported infant food real samples. All the results demonstrate that this novel strategy is of great significance for providing a rapid and reliable analytical technique for allergen proteomics.

NMR reaction monitoring in flow synthesis

PubMed Central

Gomez, M Victoria

2017-01-01

Recent advances in the use of flow chemistry with in-line and on-line analysis by NMR are presented. The use of macro- and microreactors, coupled with standard and custom made NMR probes involving microcoils, incorporated into high resolution and benchtop NMR instruments is reviewed. Some recent selected applications have been collected, including synthetic applications, the determination of the kinetic and thermodynamic parameters and reaction optimization, even in single experiments and on the μL scale. Finally, software that allows automatic reaction monitoring and optimization is discussed. PMID:28326137

NMR reaction monitoring in flow synthesis.

PubMed

Gomez, M Victoria; de la Hoz, Antonio

2017-01-01

Recent advances in the use of flow chemistry with in-line and on-line analysis by NMR are presented. The use of macro- and microreactors, coupled with standard and custom made NMR probes involving microcoils, incorporated into high resolution and benchtop NMR instruments is reviewed. Some recent selected applications have been collected, including synthetic applications, the determination of the kinetic and thermodynamic parameters and reaction optimization, even in single experiments and on the μL scale. Finally, software that allows automatic reaction monitoring and optimization is discussed.

An Optical Biosensing Strategy Based on Selective Light Absorption and Wavelength Filtering from Chromogenic Reaction

PubMed Central

Chun, Hyeong Jin; Han, Yong Duk; Park, Yoo Min; Kim, Ka Ram; Lee, Seok Jae

2018-01-01

To overcome the time and space constraints in disease diagnosis via the biosensing approach, we developed a new signal-transducing strategy that can be applied to colorimetric optical biosensors. Our study is focused on implementation of a signal transduction technology that can directly translate the color intensity signals—that require complicated optical equipment for the analysis—into signals that can be easily counted with the naked eye. Based on the selective light absorption and wavelength-filtering principles, our new optical signaling transducer was built from a common computer monitor and a smartphone. In this signal transducer, the liquid crystal display (LCD) panel of the computer monitor served as a light source and a signal guide generator. In addition, the smartphone was used as an optical receiver and signal display. As a biorecognition layer, a transparent and soft material-based biosensing channel was employed generating blue output via a target-specific bienzymatic chromogenic reaction. Using graphics editor software, we displayed the optical signal guide patterns containing multiple polygons (a triangle, circle, pentagon, heptagon, and 3/4 circle, each associated with a specified color ratio) on the LCD monitor panel. During observation of signal guide patterns displayed on the LCD monitor panel using a smartphone camera via the target analyte-loaded biosensing channel as a color-filtering layer, the number of observed polygons changed according to the concentration of the target analyte via the spectral correlation between absorbance changes in a solution of the biosensing channel and color emission properties of each type of polygon. By simple counting of the changes in the number of polygons registered by the smartphone camera, we could efficiently measure the concentration of a target analyte in a sample without complicated and expensive optical instruments. In a demonstration test on glucose as a model analyte, we could easily measure the concentration of glucose in the range from 0 to 10 mM. PMID:29509682

An Optical Biosensing Strategy Based on Selective Light Absorption and Wavelength Filtering from Chromogenic Reaction.

PubMed

Chun, Hyeong Jin; Han, Yong Duk; Park, Yoo Min; Kim, Ka Ram; Lee, Seok Jae; Yoon, Hyun C

2018-03-06

To overcome the time and space constraints in disease diagnosis via the biosensing approach, we developed a new signal-transducing strategy that can be applied to colorimetric optical biosensors. Our study is focused on implementation of a signal transduction technology that can directly translate the color intensity signals-that require complicated optical equipment for the analysis-into signals that can be easily counted with the naked eye. Based on the selective light absorption and wavelength-filtering principles, our new optical signaling transducer was built from a common computer monitor and a smartphone. In this signal transducer, the liquid crystal display (LCD) panel of the computer monitor served as a light source and a signal guide generator. In addition, the smartphone was used as an optical receiver and signal display. As a biorecognition layer, a transparent and soft material-based biosensing channel was employed generating blue output via a target-specific bienzymatic chromogenic reaction. Using graphics editor software, we displayed the optical signal guide patterns containing multiple polygons (a triangle, circle, pentagon, heptagon, and 3/4 circle, each associated with a specified color ratio) on the LCD monitor panel. During observation of signal guide patterns displayed on the LCD monitor panel using a smartphone camera via the target analyte-loaded biosensing channel as a color-filtering layer, the number of observed polygons changed according to the concentration of the target analyte via the spectral correlation between absorbance changes in a solution of the biosensing channel and color emission properties of each type of polygon. By simple counting of the changes in the number of polygons registered by the smartphone camera, we could efficiently measure the concentration of a target analyte in a sample without complicated and expensive optical instruments. In a demonstration test on glucose as a model analyte, we could easily measure the concentration of glucose in the range from 0 to 10 mM.

A General LC-MS/MS Method for Monitoring Potential β-Lactam Contamination in Drugs and Drug-Manufacturing Surfaces.

PubMed

Qiu, Chen; Zhu, Hongbin; Ruzicka, Connie; Keire, David; Ye, Hongping

2018-05-15

Penicillins and some non-penicillin β-lactams may cause potentially life-threatening allergic reactions. Thus, possible cross contamination of β-lactams in food or drugs can put people at risk. Therefore, when there is a reasonable possibility that a non-penicillin product could be contaminated by penicillin, the drug products are tested for penicillin contamination. Here, a sensitive and rapid method for simultaneous determination of multiple β-lactam antibiotics using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated. Mass spectral acquisition was performed on a Q-Exactive HF mass spectrometer in positive ion mode with parallel reaction monitoring (PRM). The method was validated for seven β-lactam antibiotics including one or two from each class and a synthetic intermediate. The quantification precision and accuracy at 200 ppb were in the range of ± 1.84 to ± 4.56 and - 5.20 to 3.44%, respectively. The limit of detection (LOD) was 0.2 ppb, and the limit of quantitation (LOQ) was 2 ppb with a linear dynamic range (LDR) of 2-2000 ppb for all eight β-lactams. From various drug products, the recoveries of eight β-lactams at 200 and 2 ppb ranged from 93.8 ± 3.2 to 112.1 ± 4.2% and 89.7 ± 4.6 to 110.6 ± 1.9%, respectively. The application of the method for detecting cross contamination of trace β-lactams (0.2 ppb) and for monitoring facility surface cleaning was also investigated. This sensitive and fast method was fit-for-purpose for detecting and quantifying trace amount of β-lactam contamination, monitoring cross contamination in manufacturing processes, and determining potency for regulatory purposes and for quality control.

Rate coefficients for the reaction of formaldehyde with HO2 radicals from fluorescence spectroscopy of HOCH2OO radicals

NASA Astrophysics Data System (ADS)

Bunkan, Arne; Amédro, Damien; Crowley, John

2017-04-01

The reaction of formaldehyde with HO2 radicals constitutes a minor, but significant sink of formaldehyde in the troposphere as well as a possible interference in other formaldehyde photooxidation experiments. HCHO + HO2 ⇌ HOCH2OO (1) Due to the difficulty of simultaneously monitoring the reactant and product concentrations while preventing interfering secondary chemistry, there is a considerable uncertainty in the literature values for the reaction rate coefficients. We have used two photon, excited fragment spectroscopy (TPEFS), originally developed for monitoring HNO3 formation in kinetic experiments, to monitor the formation of the HOCH2OO radical. Dispersed and single wavelength fluorescence emission following the 193 nm photolysis of HOCH2OO have been recorded and analysed. Characterisation of the method is presented along with rate coefficients for the reaction of HCHO with HO2 radicals at tropospheric temperatures.

Effect of perception irregularity on chain-reaction crash in low visibility

NASA Astrophysics Data System (ADS)

Nagatani, Takashi

2015-06-01

We present the dynamic model of the chain-reaction crash to take into account the irregularity of the perception-reaction time. When a driver brakes according to taillights of the forward vehicle, the perception-reaction time varies from driver to driver. We study the effect of the perception irregularity on the chain-reaction crash (multiple-vehicle collision) in low-visibility condition. The first crash may induce more collisions. We investigate how the first collision induces the chain-reaction crash numerically. We derive, analytically, the transition points and the region maps for the chain-reaction crash in traffic flow of vehicles with irregular perception times. We clarify the effect of the perception irregularity on the multiple-vehicle collision.

A multiple index integrating different levels of organization.

PubMed

Cortes, Rui; Hughes, Samantha; Coimbra, Ana; Monteiro, Sandra; Pereira, Vítor; Lopes, Marisa; Pereira, Sandra; Pinto, Ana; Sampaio, Ana; Santos, Cátia; Carrola, João; de Jesus, Joaquim; Varandas, Simone

2016-10-01

Many methods in freshwater biomonitoring tend to be restricted to a few levels of biological organization, limiting the potential spectrum of measurable of cause-effect responses to different anthropogenic impacts. We combined distinct organisational levels, covering biological biomarkers (histopathological and biochemical reactions in liver and fish gills), community based bioindicators (fish guilds, invertebrate metrics/traits and chironomid pupal exuviae) and ecosystem functional indicators (decomposition rates) to assess ecological status at designated Water Framework Directive monitoring sites, covering a gradient of human impact across several rivers in northern Portugal. We used Random Forest to rank the variables that contributed more significantly to successfully predict the different classes of ecological status and also to provide specific cut levels to discriminate each WFD class based on reference condition. A total of 59 Biological Quality Elements and functional indicators were determined using this procedure and subsequently applied to develop the integrated Multiple Ecological Level Index (MELI Index), a potentially powerful bioassessment tool. Copyright © 2016 Elsevier Inc. All rights reserved.

Multiplex High-Throughput Targeted Proteomic Assay To Identify Induced Pluripotent Stem Cells.

PubMed

Baud, Anna; Wessely, Frank; Mazzacuva, Francesca; McCormick, James; Camuzeaux, Stephane; Heywood, Wendy E; Little, Daniel; Vowles, Jane; Tuefferd, Marianne; Mosaku, Olukunbi; Lako, Majlinda; Armstrong, Lyle; Webber, Caleb; Cader, M Zameel; Peeters, Pieter; Gissen, Paul; Cowley, Sally A; Mills, Kevin

2017-02-21

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research, only the best, competent clones should be used. The standard assays for pluripotency are based on genomic approaches, which take up to 1 week to perform and incur significant cost. Therefore, there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here, we describe a novel multiplexed, high-throughput, and sensitive peptide-based multiple reaction monitoring mass spectrometry assay, allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests.

Development and application of a quantitative multiplexed small GTPase activity assay using targeted proteomics.

PubMed

Zhang, Cheng-Cheng; Li, Ru; Jiang, Honghui; Lin, Shujun; Rogalski, Jason C; Liu, Kate; Kast, Juergen

2015-02-06

Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.

Method for preparing small volume reaction containers

DOEpatents

Retterer, Scott T.; Doktycz, Mitchel J.

2017-04-25

Engineered reaction containers that can be physically and chemically defined to control the flux of molecules of different sizes and charge are disclosed. Methods for constructing small volume reaction containers through a combination of etching and deposition are also disclosed. The methods allow for the fabrication of multiple devices that possess features on multiple length scales, specifically small volume containers with controlled porosity on the nanoscale.

Energy-loss cross sections for inclusive charge-exchange reactions at intermediate energies

NASA Technical Reports Server (NTRS)

Cucinotta, Francis A.; Townsend, Lawrence W.; Dubey, Rajendra R.

1993-01-01

Charge-exchange reactions for scattering to the continuum are considered in a high-energy multiple scattering model. Calculations for (p,n) and (He-3,H-3) reactions are made and compared with experimental results for C-12, O-16, and Al-27 targets. Coherent effects are shown to lead to an important role for inelastic multiple scattering terms when light projectiles are considered.

Accurate quantification of 5 German cockroach (GCr) allergens in complex extracts using multiple reaction monitoring mass spectrometry (MRM MS).

PubMed

Mindaye, S T; Spiric, J; David, N A; Rabin, R L; Slater, J E

2017-12-01

German cockroach (GCr) allergen extracts are complex and heterogeneous products, and methods to better assess their potency and composition are needed for adequate studies of their safety and efficacy. The objective of this study was to develop an assay based on liquid chromatography and multiple reaction monitoring mass spectrometry (LC-MRM MS) for rapid, accurate, and reproducible quantification of 5 allergens (Bla g 1, Bla g 2, Bla g 3, Bla g 4, and Bla g 5) in crude GCr allergen extracts. We first established a comprehensive peptide library of allergens from various commercial extracts as well as recombinant allergens. Peptide mapping was performed using high-resolution MS, and the peptide library was then used to identify prototypic and quantotypic peptides to proceed with MRM method development. Assay development included a systematic optimization of digestion conditions (buffer, digestion time, and trypsin concentration), chromatographic separation, and MS parameters. Robustness and suitability were assessed following ICH (Q2 [R1]) guidelines. The method is precise (RSD < 10%), linear over a wide range (r > 0.99, 0.01-1384 fmol/μL), and sensitive (LLOD and LLOQ <1 fmol/μL). Having established the parameters for LC-MRM MS, we quantified allergens from various commercial GCr extracts and showed considerable variability that may impact clinical efficacy. Our data demonstrate that the LC-MRM MS method is valuable for absolute quantification of allergens in GCr extracts and likely has broader applicability to other complex allergen extracts. Definitive quantification provides a new standard for labelling of allergen extracts, which will inform patient care, enable personalized therapy, and enhance the efficacy of immunotherapy for environmental and food allergies. © 2017 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Multi-targeted interference-free determination of ten β-blockers in human urine and plasma samples by alternating trilinear decomposition algorithm-assisted liquid chromatography-mass spectrometry in full scan mode: comparison with multiple reaction monitoring.

PubMed

Gu, Hui-Wen; Wu, Hai-Long; Yin, Xiao-Li; Li, Yong; Liu, Ya-Juan; Xia, Hui; Zhang, Shu-Rong; Jin, Yi-Feng; Sun, Xiao-Dong; Yu, Ru-Qin; Yang, Peng-Yuan; Lu, Hao-Jie

2014-10-27

β-blockers are the first-line therapeutic agents for treating cardiovascular diseases and also a class of prohibited substances in athletic competitions. In this work, a smart strategy that combines three-way liquid chromatography-mass spectrometry (LC-MS) data with second-order calibration method based on alternating trilinear decomposition (ATLD) algorithm was developed for simultaneous determination of ten β-blockers in human urine and plasma samples. This flexible strategy proved to be a useful tool to solve the problems of overlapped peaks and uncalibrated interferences encountered in quantitative LC-MS, and made the multi-targeted interference-free qualitative and quantitative analysis of β-blockers in complex matrices possible. The limits of detection were in the range of 2.0×10(-5)-6.2×10(-3) μg mL(-1), and the average recoveries were between 90 and 110% with standard deviations and average relative prediction errors less than 10%, indicating that the strategy could provide satisfactory prediction results for ten β-blockers in human urine and plasma samples only using liquid chromatography hyphenated single-quadrupole mass spectrometer in full scan mode. To further confirm the feasibility and reliability of the proposed method, the same batch samples were analyzed by multiple reaction monitoring (MRM) method. T-test demonstrated that there are no significant differences between the prediction results of the two methods. Considering the advantages of fast, low-cost, high sensitivity, and no need of complicated chromatographic and tandem mass spectrometric conditions optimization, the proposed strategy is expected to be extended as an attractive alternative method to quantify analyte(s) of interest in complex systems such as cells, biological fluids, food, environment, pharmaceuticals and other complex samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Multiple reaction monitoring with multistage fragmentation (MRM3) detection enhances selectivity for LC-MS/MS analysis of plasma free metanephrines.

PubMed

Wright, Michael J; Thomas, Rebecca L; Stanford, Phoebe E; Horvath, Andrea R

2015-03-01

LC-MS/MS with multiple reaction monitoring (MRM) is a powerful tool for quantifying target analytes in complex matrices. However, the technique lacks selectivity when plasma free metanephrines are measured. We propose the use of multistage fragmentation (MRM(3)) to improve the analytical selectivity of plasma free metanephrine measurement. Metanephrines were extracted from plasma with weak cation exchange solid-phase extraction before separation by hydrophilic interaction liquid chromatography. We quantified normetanephrine and metanephrine by either MRM or MRM(3) transitions m/z 166→134→79 and m/z 180→149→121, respectively. Over a 6-month period, approximately 1% (n = 21) of patient samples showed uncharacterized coeluting substances that interfered with the routine assay, resulting in an inability to report results. Quantification with MRM(3) removed these interferences and enabled measurement of the target compounds. For patient samples unaffected by interferences, Deming regression analysis demonstrated a correlation between MRM(3) and MRM methods of y = 1.00x - 0.00 nmol/L for normetanephrine and y = 0.99x + 0.03 nmol/L for metanephrine. Between the MRM(3) method and the median of all LC-MS/MS laboratories enrolled in a quality assurance program, the correlations were y = 0.97x + 0.03 nmol/L for normetanephrine and y = 1.03x - 0.04 nmol/L for metanephrine. Imprecision for the MRM(3) method was 6.2%-7.0% for normetanephrine and 6.1%-9.9% for metanephrine (n = 10). The lower limits of quantification for the MRM(3) method were 0.20 nmol/L for normetanephrine and 0.16 nmol/L for metanephrine. The use of MRM(3) technology improves the analytical selectivity of plasma free metanephrine quantification by LC-MS/MS while demonstrating sufficient analytical sensitivity and imprecision. © 2014 American Association for Clinical Chemistry.

A retrospective study on sequential desensitization-rechallenge for antituberculosis drug allergy

PubMed Central

Chia, Faith Li-Ann; Tan, Sze-Chin; Tan, Teck-Choon; Leong, Khai-Pang; Tan, Justina Wei-Lyn; Tang, Chwee-Ying; Hou, Jin-Feng; Chan, Grace Yin-Lai; Chng, Hiok-Hee

2014-01-01

Background Antituberculosis (anti-TB) drug allergy often involves multiple concurrently administered drugs which subsequently need to be reinitiated as no better alternatives exist. Objective To describe the results of tailored sequential desensitization-rechallenge (D-R) for anti-TB drug allergy. Methods Consecutive patients who had undergone D-R to anti-TB drugs between 1 September 1997 and 31 January 2012 were recruited. Following resolution of the acute reaction, anti-TB drug was restarted at 1:6,000 to 1:3 of the final daily dose (FDD), with gradual single or multiple step daily dose escalation to the FDD. Subsequent drugs were sequentially added ≥3 days later when the preceding drug was tolerated. Full blood count and liver function tests were monitored prior to addition of each new drug. Results There were 11 patients of whom 10 were male, predominantly Chinese (8 patients). Regimens comprised at least 3 drugs: isoniazid (INH), rifampicin (RIF), ethambutol (EMB), pyrazinamide (PZA), or streptomycin. All patients had nonimmediate reactions, with cutaneous eruptions, where maculopapular exanthema (MPE) was the most common (8 patients). Drug-induced hypersensitivity syndrome (DIHS) occurred in 6 patients, and Stevens Johnson syndrome (SJS) in 2 patients. D-R to INH was successful in 7/9 patients (77.8%) and to RIF/EMB/PZA/streptomycin in all. Of the 2 patients who failed INH D-R, 1 developed fever and MPE on day 3, the other MPE on day 8. D-R with INH and RIF respectively was successful in 2 patients with SJS. Among DIHS patients, 1 failed D-R with INH (fever and MPE on day 3). There were 23/25 (92%) successful D-R among the 11 patients. All patients completed TB treatment of ≥5 months' duration with no cases of drug-resistant TB. Conclusion Tailored sequential TB drug D-R is successful where no better alternative therapies are available, with careful dose escalation and close monitoring, and after a careful risk-benefit assessment. PMID:25097851

Analysis of trans-2,6-difluoro-4'-(N,N-dimethylamino)stilbene (DFS) in biological samples by liquid chromatography-tandem mass spectrometry: metabolite identification and pharmacokinetics.

PubMed

Yeo, Samuel Chao Ming; Sviripa, Vitaliy M; Huang, Meng; Kril, Liliia; Watt, David S; Liu, Chunming; Lin, Hai-Shu

2015-09-01

The metabolism of a promising antineoplastic agent, trans-2,6-difluoro-4'-(N,N-dimethylamino)stilbene (DFS), was studied in mouse, rat, and human liver microsomes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the multiple reaction monitoring-information-dependent acquisition-enhanced product ion scan (MRM-IDA-EPI) method. Ten putative metabolites were identified and the structures of four metabolites were confirmed using authentic standards. Since trans-2,6-difluoro-4'-(N-methylamino)stilbene (DMDFS, M1) was present in all species as metabolite and displayed in vitro growth inhibition superior to DFS, its pharmacokinetic profiles were examined in Sprague-Dawley rats using DFS as a comparator. A reliable LC-MS/MS multiple reaction monitoring (MRM) method was subsequently developed and validated for the simultaneous quantification of both DFS and DMDFS in rat plasma for this purpose. Upon intravenous administration (4 mg/kg), DFS had a moderate clearance (Cl = 62.7 ± 23.2 mL/min/kg), terminal elimination half-life (t 1/2 λZ  = 299 ± 73 min), and mean transit time (MTT = 123 ± 14 min) with demethylation metabolism accounting for about 10 % of its total clearance. DMDFS possessed an intravenous pharmacokinetic profile similar to DFS. During oral dosing (10 mg/kg) where both DFS and DMDFS were absorbed rapidly, the oral bioavailability of DFS was approximately 2-fold greater than that of DMDFS (DFS: F = 42.1 ± 12.8 %; DMDFS: F = 18.7 ± 3.9 %). Interestingly, the DMDFS exposure after oral dosing of DFS (10 mg/kg) was comparable to that after oral administration of DMDFS (10 mg/kg) alone. As DFS displayed potent anticancer activities and excellent pharmacokinetic profiles, it appears to be a favorable candidate for further pharmaceutical development.

Unexpected peaks in tandem mass spectra due to reaction of product ions with residual water in mass spectrometer collision cells.

PubMed

Neta, Pedatsur; Farahani, Mahnaz; Simón-Manso, Yamil; Liang, Yuxue; Yang, Xiaoyu; Stein, Stephen E

2014-12-15

Certain product ions in electrospray ionization tandem mass spectrometry are found to react with residual water in the collision cell. This reaction often leads to the formation of ions that cannot be formed directly from the precursor ions, and this complicates the mass spectra and may distort MRM (multiple reaction monitoring) results. Various drugs, pesticides, metabolites, and other compounds were dissolved in acetonitrile/water/formic acid and studied by electrospray ionization mass spectrometry to record their MS(2) and MS(n) spectra in several mass spectrometers (QqQ, QTOF, IT, and Orbitrap HCD). Certain product ions were found to react with residual water in collision cells. The reaction was confirmed by MS(n) studies and the rate of reaction was determined in the IT instrument using zero collision energy and variable activation times. Examples of product ions reacting with water include phenyl and certain substituted phenyl cations, benzoyl-type cations formed from protonated folic acid and similar compounds by loss of the glutamate moiety, product ions formed from protonated cyclic siloxanes by loss of methane, product ions formed from organic phosphates, and certain negative ions. The reactions of product ions with residual water varied greatly in their rate constant and in the extent of reaction (due to isomerization). Various types of product ions react with residual water in mass spectrometer collision cells. As a result, tandem mass spectra may contain unexplained peaks and MRM results may be distorted by the occurrence of such reactions. These often unavoidable reactions must be taken into account when annotating peaks in tandem mass spectra and when interpreting MRM results. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.

Exploration of kinetic and multiple-ion-fluids effects in D3He and T3He gas-filled ICF implosions using multiple nuclear reaction histories

NASA Astrophysics Data System (ADS)

Sio, Hong; Rinderknecht, Hans; Rosenberg, Michael; Zylstra, Alex; Séguin, Fredrick; Gatu Johnson, Maria; Li, Chikang; Petrasso, Richard; Hoffman, Nelson; Kagan, Krigory; Molvig, Kim; Amendt, Peter; Bellei, Claudio; Wilks, Scott; Stoeckl, Christian; Glebov, Vladimir; Betti, Riccardo; Sangster, Thomas; Katz, Joseph

2014-10-01

To explore kinetic and multi-ion-fluid effects in D3He and T3He gas-filled shock-driven implosions, multiple nuclear reaction histories were measured using the upgraded Particle Temporal Diagnostic (PTD) on OMEGA. For D3He gas-filled implosions, the relative timing of the DD and D3He reaction histories were measured with 20 ps precision. For T3He gas-filled implosions (with 1-2% deuterium), the relative timing of the DT and D3He reaction histories were measured with 10 ps precision. The observed differences between the reaction histories on these two OMEGA experiments are contrasted to 1-D single-ion hydro simulations for different gas-fill pressure and gas mixture. This work is supported in part by the U.S. DOE, LLNL, LLE, and NNSA SSGF.

MALDI MS-based Composition Analysis of the Polymerization Reaction of Toluene Diisocyanate (TDI) and Ethylene Glycol (EG).

PubMed

Ahn, Yeong Hee; Lee, Yeon Jung; Kim, Sung Ho

2015-01-01

This study describes an MS-based analysis method for monitoring changes in polymer composition during the polyaddition polymerization reaction of toluene diisocyanate (TDI) and ethylene glycol (EG). The polymerization was monitored as a function of reaction time using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). The resulting series of polymer adducts terminated with various end-functional groups were precisely identified and the relative compositions of those series were estimated. A new MALDI MS data interpretation method was developed, consisting of a peak-resolving algorithm for overlapping peaks in MALDI MS spectra, a retrosynthetic analysis for the generation of reduced unit mass peaks, and a Gaussian fit-based selection of the most prominent polymer series among the reconstructed unit mass peaks. This method of data interpretation avoids errors originating from side reactions due to the presence of trace water in the reaction mixture or MALDI analysis. Quantitative changes in the relative compositions of the resulting polymer products were monitored as a function of reaction time. These results demonstrate that the mass data interpretation method described herein can be a powerful tool for estimating quantitative changes in the compositions of polymer products arising during a polymerization reaction.

Continuous monitoring of enzymatic activity within native electrophoresis gels: Application to mitochondrial oxidative phosphorylation complexes

PubMed Central

Covian, Raul; Chess, David; Balaban, Robert S.

2012-01-01

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction media recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase where catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. PMID:22975200

Continuous monitoring of enzymatic activity within native electrophoresis gels: application to mitochondrial oxidative phosphorylation complexes.

PubMed

Covian, Raul; Chess, David; Balaban, Robert S

2012-12-01

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light-scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze the enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction medium recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high-resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase in which catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed. Published by Elsevier Inc.

Label-free monitoring of interaction between DNA and oxaliplatin in aqueous solution by terahertz spectroscopy

NASA Astrophysics Data System (ADS)

Wu, Xiaojun; E, Yiwen; Xu, Xinlong; Wang, Li

2012-07-01

We demonstrated the feasibility of applying terahertz time-domain spectroscopy (THz-TDS) to monitor the molecular reactions in aqueous solutions of anticancer drug oxaliplatin with λ-DNA and macrophages DNA. The reaction time dependent refractive index and absorption coefficient were extracted and analyzed. The reaction half-decaying time of about 4.0 h for λ-DNA and 12.9 h for M-DNA was established. The results suggest that the THz-TDS detection could be an effective label-free technique to sense the molecular reaction in aqueous solutions and could be very useful in biology, medicine, and pharmacy industry.

An ultrahigh-performance liquid chromatography method with electrospray ionization tandem mass spectrometry for simultaneous quantification of five phytohormones in medicinal plant Glycyrrhiza uralensis under abscisic acid stress.

PubMed

Xiang, Yu; Song, Xiaona; Qiao, Jing; Zang, Yimei; Li, Yanpeng; Liu, Yong; Liu, Chunsheng

2015-07-01

An efficient simplified method was developed to determine multiple classes of phytohormones simultaneously in the medicinal plant Glycyrrhiza uralensis. Ultrahigh-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) with multiple reaction monitoring (MRM) in negative mode was used for quantification. The five studied phytohormones are gibberellic acid (GA3), abscisic acid (ABA), jasmonic acid (JA), indole-3-acetic acid, and salicylic acid (SA). Only 100 mg of fresh leaves was needed, with one purification step based on C18 solid-phase extraction. Cinnamic acid was chosen as the internal standard instead of isotope-labeled internal standards. Under the optimized conditions, the five phytohormones with internal standard were separated within 4 min, with good linearities and high sensitivity. The validated method was applied to monitor the spatial and temporal changes of the five phytohormones in G. uralensis under ABA stress. The levels of GA3, ABA, JA, and SA in leaves of G. uralensis were increased at different times and with different tendencies in the reported stress mode. These changes in phytohormone levels are discussed in the context of a possible feedback regulation mechanism. Understanding this mechanism will provide a good chance of revealing the mutual interplay between different biosynthetic routes, which could further help elucidate the mechanisms of effective composition accumulation in medicinal plants.

Screening and identification of steroidal saponins from Anemarrhena asphodeloides employing UPLC tandem triple quadrupole linear ion trap mass spectrometry.

PubMed

Xia, Yong-Gang; Guo, Xin-Dong; Liang, Jun; Yang, Bing-You; Kuang, Hai-Xue

2017-09-01

This study presents a practical and valid strategy for the screening and structural characterization of Anemarrhena asphodeloides Bge steroidal saponins (SSs) using ultra-high performance liquid chromatography coupled with triple quadrupole linear ion trap mass spectrometry. The whole analytical protocols integrate four-step procedures in the positive mode: (1) rational deduction of mass fragmentation pathways of A. asphodeloides SSs; (2) untargeted screening of potential A. asphodeloides SSs by multiple-ion monitoring-information-dependent-acquiring-enhanced product ion (MIM-IDA-EPI) scan through reverse phase liquid chromatography; (3) comprehensive construction of an ammoniated precursor ion database by combining untargeted MIM-IDA-EPI scans and data literature; and (4) structural interpretation of targeted A. asphodeloides SSs using MIM-IDA-EPI and multiple reaction monitoring (MRM)-IDA-EPI with an energy-resolved technique. The protocols were used to analyze SSs in A. asphodeloides; of the 87 detected SSs that were unambiguously characterized or tentatively identified, 19 compounds were the first to be reported from A. asphodeloides and 13 ones were characterized as potential new compounds. Accuracy of the analytical procedure was demonstrated by structural identification of three SSs by NMR spectroscopy. The proposed schemes hold an excellent promise in the structural prediction and interpretation of complex SSs from plant medicines by mass spectrometry. Copyright © 2017 Elsevier Inc. All rights reserved.

Population Structure of Invasive Streptococcus pneumoniae in the Netherlands in the Pre-Vaccination Era Assessed by MLVA and Capsular Sequence Typing

PubMed Central

Elberse, Karin E. M.; van de Pol, Ingrid; Witteveen, Sandra; van der Heide, Han G. J.; Schot, Corrie S.; van Dijk, Anita; van der Ende, Arie; Schouls, Leo M.

2011-01-01

The introduction of nationwide pneumococcal vaccination may lead to serotype replacement and the emergence of new variants that have expanded their genetic repertoire through recombination. To monitor alterations in the pneumococcal population structure, we have developed and utilized Capsular Sequence Typing (CST) in addition to Multiple-Locus Variable number tandem repeat Analysis (MLVA). To assess the serotype of each isolate CST was used. Based on the determination of the partial sequence of the capsular wzh gene, this method assigns a capsular type of an isolate within a single PCR reaction using multiple primersets. The genetic background of pneumococcal isolates was assessed by MLVA. MLVA and CST were used to create a snapshot of the Dutch pneumococcal population causing invasive disease before the introduction of the 7-valent pneumococcal conjugate vaccine in the Netherlands in 2006. A total of 1154 clinical isolates collected and serotyped by the Netherlands Reference Laboratory for Bacterial Meningitis were included in the snapshot. The CST was successful in discriminating most serotypes present in our collection. MLVA demonstrated that isolates belonging to some serotypes had a relatively high genetic diversity whilst other serotypes had a very homogeneous genetic background. MLVA and CST appear to be valuable tools to determine the population structure of pneumococcal isolates and are useful in monitoring the effects of pneumococcal vaccination. PMID:21637810

Investigation of avian influenza infection in wild birds in Ismailia and Damietta cities, Egypt

PubMed Central

Fadel, Hanaa Mohamed; Afifi, Rabab

2017-01-01

Aim: This study was carried out to monitor avian influenza (AI) infection in wild birds in Egypt. Materials and Methods: A total of 135 wild birds were examined for the presence of H5, H7, and H9 hemagglutination inhibition antibodies. Organs and swab samples of 75 birds were screened by multiplex real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) to detect AI subtypes H5, H7, and H9 matrix genes. Results: The highest seropositive result was recorded in cattle egrets (90.9%) followed by crows (88.6%), semi-captive pigeons (44.8%), and moorhens (39.1%). In cattle egrets, semi-captive pigeons and moorhens, H5 antibodies predominated. In crows, H9 antibodies predominated. Multiple infections with two or three virus subtypes were highest in crows (6/39, 15.4%) followed by cattle egrets (3/30, 10%) and moorhens’ (1/9, 11.1%) positive samples. Multiplex RRT-PCR results revealed two positive samples in cattle egrets and moorhens. Conclusion: The results indicated high seropositive rates against AI virus subtypes H5 and H9 in the examined wild birds. Multiple infections with more than one AI virus (AIV) subtypes were detected in some birds. This requires a collaboration of efforts to monitor AIV infection in wild birds and implement suitable early intervention measures. PMID:28717324

Identifying Understudied Nuclear Reactions by Text-mining the EXFOR Experimental Nuclear Reaction Library

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hirdt, J.A.; Brown, D.A., E-mail: dbrown@bnl.gov

The EXFOR library contains the largest collection of experimental nuclear reaction data available as well as the data's bibliographic information and experimental details. We text-mined the REACTION and MONITOR fields of the ENTRYs in the EXFOR library in order to identify understudied reactions and quantities. Using the results of the text-mining, we created an undirected graph from the EXFOR datasets with each graph node representing a single reaction and quantity and graph links representing the various types of connections between these reactions and quantities. This graph is an abstract representation of the connections in EXFOR, similar to graphs of socialmore » networks, authorship networks, etc. We use various graph theoretical tools to identify important yet understudied reactions and quantities in EXFOR. Although we identified a few cross sections relevant for shielding applications and isotope production, mostly we identified charged particle fluence monitor cross sections. As a side effect of this work, we learn that our abstract graph is typical of other real-world graphs.« less

Identifying Understudied Nuclear Reactions by Text-mining the EXFOR Experimental Nuclear Reaction Library

NASA Astrophysics Data System (ADS)

Hirdt, J. A.; Brown, D. A.

2016-01-01

The EXFOR library contains the largest collection of experimental nuclear reaction data available as well as the data's bibliographic information and experimental details. We text-mined the REACTION and MONITOR fields of the ENTRYs in the EXFOR library in order to identify understudied reactions and quantities. Using the results of the text-mining, we created an undirected graph from the EXFOR datasets with each graph node representing a single reaction and quantity and graph links representing the various types of connections between these reactions and quantities. This graph is an abstract representation of the connections in EXFOR, similar to graphs of social networks, authorship networks, etc. We use various graph theoretical tools to identify important yet understudied reactions and quantities in EXFOR. Although we identified a few cross sections relevant for shielding applications and isotope production, mostly we identified charged particle fluence monitor cross sections. As a side effect of this work, we learn that our abstract graph is typical of other real-world graphs.

{sup 40}Ca+{sup 40}Ca reaction at {ital E}{sub lab}=35 MeV/nucleon: Filters and signatures to distinguish nearly central from peripheral collisions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pawl /owski, P.; Brzychczyk, J.; Benchekrovn, D.

1996-07-01

Multifragment emission of charged particles from the {sup 40}Ca + {sup 40}Ca reaction at 35 MeV/nucleon has been investigated. Multiplicity as well as event shape filters were used to distinguish nearly central from peripheral collisions. A correlation between higher multiplicities and random events from two reactions occurring in one beam burst is discussed. Signatures of different reaction scenarios are investigated using simulation techniques. {copyright} {ital 1996 The American Physical Society.}

Reaction schemes visualized in network form: the syntheses of strychnine as an example.

PubMed

Proudfoot, John R

2013-05-24

Representation of synthesis sequences in a network form provides an effective method for the comparison of multiple reaction schemes and an opportunity to emphasize features such as reaction scale that are often relegated to experimental sections. An example of data formatting that allows construction of network maps in Cytoscape is presented, along with maps that illustrate the comparison of multiple reaction sequences, comparison of scaffold changes within sequences, and consolidation to highlight common key intermediates used across sequences. The 17 different synthetic routes reported for strychnine are used as an example basis set. The reaction maps presented required a significant data extraction and curation, and a standardized tabular format for reporting reaction information, if applied in a consistent way, could allow the automated combination of reaction information across different sources.

[Necessity of applying pharmacovigilance in post-marketing safety monitoring of traditional Chinese medicine injections].

PubMed

Wang, Hai-Nan; Chen, Wen; Fu, Zheng; Du, Wen-min; He, Jia

2008-03-01

Traditional Chinese medicine (TCM) injection has become one of the hotspots in the new TCM research and development. The serious adverse drug reactions happened in clinical have arosed attention widely in the whole society. It's very urgent to monitor the post-marketing safety of TCM injections. This paper elucidated the pharmacovigilance's necessity in the post-marketing safety monitoring of TCM injections, basing on the reason of safety problem of TCM injections and the future developing trend of adverse drug reaction monitoring. Also, this paper introduced the rapid signal detection method of spontaneous reporting system database by data mining technology.

Multiple Reaction Equilibria--With Pencil and Paper: A Class Problem on Coal Methanation.

ERIC Educational Resources Information Center

Helfferich, Friedrich G.

1989-01-01

Points out a different and much simpler approach for the study of equilibria of multiple and heterogeneous chemical reactions. A simulation on coal methanation is used to teach the technique. An example and the methodology used are provided. (MVL)

Desktop NMR spectroscopy for real-time monitoring of an acetalization reaction in comparison with gas chromatography and NMR at 9.4 T.

PubMed

Singh, Kawarpal; Danieli, Ernesto; Blümich, Bernhard

2017-12-01

Monitoring of chemical reactions in real-time is in demand for process control. Different methods such as gas chromatography (GC), mass spectroscopy, infrared spectroscopy, and nuclear magnetic resonance (NMR) are used for that purpose. The current state-of-the-art compact NMR systems provide a useful method to employ with various reaction conditions for studying chemical reactions inside the fume hood at the chemical workplace. In the present study, an acetalization reaction was investigated with compact NMR spectroscopy in real-time. Acetalization is used for multistep synthesis of the variety of organic compounds to protect particular chemical groups. A compact 1 T NMR spectrometer with a permanent magnet was employed to monitor the acid catalyzed acetalization of the p-nitrobenzaldehyde with ethylene glycol. The concentrations of both reactant and product were followed by peak integrals in single-scan 1 H NMR spectra as a function of time. The reaction conditions were varied in terms of temperature, agitation speed, catalyst loading, and feed concentrations in order to determine the activation energy with the help of a pseudo-homogeneous kinetic model. For low molar ratios of aldehyde and glycol, the equilibrium conversions were lower than for the stoichiometric ratio. Increasing catalyst concentration leads to faster conversion. The data obtained with low-field NMR spectroscopy were compared with data from GC and NMR spectroscopy at 9.4 T acquired in batch mode by extracting samples at regular time intervals. The reaction kinetics followed by either method agreed well. The activation energies for forward and backward reactions were determined by real-time monitoring with compact NMR at 1 T were 48 ± 5 and 60 ± 4 kJ/mol, respectively. The activation energies obtained with gas chromatography for forward and backward reactions were 48 ± 4 and 51 ± 4 kJ/mol. The equilibrium constant decreases with increasing temperature as expected for an exothermic reaction. The impact of dense sampling with online NMR and sparse sampling with GC was observed on the kinetic outcome using the same kinetic model. Graphical abstract Acetalization reaction kinetics were monitored with real-time desktop NMR spectroscopy at 1 T. Each data point was obtained at regular intervals with a single shot in 15 s. The kinetics was compared with sparsely sampled data obtained with GC and NMR at 9.4 T.

Pyrosequencing for Microbial Identification and Characterization

PubMed Central

Cummings, Patrick J.; Ahmed, Ray; Durocher, Jeffrey A.; Jessen, Adam; Vardi, Tamar; Obom, Kristina M.

2013-01-01

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns. PMID:23995536

Measurement of the 7Li(γ,t)4He reaction between 4 and 11 MeV

NASA Astrophysics Data System (ADS)

Pain, Steven; Matei, Catalin; Munch, Michael; Brune, Carl; Febbraro, Michael; Karwowski, Hugon; Walter, David; P-10-16 Experiment Collaboration

2017-09-01

The discrepancy in the primordial 7Li abundance, as derived from stellar observations and nucleosynthesis calculations at WMAP baryonic density, is sensitive to alpha capture rates on 3He and 3H. The 3He(α, γ)7Be reaction has been well studied over a wide range of energies, but for 3H(α, γ)7Li discrepancies exist in measurements below ECM = 1 MeV, and limited data above 1.2 MeV do not sufficiently constrain the contribution from higher-lying resonances at astrophysical energies. To contribute to the understanding of this process we have measured cross sections and angular distributions for the time-reversed 7Li(γ, α)3H reaction. The measurement was performed at the HIGS facility at the Triangle Universities Nuclear Laboratory (TUNL) using quasi-monoenergetic ( 3 % resolution) photon energies between 4 and 11 MeV. Tritons and alpha particles were detected in silicon detectors of SIDAR surrounding the 7Li target, and the beam intensity was monitored using multiple techniques. Details of the measurement, including the challenges of charged-particle measurements with gamma-ray beams, and preliminary results will be presented. This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Nuclear Physics.

Pyrosequencing for microbial identification and characterization.

PubMed

Cummings, Patrick J; Ahmed, Ray; Durocher, Jeffrey A; Jessen, Adam; Vardi, Tamar; Obom, Kristina M

2013-08-22

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Comparison of Modeling Methods to Determine Liver-to-blood Inocula and Parasite Multiplication Rates During Controlled Human Malaria Infection

PubMed Central

Douglas, Alexander D.; Edwards, Nick J.; Duncan, Christopher J. A.; Thompson, Fiona M.; Sheehy, Susanne H.; O'Hara, Geraldine A.; Anagnostou, Nicholas; Walther, Michael; Webster, Daniel P.; Dunachie, Susanna J.; Porter, David W.; Andrews, Laura; Gilbert, Sarah C.; Draper, Simon J.; Hill, Adrian V. S.; Bejon, Philip

2013-01-01

Controlled human malaria infection is used to measure efficacy of candidate malaria vaccines before field studies are undertaken. Mathematical modeling using data from quantitative polymerase chain reaction (qPCR) parasitemia monitoring can discriminate between vaccine effects on the parasite's liver and blood stages. Uncertainty regarding the most appropriate modeling method hinders interpretation of such trials. We used qPCR data from 267 Plasmodium falciparum infections to compare linear, sine-wave, and normal-cumulative-density-function models. We find that the parameters estimated by these models are closely correlated, and their predictive accuracy for omitted data points was similar. We propose that future studies include the linear model. PMID:23570846

Scout-MRM: Multiplexed Targeted Mass Spectrometry-Based Assay without Retention Time Scheduling Exemplified by Dickeya dadantii Proteomic Analysis during Plant Infection.

PubMed

Rougemont, Blandine; Bontemps Gallo, Sébastien; Ayciriex, Sophie; Carrière, Romain; Hondermarck, Hubert; Lacroix, Jean Marie; Le Blanc, J C Yves; Lemoine, Jérôme

2017-02-07

Targeted mass spectrometry of a surrogate peptide panel is a powerful method to study the dynamics of protein networks, but chromatographic time scheduling remains a major limitation for dissemination and implementation of robust and large multiplexed assays. We unveil a Multiple Reaction Monitoring method (Scout-MRM) where the use of spiked scout peptides triggers complex transition lists, regardless of the retention time of targeted surrogate peptides. The interest of Scout-MRM method regarding the retention time independency, multiplexing capability, reproducibility, and putative interest in facilitating method transfer was illustrated by a 782-peptide-plex relative assay targeting 445 proteins of the phytopathogen Dickeya dadantii during plant infection.

EIMS Fragmentation Pathways and MRM Quantification of 7α/β-Hydroxy-Dehydroabietic Acid TMS Derivatives

NASA Astrophysics Data System (ADS)

Rontani, Jean-François; Aubert, Claude; Belt, Simon T.

2015-09-01

EI mass fragmentation pathways of TMS derivatives οf 7α/β-hydroxy-dehydroabietic acids resulting from NaBH4-reduction of oxidation products of dehydroabietic acid (a component of conifers) were investigated and deduced by a combination of (1) low energy CID-GC-MS/MS, (2) deuterium labeling, (3) different derivatization methods, and (4) GC-QTOF accurate mass measurements. Having identified the main fragmentation pathways, the TMS-derivatized 7α/β-hydroxy-dehydroabietic acids could be quantified in multiple reaction monitoring (MRM) mode in sea ice and sediment samples collected from the Arctic. These newly characterized transformation products of dehydroabietic acid constitute potential tracers of biotic and abiotic degradation of terrestrial higher plants in the environment.

Glu-Phe from onion (Allium Cepa L.) attenuates lipogenesis in hepatocytes.

PubMed

Lee, Yu Geon; Cho, Jeong-Yong; Hwang, Eom Ji; Jeon, Tae-Il; Moon, Jae-Hak

2017-07-01

A Glu-Phe (EF) was isolated from onion (Allium cepa L. cv. Sunpower). The chemical structure of EF was determined by nuclear magnetic resonance and electrospray ionization-mass (ESI-MS) spectroscopy. We showed that EF reduced lipid accumulation in mouse hepatocytes by inhibiting the expression of sterol regulatory element-binding protein-1c (SREBP-1c) and its lipogenic target genes. We also found that AMP-activated protein kinase (AMPK) was required for the inhibitory effect of EF on lipid accumulation in mouse hepatocytes. Furthermore, EF was qualified in nine onion cultivars by selective multiple reaction-monitoring detection of liquid chromatography-ESI-MS. These results suggest that EF could contribute to the beneficial effect of onion supplement in maintaining hepatic lipid homeostasis.

Clinical development of imatinib: an anticancer drug

PubMed Central

Goswami, Dipanjan; Gurule, Sanjay; Lahiry, Abhiroop; Anand, Amit; Khuroo, Arshad; Monif, Tausif

2016-01-01

Background: A novel and accurate high-throughput tandem mass spectroscopic method has been developed and validated for determination of imatinib, a protein-tyrosine kinase inhibitor against chronic myeloid leukemia. Materials & methods: Chromatographic separation was carried on XTerra® RP18 column (150 mm × 4.6 mm, 5 µm particle size) manufactured by Waters Corporation, MA, USA. The detection was performed on a triple quadruple tandem mass spectrometer by multiple reactions monitoring mode via electrospray ionization source. Results: The selective and sensitive method was linear in the concentration range of 9.57–4513.29 ng/ml and reported no matrix effect. Conclusion: The mean Cmax was found to be 10–15% lower in European subjects as compared with Indian subjects. PMID:28031942

Chain-reaction crash in traffic flow controlled by taillights

NASA Astrophysics Data System (ADS)

Nagatani, Takashi

2015-02-01

We study the chain-reaction crash (multiple-vehicle collision) in low-visibility condition on a road. In the traffic situation, drivers brake according to taillights of the forward vehicle. The first crash may induce more collisions. We investigate whether or not the first collision induces the chain-reaction crash, numerically and analytically. The dynamic transitions occur from no collisions through a single collision, double collisions and triple collisions, to multiple collisions with decreasing the headway. Also, we find that the dynamic transition occurs from the finite chain reaction to the infinite chain reaction when the headway is less than the critical value. We derive, analytically, the transition points and the region maps for the chain-reaction crash in traffic flow controlled by taillights.

REVIEW ARTICLE: Industrial applications of temperature and species concentration monitoring using laser diagnostics

NASA Astrophysics Data System (ADS)

Deguchi, Y.; Noda, M.; Fukuda, Y.; Ichinose, Y.; Endo, Y.; Inada, M.; Abe, Y.; Iwasaki, S.

2002-10-01

Industrial applications of laser diagnostics have been demonstrated for the purpose of clarifying combustor chemical reaction mechanisms, as well as temperature and harmful substance monitoring in large-scale burners and commercial plant exhaust ducts, and the combustion control of commercial plants. Laser induced fluorescence (LIF), laser induced breakdown spectroscopy (LIBS), and tunable diode laser absorption spectroscopy (TDLAS) have accordingly been applied in various industrial fields. In this study, temperature and species concentration were measured inside gas turbine combustors, a diesel engine, and a large-scale industrial burner using LIF. This technique introduces a new tool with respect to practical combustors for the analysis of NO formation characteristics, turbulent flame front structure, and differences between standard and improved combustors. On-line monitoring of trace elements to the ppb level was also successfully demonstrated using LIBS. The automated LIBS unit was found to be capable of monitoring trace element concentration fluctuations at ppb levels with a 1 min detection time under actual plant conditions. In addition, real-time measurement of O2 and CO concentrations in a commercial incinerator furnace was performed using TDLAS to improve the combustion control. By using the multiple-point laser measurement results to control secondary air allocation, higher secondary combustion efficiency was achieved, and CO concentration (considered to be a substitute indicator for dioxins) was reduced from 11.9 to 8.0 ppm.

Portable formaldehyde monitoring device using porous glass sensor and its applications in indoor air quality studies.

PubMed

Maruo, Yasuko Yamada; Nakamura, Jiro

2011-09-30

We have developed a portable device for formaldehyde monitoring with both high sensitivity and high temporal resolution, and carried out indoor air formaldehyde concentration analysis. The absorbance difference of the sensor element was measured in the monitoring device at regular intervals of, for example, one hour or 30 min, and the result was converted into the formaldehyde concentration. This was possible because we found that the lutidine derivative that was formed as a yellow product of the reaction between 1-phenyl-1,3-butandione and formaldehyde was stable in porous glass for at least six months. We estimated the reaction rate and to be 0.049 min(-1) and the reaction occurred quickly enough for us to monitor hourly changes in the formaldehyde concentration. The detection limit was 5 μg m(-3) h. We achieved hourly formaldehyde monitoring using the developed device under several indoor conditions, and estimated the air exchange rate and formaldehyde adsorption rate, which we adopted as a new term in the mass balance equation for formaldehyde, in one office. Copyright © 2011 Elsevier B.V. All rights reserved.

Optofluidic UV-Vis spectrophotometer for online monitoring of photocatalytic reactions

NASA Astrophysics Data System (ADS)

Wang, Ning; Tan, Furui; Zhao, Yu; Tsoi, Chi Chung; Fan, Xudong; Yu, Weixing; Zhang, Xuming

2016-06-01

On-chip integration of optical detection units into the microfluidic systems for online monitoring is highly desirable for many applications and is also well in line with the spirit of optofluidics technology-fusion of optics and microfluidics for advanced functionalities. This paper reports the construction of a UV-Vis spectrophotometer on a microreactor, and demonstrates the online monitoring of the photocatalytic degradations of methylene blue and methyl orange under different flow rates and different pH values by detecting the intensity change and/or the peak shift. The integrated device consists of a TiO2-coated glass substrate, a PDMS micro-sized reaction chamber and two flow cells. By comparing with the results of commercial equipment, we have found that the measuring range and the sensitivity are acceptable, especially when the transmittance is in the range of 0.01-0.9. This integrated optofluidic device can significantly cut down the test time and the sample volume, and would provide a versatile platform for real-time characterization of photochemical performance. Moreover, its online monitoring capability may enable to access the usually hidden information in biochemical reactions like intermediate products, time-dependent processes and reaction kinetics.

Rapid identification of antibiotic resistance using droplet microfluidics.

PubMed

Keays, Marie C; O'Brien, Mark; Hussain, Anam; Kiely, Patrick A; Dalton, Tara

2016-04-02

Culturing bacteria and monitoring bacterial cell growth is a critical issue when dealing with patients who present with bacterial infections. One of the main challenges that arises is the time taken to identify the particular strain of bacteria and consequently, decide the correct treatment. In the majority of cases, broad spectrum antibiotics are used to target infections when a narrow spectrum drug would be more appropriate. The efficient monitoring of bacterial growth and potential antibiotic resistance is necessary to identify the best treatment options for patients. Minturising the reactions into microfluidic droplets offers a novel method to rapidy analyze bacteria. Microfluidics facilitates low volume reactions that provide a unique system where each droplet reaction acts as an individual bioreactor. Here, we designed and built a novel platform that allowed us to create and monitor E.coli microfluidic droplet cultures. Optical capacity was built in and measurements of bacterial cultures were captured facilitating the continuous monitoring of individual reactions. The capacity of the instrument was demonstrated by the application of treatments to both bacteria and drug resistant strains of bacteria. We were able to detect responses within one hour in the droplet cultures, demonstrating the capacity of this workflow to the culture and rapid characterization of bacterial strains.

Optofluidic UV-Vis spectrophotometer for online monitoring of photocatalytic reactions

PubMed Central

Wang, Ning; Tan, Furui; Zhao, Yu; Tsoi, Chi Chung; Fan, Xudong; Yu, Weixing; Zhang, Xuming

2016-01-01

On-chip integration of optical detection units into the microfluidic systems for online monitoring is highly desirable for many applications and is also well in line with the spirit of optofluidics technology–fusion of optics and microfluidics for advanced functionalities. This paper reports the construction of a UV-Vis spectrophotometer on a microreactor, and demonstrates the online monitoring of the photocatalytic degradations of methylene blue and methyl orange under different flow rates and different pH values by detecting the intensity change and/or the peak shift. The integrated device consists of a TiO2-coated glass substrate, a PDMS micro-sized reaction chamber and two flow cells. By comparing with the results of commercial equipment, we have found that the measuring range and the sensitivity are acceptable, especially when the transmittance is in the range of 0.01–0.9. This integrated optofluidic device can significantly cut down the test time and the sample volume, and would provide a versatile platform for real-time characterization of photochemical performance. Moreover, its online monitoring capability may enable to access the usually hidden information in biochemical reactions like intermediate products, time-dependent processes and reaction kinetics. PMID:27352840

Optofluidic UV-Vis spectrophotometer for online monitoring of photocatalytic reactions.

PubMed

Wang, Ning; Tan, Furui; Zhao, Yu; Tsoi, Chi Chung; Fan, Xudong; Yu, Weixing; Zhang, Xuming

2016-06-29

On-chip integration of optical detection units into the microfluidic systems for online monitoring is highly desirable for many applications and is also well in line with the spirit of optofluidics technology-fusion of optics and microfluidics for advanced functionalities. This paper reports the construction of a UV-Vis spectrophotometer on a microreactor, and demonstrates the online monitoring of the photocatalytic degradations of methylene blue and methyl orange under different flow rates and different pH values by detecting the intensity change and/or the peak shift. The integrated device consists of a TiO2-coated glass substrate, a PDMS micro-sized reaction chamber and two flow cells. By comparing with the results of commercial equipment, we have found that the measuring range and the sensitivity are acceptable, especially when the transmittance is in the range of 0.01-0.9. This integrated optofluidic device can significantly cut down the test time and the sample volume, and would provide a versatile platform for real-time characterization of photochemical performance. Moreover, its online monitoring capability may enable to access the usually hidden information in biochemical reactions like intermediate products, time-dependent processes and reaction kinetics.

Excitation functions of nuclear reactions induced by alpha particles up to 42 MeV on natTi for monitoring purposes and TLA

NASA Astrophysics Data System (ADS)

Hermanne, A.; Sonck, M.; Takács, S.; Szelecsényi, F.; Tárkányi, F.

1999-05-01

Excitation functions for the reactions induced by alpha particles on natTi foils and leading to the formation of 44m,44g,46,47,48Sc; 48,51Cr and 48V were determined using the stacked foil technique for energies from the respective reaction thresholds up to 42 MeV. The new experimental values are compared to earlier literature values and generally good accordance is found. It appears that the natTi(α,x) 51Cr reaction is particularly useful for monitoring α-beams in the 10-20 MeV region while for energies above 20 MeV the natTi(α,x) 47Sc reaction or the natTi(α,x) 48V reaction are more suited. The excitation functions established can be used to determine calibration curves for thin layer activation (TLA) as well.

Modulation of MMP-2 and MMP-9 in Churg-Strauss syndrome respiratory mucosa: potential monitoring parameters.

PubMed

Leone, A; Uzzo, M L; Gerbino, A; Tortorici, S; Tralongo, P; Cappello, F; Incandela, S; Spatola, G F; Jurjus, A R

2014-01-01

Churg-Strauss (CSS) syndrome is rare and of unknown etiology. It is associated with vasculitis, blood eosinophilia and granulomatosis, and affects multiple organs and systems at various stages of the disease. Specific diagnostic and monitoring tests are not yet available. This study aims to assess the changes in MMP-2 and MMP-9 along with the histopathological alterations in two cases of CSS, as possible potential diagnostic and monitoring criteria. Two adult male patients were diagnosed with CSS in the otorhinolaryngology clinic in the University of Palermo, based on multiple clinical and histopathologic criteria. Biopsies of respiratory mucosa were taken after the consent of the patients, processed for routine histopathology and immunohistochemistry as well as quantitative polymerase chain reaction (qPCR). Similar biopsies were also taken from a non- CSS patient. The Assessment of MMP-2 and MMP-9 was performed using both immunohistochemistry and qPCR techniques. Histopathological alterations in the respiratory mucosa were consistent with vasculitis and granulomatous tissue formation, in addition to inflammatory cell infiltration with abundance of eosinophils. Immunohistochemistry assay performed on the samples derived from the two CSS patients showed a relative and remarkable increase of both MMP-2 and MMP-9 compared to controls. Such an increase was consistent with the qPCR results which depicted a significant increase between 20 and 30% for both MMP-2 and MMP-9, respectively. Since the secretion of MMPs is an essential step in angiogenesis, could these enzymatic factors be used as parameters to diagnose or monitor the evolution of CSS? The small number of samples analyzed in this study does not allow us to suggest a general statement correlating the increase in expression of MMP-2 and MMP-9 to the appearance or evolution of vasculitis; it is only speculative.

Isolating the impact of septic systems on fecal pollution in streams of suburban watersheds in Georgia, United States.

PubMed

Sowah, Robert A; Habteselassie, Mussie Y; Radcliffe, David E; Bauske, Ellen; Risse, Mark

2017-01-01

The presence of multiple sources of fecal pollution at the watershed level presents challenges to efforts aimed at identifying the influence of septic systems. In this study multiple approaches including targeted sampling and monitoring of host-specific Bacteroidales markers were used to identify the impact of septic systems on microbial water quality. Twenty four watersheds with septic density ranging from 8 to 373 septic units/km 2 were monitored for water quality under baseflow conditions over a 3-year period. The levels of the human-associated HF183 marker, as well as total and ruminant Bacteroidales, were quantified using quantitative polymerase chain reaction. Human-associated Bacteroidales yield was significantly higher in high density watersheds compared to low density areas and was negatively correlated (r = -0.64) with the average distance of septic systems to streams in the spring season. The human marker was also positively correlated with the total Bacteroidales marker, suggesting that the human source input was a significant contributor to total fecal pollution in the study area. Multivariable regression analysis indicates that septic systems, along with forest cover, impervious area and specific conductance could explain up to 74% of the variation in human fecal pollution in the spring season. The results suggest septic system impact through contributions to groundwater recharge during baseflow or failing septic system input, especially in areas with >87 septic units/km 2 . This study supports the use of microbial source tracking approaches along with traditional fecal indicator bacteria monitoring and land use characterization in a tiered approach to isolate the influence of septic systems on water quality in mixed-use watersheds. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enriching the biological space of natural products and charting drug metabolites, through real time biotransformation monitoring: The NMR tube bioreactor.

PubMed

Chatzikonstantinou, Alexandra V; Chatziathanasiadou, Maria V; Ravera, Enrico; Fragai, Marco; Parigi, Giacomo; Gerothanassis, Ioannis P; Luchinat, Claudio; Stamatis, Haralambos; Tzakos, Andreas G

2018-01-01

Natural products offer a wide range of biological activities, but they are not easily integrated in the drug discovery pipeline, because of their inherent scaffold intricacy and the associated complexity in their synthetic chemistry. Enzymes may be used to perform regioselective and stereoselective incorporation of functional groups in the natural product core, avoiding harsh reaction conditions, several protection/deprotection and purification steps. Herein, we developed a three step protocol carried out inside an NMR-tube. 1st-step: STD-NMR was used to predict the: i) capacity of natural products as enzyme substrates and ii) possible regioselectivity of the biotransformations. 2nd-step: The real-time formation of multiple-biotransformation products in the NMR-tube bioreactor was monitored in-situ. 3rd-step: STD-NMR was applied in the mixture of the biotransformed products to screen ligands for protein targets. Herein, we developed a simple and time-effective process, the "NMR-tube bioreactor", that is able to: (i) predict which component of a mixture of natural products can be enzymatically transformed, (ii) monitor in situ the transformation efficacy and regioselectivity in crude extracts and multiple substrate biotransformations without fractionation and (iii) simultaneously screen for interactions of the biotransformation products with pharmaceutical protein targets. We have developed a green, time-, and cost-effective process that provide a simple route from natural products to lead compounds for drug discovery. This process can speed up the most crucial steps in the early drug discovery process, and reduce the chemical manipulations usually involved in the pipeline, improving the environmental compatibility. Copyright © 2017 Elsevier B.V. All rights reserved.

An optics-based variable-temperature assay system for characterizing thermodynamics of biomolecular reactions on solid support

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fei, Yiyan; Landry, James P.; Zhu, X. D., E-mail: xdzhu@physics.ucdavis.edu

A biological state is equilibrium of multiple concurrent biomolecular reactions. The relative importance of these reactions depends on physiological temperature typically between 10 °C and 50 °C. Experimentally the temperature dependence of binding reaction constants reveals thermodynamics and thus details of these biomolecular processes. We developed a variable-temperature opto-fluidic system for real-time measurement of multiple (400–10 000) biomolecular binding reactions on solid supports from 10 °C to 60 °C within ±0.1 °C. We illustrate the performance of this system with investigation of binding reactions of plant lectins (carbohydrate-binding proteins) with 24 synthetic glycans (i.e., carbohydrates). We found that the lectin-glycan reactions in general can be enthalpy-driven,more » entropy-driven, or both, and water molecules play critical roles in the thermodynamics of these reactions.« less

An optics-based variable-temperature assay system for characterizing thermodynamics of biomolecular reactions on solid support

NASA Astrophysics Data System (ADS)

Fei, Yiyan; Landry, James P.; Li, Yanhong; Yu, Hai; Lau, Kam; Huang, Shengshu; Chokhawala, Harshal A.; Chen, Xi; Zhu, X. D.

2013-11-01

A biological state is equilibrium of multiple concurrent biomolecular reactions. The relative importance of these reactions depends on physiological temperature typically between 10 °C and 50 °C. Experimentally the temperature dependence of binding reaction constants reveals thermodynamics and thus details of these biomolecular processes. We developed a variable-temperature opto-fluidic system for real-time measurement of multiple (400-10 000) biomolecular binding reactions on solid supports from 10 °C to 60 °C within ±0.1 °C. We illustrate the performance of this system with investigation of binding reactions of plant lectins (carbohydrate-binding proteins) with 24 synthetic glycans (i.e., carbohydrates). We found that the lectin-glycan reactions in general can be enthalpy-driven, entropy-driven, or both, and water molecules play critical roles in the thermodynamics of these reactions.

Direct observation of sequential oxidations of a titania-bound molecular proxy catalyst generated through illumination of molecular sensitizers

NASA Astrophysics Data System (ADS)

Chen, Hsiang-Yun; Ardo, Shane

2018-01-01

Natural photosynthesis uses the energy in sunlight to oxidize or reduce reaction centres multiple times, therefore preparing each reaction centre for a multiple-electron-transfer reaction that will ultimately generate stable reaction products. This process relies on multiple chromophores per reaction centre to quickly generate the active state of the reaction centre and to outcompete deleterious charge recombination. Using a similar design principle, we report spectroscopic evidence for the generation of a twice-oxidized TiO2-bound molecular proxy catalyst after low-intensity visible-light excitation of co-anchored molecular Ru(II)-polypyridyl dyes. Electron transfer from an excited dye to TiO2 generated a Ru(III) state that subsequently and repeatedly reacted with neighbouring Ru(II) dyes via self-exchange electron transfer to ultimately oxidize a distant co-anchored proxy catalyst before charge recombination. The largest yield for twice-oxidized proxy catalysts occurred when they were present at low coverage, suggesting that large dye/electrocatalyst ratios are also desired in dye-sensitized photoelectrochemical cells.

Extending the Limits of Quantitative Proteome Profiling with Data-Independent Acquisition and Application to Acetaminophen-Treated Three-Dimensional Liver Microtissues*

PubMed Central

Bruderer, Roland; Bernhardt, Oliver M.; Gandhi, Tejas; Miladinović, Saša M.; Cheng, Lin-Yang; Messner, Simon; Ehrenberger, Tobias; Zanotelli, Vito; Butscheid, Yulia; Escher, Claudia; Vitek, Olga; Rinner, Oliver; Reiter, Lukas

2015-01-01

The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)1-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling. PMID:25724911

Shape-Independent Model of Monitor Neutron Activation Analysis

NASA Astrophysics Data System (ADS)

Yusuf, Siaka Ojo

The technique of monitor neutron activation analysis has been improved by developing a shape-independent model to solve the problem of the treatment of the epithermal reaction contribution to the reaction rate in reactor neutron activation analysis. It is a form of facility characterization in which differential approximations to neither the neutron flux distribution as a function of energy nor the reaction cross section as a function of energy are necessary. The model predicts a linear relationship when the k-factors (ratios of reaction rates of two nuclides at a given irradiation position) for element x, k _{c} (x), is plotted against the k-factor for the monitor, k_{c} (m). The slope of this line, B(x,c,m) is measured for each element x to provide the calibration of the irradiation facility for monitor activation analysis. In this thesis, scandium was chosen as the comparator and antimony as the epithermal monitor. B(x, Sc, Sb) has been accurately measured for a number of nuclides in three different reactors. The measurement was done by irradiating filter papers containing binary mixture of the elements x and the flux monitor Sc at the various irradiation positions in these three reactors. The experiment was designed in such a way that systematic errors due to mass ratios and efficiency ratios cancel out. Also, rate related errors and backgrounds were kept at negligible values. The results show that B(x,c,m) depends not only on x, c, and m, but also on the type of moderator used for the reactor. We want this new approach to be adopted at all laboratories where routine analysis of multi-element samples are done with the monitor method since the choices of c and m are flexible.

Comprehensive quantitative analysis of central carbon and amino-acid metabolism in Saccharomyces cerevisiae under multiple conditions by targeted proteomics

PubMed Central

Costenoble, Roeland; Picotti, Paola; Reiter, Lukas; Stallmach, Robert; Heinemann, Matthias; Sauer, Uwe; Aebersold, Ruedi

2011-01-01

Decades of biochemical research have identified most of the enzymes that catalyze metabolic reactions in the yeast Saccharomyces cerevisiae. The adaptation of metabolism to changing nutritional conditions, in contrast, is much less well understood. As an important stepping stone toward such understanding, we exploit the power of proteomics assays based on selected reaction monitoring (SRM) mass spectrometry to quantify abundance changes of the 228 proteins that constitute the central carbon and amino-acid metabolic network in the yeast Saccharomyces cerevisiae, at five different metabolic steady states. Overall, 90% of the targeted proteins, including families of isoenzymes, were consistently detected and quantified in each sample, generating a proteomic data set that represents a nutritionally perturbed biological system at high reproducibility. The data set is near comprehensive because we detect 95–99% of all proteins that are required under a given condition. Interpreted through flux balance modeling, the data indicate that S. cerevisiae retains proteins not necessarily used in a particular environment. Further, the data suggest differential functionality for several metabolic isoenzymes. PMID:21283140

Trimethylation enhancement using diazomethane (TrEnDi): rapid on-column quaternization of peptide amino groups via reaction with diazomethane significantly enhances sensitivity in mass spectrometry analyses via a fixed, permanent positive charge.

PubMed

Wasslen, Karl V; Tan, Le Hoa; Manthorpe, Jeffrey M; Smith, Jeffrey C

2014-04-01

Defining cellular processes relies heavily on elucidating the temporal dynamics of proteins. To this end, mass spectrometry (MS) is an extremely valuable tool; different MS-based quantitative proteomics strategies have emerged to map protein dynamics over the course of stimuli. Herein, we disclose our novel MS-based quantitative proteomics strategy with unique analytical characteristics. By passing ethereal diazomethane over peptides on strong cation exchange resin within a microfluidic device, peptides react to contain fixed, permanent positive charges. Modified peptides display improved ionization characteristics and dissociate via tandem mass spectrometry (MS(2)) to form strong a2 fragment ion peaks. Process optimization and determination of reactive functional groups enabled a priori prediction of MS(2) fragmentation patterns for modified peptides. The strategy was tested on digested bovine serum albumin (BSA) and successfully quantified a peptide that was not observable prior to modification. Our method ionizes peptides regardless of proton affinity, thus decreasing ion suppression and permitting predictable multiple reaction monitoring (MRM)-based quantitation with improved sensitivity.

Ethylene Glycol Quenching of Nitrogenase Catalysis: An Electron Paramagnetic Resonance Spectroscopic Study of Nitrogenase Turnover States and CO Bonding.

PubMed

Hales, Brian J

2015-07-14

Most hydrophilic organic solvents inhibit enzymatic activity. Nitrogenase is shown to be approximately 3 times more sensitive to organic inhibition than most other soluble enzymes. Ethylene glycol (EG) is demonstrated to rapidly inhibit nitrogenase activity without uncoupling ATP hydrolysis. Our data suggest the mechanism of inhibition is EG's blocking of binding of MgATP to the nitrogenase Fe protein. EG quenching allows, for the first time, the observation of the relaxation of the intermediate reaction states at room temperature. Electron paramagnetic resonance (EPR) spectroscopy is used to monitor the room-temperature decay of the nitrogenase turnover states following EG quenching of catalytic activity. The return of the intermediate states to the resting state occurs in multiple phases over 2 h. During the initial stage, nitrogenase still possesses the ability to generate CO-induced EPR signals even though catalytic activity has ceased. During the last phase of relaxation, the one-electron reduced state of the MoFe protein (E1) relaxes to the resting state (E0) in a slow first-order reaction.

Measuring vigilance decrement using computer vision assisted eye tracking in dynamic naturalistic environments.

PubMed

Bodala, Indu P; Abbasi, Nida I; Yu Sun; Bezerianos, Anastasios; Al-Nashash, Hasan; Thakor, Nitish V

2017-07-01

Eye tracking offers a practical solution for monitoring cognitive performance in real world tasks. However, eye tracking in dynamic environments is difficult due to high spatial and temporal variation of stimuli, needing further and thorough investigation. In this paper, we study the possibility of developing a novel computer vision assisted eye tracking analysis by using fixations. Eye movement data is obtained from a long duration naturalistic driving experiment. Source invariant feature transform (SIFT) algorithm was implemented using VLFeat toolbox to identify multiple areas of interest (AOIs). A new measure called `fixation score' was defined to understand the dynamics of fixation position between the target AOI and the non target AOIs. Fixation score is maximum when the subjects focus on the target AOI and diminishes when they gaze at the non-target AOIs. Statistically significant negative correlation was found between fixation score and reaction time data (r =-0.2253 and p<;0.05). This implies that with vigilance decrement, the fixation score decreases due to visual attention shifting away from the target objects resulting in an increase in the reaction time.

Microfluidic chemical reaction circuits

DOEpatents

Lee, Chung-cheng [Irvine, CA; Sui, Guodong [Los Angeles, CA; Elizarov, Arkadij [Valley Village, CA; Kolb, Hartmuth C [Playa del Rey, CA; Huang, Jiang [San Jose, CA; Heath, James R [South Pasadena, CA; Phelps, Michael E [Los Angeles, CA; Quake, Stephen R [Stanford, CA; Tseng, Hsian-rong [Los Angeles, CA; Wyatt, Paul [Tipperary, IE; Daridon, Antoine [Mont-Sur-Rolle, CH

2012-06-26

New microfluidic devices, useful for carrying out chemical reactions, are provided. The devices are adapted for on-chip solvent exchange, chemical processes requiring multiple chemical reactions, and rapid concentration of reagents.

Experimental investigations of mechanical and reaction responses for drop-weight impacted energetic particles

NASA Astrophysics Data System (ADS)

Bao, Xiao-Wei; Wu, Yan-Qing; Wang, Ming-Yang; Huang, Feng-Lei

2017-02-01

Low-velocity drop-weight impact experiments on individual and multiple Cyclotetramethylene tetranitramine (HMX) energetic particles were performed using a modified drop-weight machine equipped with high-speed photography components. Multiple particles experienced more severe burning reactions than an individual particle. Comparisons between impacted salt and HMX particle show that jetting in HMX is mainly due to the motion of fragmented particles driven by gaseous reaction products. Velocity of jetting, flame propagation, and area expansion were measured via image processing, making it possible to quantify the chemical reaction or mechanical deformation violence at different stages.

Free and Protein-Bound Maillard Reaction Products in Beer: Method Development and a Survey of Different Beer Types.

PubMed

Hellwig, Michael; Witte, Sophia; Henle, Thomas

2016-09-28

The Maillard reaction is important for beer color and flavor, but little is known about the occurrence of individual glycated amino acids in beer. Therefore, seven Maillard reaction products (MRPs), namely, fructosyllysine, maltulosyllysine, pyrraline, formyline, maltosine, MG-H1, and argpyrimidine, were synthesized and quantitated in different types of beer (Pilsner, dark, bock, wheat, and nonalcoholic beers) by HPLC-ESI-MS/MS in the multiple reaction monitoring mode through application of the standard addition method. Free MRPs were analyzed directly. A high molecular weight fraction was isolated by dialysis and hydrolyzed enzymatically prior to analysis. Maltulosyllysine was quantitated for the first time in food. The most important free MRPs in beer are fructosyllysine (6.8-27.0 mg/L) and maltulosyllysine (3.7-21.8 mg/L). Beer contains comparatively high amounts of late-stage free MRPs such as pyrraline (0.2-1.6 mg/L) and MG-H1 (0.3-2.5 mg/L). Minor amounts of formyline (4-230 μg/L), maltosine (6-56 μg/L), and argpyrimidine (0.1-4.1 μg/L) were quantitated. Maltulosyllysine was the most significant protein-bound MRP, but both maltulosyllysine and fructosyllysine represent only 15-60% of the total protein-bound lysine-derived Amadori products. Differences in the patterns of protein-bound and free individual MRPs and the ratios between them were identified, which indicate differences in their chemical, biochemical, and microbiological stabilities during the brewing process.

Monitoring Acidophilic Microbes with Real-Time Polymerase Chain Reaction (PCR) Assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frank F. Roberto

2008-08-01

Many techniques that are used to characterize and monitor microbial populations associated with sulfide mineral bioleaching require the cultivation of the organisms on solid or liquid media. Chemolithotrophic species, such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, or thermophilic chemolithotrophs, such as Acidianus brierleyi and Sulfolobus solfataricus can grow quite slowly, requiring weeks to complete efforts to identify and quantify these microbes associated with bioleach samples. Real-time PCR (polymerase chain reaction) assays in which DNA targets are amplified in the presence of fluorescent oligonucleotide primers, allowing the monitoring and quantification of the amplification reactions as they progress, provide a means ofmore » rapidly detecting the presence of microbial species of interest, and their relative abundance in a sample. This presentation will describe the design and use of such assays to monitor acidophilic microbes in the environment and in bioleaching operations. These assays provide results within 2-3 hours, and can detect less than 100 individual microbial cells.« less

Reformulation and solution of the master equation for multiple-well chemical reactions.

PubMed

Georgievskii, Yuri; Miller, James A; Burke, Michael P; Klippenstein, Stephen J

2013-11-21

We consider an alternative formulation of the master equation for complex-forming chemical reactions with multiple wells and bimolecular products. Within this formulation the dynamical phase space consists of only the microscopic populations of the various isomers making up the reactive complex, while the bimolecular reactants and products are treated equally as sources and sinks. This reformulation yields compact expressions for the phenomenological rate coefficients describing all chemical processes, i.e., internal isomerization reactions, bimolecular-to-bimolecular reactions, isomer-to-bimolecular reactions, and bimolecular-to-isomer reactions. The applicability of the detailed balance condition is discussed and confirmed. We also consider the situation where some of the chemical eigenvalues approach the energy relaxation time scale and show how to modify the phenomenological rate coefficients so that they retain their validity.

Fall risk factors analysis based on sample entropy of plantar kinematic signal during stance phase.

PubMed

Shengyun Liang; Huiyu Jia; Zilong Li; Huiqi Li; Xing Gao; Zuchang Ma; Yingnan Ma; Guoru Zhao

2016-08-01

Falls are a multi-causal phenomenon with a complex interaction. The aim of our research is to study the effect of multiple variables for potential risk of falls and construct an elderly fall risk assessment model based on demographics data and gait characteristics. A total of 101 subjects, whom belong to Malianwa Street, aged above 50 years old and participated in questionnaire survey. Participants were classified into three groups (high, medium and low risk group) according to the score of elderly fall risk assessment scale. In addition, the data of ground reaction force (GRF) and ground reaction moment (GRM) was record when they performed walking at comfortable state. The demographic variables, sample entropy of GRF and GRM, and impulse difference of bilateral foot were considered as potential explanatory variables of risk assessment model. Firstly, we investigated whether different groups could present difference in every variable. Statistical differences were found for the following variables: age (p=2.28e-05); impulse difference (p=0.02036); sample entropy of GRF in vertical direction (p=0.0144); sample entropy of GRM in anterior-posterior direction (p=0.0387). Finally, the multiple regression analysis results indicated that age, impulse difference and sample entropy of resultant GRM could identify individuals who had different levels of fall risk. Therefore, those results could potentially be useful in the fall risk assessment and monitor the state of physical function in elderly population.

Gold patterned biochips for on-chip immuno-MALDI-TOF MS: SPR imaging coupled multi-protein MS analysis.

PubMed

Kim, Young Eun; Yi, So Yeon; Lee, Chang-Soo; Jung, Yongwon; Chung, Bong Hyun

2012-01-21

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of immuno-captured target protein efficiently complements conventional immunoassays by offering rich molecular information such as protein isoforms or modifications. Direct immobilization of antibodies on MALDI solid support enables both target enrichment and MS analysis on the same plate, allowing simplified and potentially multiplexing protein MS analysis. Reliable on-chip immuno-MALDI-TOF MS for multiple biomarkers requires successful adaptation of antibody array biochips, which also must accommodate consistent reaction conditions on antibody arrays during immuno-capture and MS analysis. Here we developed a facile fabrication process of versatile antibody array biochips for reliable on-chip MALDI-TOF-MS analysis of multiple immuno-captured proteins. Hydrophilic gold arrays surrounded by super-hydrophobic surfaces were formed on a gold patterned biochip via spontaneous chemical or protein layer deposition. From antibody immobilization to MALDI matrix treatment, this hydrophilic/phobic pattern allowed highly consistent surface reactions on each gold spot. Various antibodies were immobilized on these gold spots both by covalent coupling or protein G binding. Four different protein markers were successfully analyzed on the present immuno-MALDI biochip from complex protein mixtures including serum samples. Tryptic digests of captured PSA protein were also effectively detected by on-chip MALDI-TOF-MS. Moreover, the present MALDI biochip can be directly applied to the SPR imaging system, by which antibody and subsequent antigen immobilization were successfully monitored.

Reaction Order Ambiguity in Integrated Rate Plots

ERIC Educational Resources Information Center

Lee, Joe

2008-01-01

Integrated rate plots are frequently used in reaction kinetics to determine orders of reactions. It is often emphasised, when using this methodology in practice, that it is necessary to monitor the reaction to a substantial fraction of completion for these plots to yield unambiguous orders. The present article gives a theoretical and statistical…

The reaction of H2O2 with NO2 and NO

NASA Technical Reports Server (NTRS)

Gray, D.; Lissi, E.; Heicklen, J.

1972-01-01

The reactions of NO and NO2 with H2O2 have been examined at 25 C. Reaction mixtures were monitored by continuously bleeding through a pinhole into a monopole mass spectrometer. NO2 was also monitored by its optical absorption in the visible part of the spectrum. Reaction mixtures containing initially 1.5 - 2.5 torr of NO2 and 0.8 - 1.4 torr of H2O2 or 1 - 12 torr of NO and 0.5 - 1.5 torr of H2O2 were studied. The H2O2 - NO reaction was complex. There was an induction period followed by a marked acceleration in reactant removal. The final products of the reaction, NO2, probably H2O, and possibly HONO2 were produced mainly after all the H2O2 was removed. The HONO intermediate was shown to disproportionate to NO2 + NO + H2O in a relatively slow first order reaction. The acceleration in H2O2 removal after the NO - H2O2 reaction is started is caused by NO2 catalysis.

Interfacing supercritical fluid reaction apparatus with on-line liquid chromatography: monitoring the progress of a synthetic organic reaction performed in supercritical fluid solution.

PubMed

Ramsey, Edward D; Li, Ben; Guo, Wei; Liu, Jing Y

2015-04-03

An interface has been developed that connects a supercritical fluid reaction (SFR) vessel directly on-line to a liquid chromatograph. The combined SFR-LC system has enabled the progress of the esterification reaction between phenol and benzoyl chloride to synthesize phenyl benzoate in supercritical fluid carbon dioxide solution to be dynamically monitored. This was achieved by the periodic SFR-LC analysis of samples directly withdrawn from the esterification reaction mixture. Using the series of SFR-LC analysis results obtained for individual esterification reactions, the reaction progress profile for each esterification reaction was obtained by expressing the measured yield of phenyl benzoate as a function of reaction time. With reaction temperature fixed at 75°C, four sets (n=3) of SFR-LC reaction progress profiles were obtained at four different SFR pressures ranging from 13.79 to 27.58 MPa. The maximum SFR yield obtained for phenyl benzoate using a standard set of reactant concentrations was 85.2% (R.S.D. 4.2%) when the reaction was performed at 13.79 MPa for 90 min. In comparison, a phenyl benzoate yield of less than 0.3% was obtained using the same standard reactant concentrations after 90 min reaction time at 75°C using either: heptane, ethyl acetate or acetonitrile as conventional organic reaction solvents. Copyright © 2015 Elsevier B.V. All rights reserved.

Method and apparatus for a catalytic firebox reactor

DOEpatents

Smith, Lance L.; Etemad, Shahrokh; Ulkarim, Hasan; Castaldi, Marco J.; Pfefferle, William C.

2001-01-01

A catalytic firebox reactor employing an exothermic catalytic reaction channel and multiple cooling conduits for creating a partially reacted fuel/oxidant mixture. An oxidation catalyst is deposited on the walls forming the boundary between the multiple cooling conduits and the exothermic catalytic reaction channel, on the side of the walls facing the exothermic catalytic reaction channel. This configuration allows the oxidation catalyst to be backside cooled by any fluid passing through the cooling conduits. The heat of reaction is added to both the fluid in the exothermic catalytic reaction channel and the fluid passing through the cooling conduits. After discharge of the fluids from the exothermic catalytic reaction channel, the fluids mix to create a single combined flow. A further innovation in the reactor incorporates geometric changes in the exothermic catalytic reaction channel to provide streamwise variation of the velocity of the fluids in the reactor.

Study of deuteron induced reactions on natural iron and copper and their use for monitoring beam parameters and for thin layer activation technique

NASA Astrophysics Data System (ADS)

Takács, S.; Tárkányi, F.; Sonck, M.; Hermanne, A.; Sudár, S.

1997-02-01

Excitation functions of deuteron induced nuclear reactions on natural iron and copper have been studied in the frame of a systematic investigation of charged particle induced nuclear reactions on metals for different applications. The excitation functions were measured up to 20 MeV deuteron energy by using stacked foil technique and activation method. The measured and the evaluated literature data showed that some reaction can be recommended for monitoring deuteron beams, and the excitation functions can be used to determine calibration curves for Thin Layer Activation Technique (TLA). Cross sections calculated by statistical model theory, STAPRE, taking into account preequilibrium effect are in reasonable agreement with the experimental results.

Determination of statins by gas chromatography - EI/MRM - Tandem mass spectrometry: fermentation of pine samples with Pleurotus ostreatus.

PubMed

Sirén, Heli; Kaijanen, Laura; Kaartinen, Sini; Väre, Monna; Riikonen, Päivi; Jernström, Eeva

2014-06-01

Statins were separated and quantified with gas chromatography-mass spectrometry (GC-EI-MS/MS) using total ion monitoring (TIC) and multiple reactions monitoring (MRM). The MRM method in statins determination has a novelty value, since there are no previous studies on their simultaneous analysis in environmental or plant samples. The method development and optimization was challenging due to the physicochemical similarities of the silylated lovastatin, simvastatin, pravastatin, fluvastatin, and atorvastatin. The results showed that the use of MRM decreased their detection and quantification limits by factors of 2-10 compared to that obtained in TIC monitoring. The concentration calibration was made between 247.5ng/L and 9900ng/L. Limits of detection and quantification were between 50ng/L (lovastatin)-500ng/L (pravastatin) and 250ng/L (lovastatin)-1000ng/L (pravastatin), respectively. Based on the MRM results, the wood bark and phloem samples contained lovastatin, lovastatin-lactone, simvastatin, simvastatin-lactone, and pravastatin. Their concentrations were 250-3000μg/L, i.e. 4.2-50mg/kg in phloem and bark. However, they were not detected in fluids made with Pleurotus ostreatus fermentation of wood core. Copyright © 2014 Elsevier B.V. All rights reserved.

A unified understanding of (γ, n) and (n, γ) reactions and direct neutron-multiplicity sorting

NASA Astrophysics Data System (ADS)

Utsunomiya, Hiroaki; Goriely, Stephane; m, Therese Renstrø; Katayama, Seitaro; Gheorghe, Ioana; Filipescu, Dan; Belyshev, Sergey; Varlamov, Vladimir

2017-09-01

We discuss the γ-ray strength function toward a unified understanding of (γ,n) and (n,γ) reactions and propose a novel technique of direct neutron-multiplicity sorting to resolve the long-standing discrepancy between the Livermore and Scalya data of partial photoneutron cross sections.

Facilitating High School Students' Use of Multiple Representations to Describe and Explain Simple Chemical Reactions

ERIC Educational Resources Information Center

Chandrasegaran, A. L.; Treagust, David F.; Mocerino, Mauro

2011-01-01

This study involved the evaluation of the efficacy of a planned instructional program to facilitate understanding of the macroscopic, submicroscopic and symbolic representational systems when describing and explaining chemical reactions by sixty-five Grade 9 students in a Singapore secondary school. A two-tier multiple-choice diagnostic instrument…

A Targeted MRM Approach for Tempo-Spatial Proteomics Analyses.

PubMed

Moradian, Annie; Porras-Yakushi, Tanya R; Sweredoski, Michael J; Hess, Sonja

2016-01-01

When deciding to perform a quantitative proteomics analysis, selectivity, sensitivity, and reproducibility are important criteria to consider. The use of multiple reaction monitoring (MRM) has emerged as a powerful proteomics technique in that regard since it avoids many of the problems typically observed in discovery-based analyses. A prerequisite for such a targeted approach is that the protein targets are known, either as a result of previous global proteomics experiments or because a specific hypothesis is to be tested. When guidelines that have been established in the pharmaceutical industry many decades ago are taken into account, setting up an MRM assay is relatively straightforward. Typically, proteotypic peptides with favorable mass spectrometric properties are synthesized with a heavy isotope for each protein that is to be monitored. Retention times and calibration curves are determined using triple-quadrupole mass spectrometers. The use of iRT peptide standards is both recommended and fully integrated into the bioinformatics pipeline. Digested biological samples are mixed with the heavy and iRT standards and quantified. Here we present a generic protocol for the development of an MRM assay.

Quantitation of human milk proteins and their glycoforms using multiple reaction monitoring (MRM).

PubMed

Huang, Jincui; Kailemia, Muchena J; Goonatilleke, Elisha; Parker, Evan A; Hong, Qiuting; Sabia, Rocchina; Smilowitz, Jennifer T; German, J Bruce; Lebrilla, Carlito B

2017-01-01

Human milk plays a substantial role in the child growth, development and determines their nutritional and health status. Despite the importance of the proteins and glycoproteins in human milk, very little quantitative information especially on their site-specific glycosylation is known. As more functions of milk proteins and other components continue to emerge, their fine-detailed quantitative information is becoming a key factor in milk research efforts. The present work utilizes a sensitive label-free MRM method to quantify seven milk proteins (α-lactalbumin, lactoferrin, secretory immunoglobulin A, immunoglobulin G, immunoglobulin M, α1-antitrypsin, and lysozyme) using their unique peptides while at the same time, quantifying their site-specific N-glycosylation relative to the protein abundance. The method is highly reproducible, has low limit of quantitation, and accounts for differences in glycosylation due to variations in protein amounts. The method described here expands our knowledge about human milk proteins and provides vital details that could be used in monitoring the health of the infant and even the mother. Graphical Abstract The glycopeptides EICs generated from QQQ.

Quantitation of human milk proteins and their glycoforms using multiple reaction monitoring (MRM)

PubMed Central

Huang, Jincui; Kailemia, Muchena J.; Goonatilleke, Elisha; Parker, Evan A.; Hong, Qiuting; Sabia, Rocchina; Smilowitz, Jennifer T.; German, J. Bruce

2017-01-01

Human milk plays a substantial role in the child growth, development and determines their nutritional and health status. Despite the importance of the proteins and glycoproteins in human milk, very little quantitative information especially on their site-specific glycosylation is known. As more functions of milk proteins and other components continue to emerge, their fine-detailed quantitative information is becoming a key factor in milk research efforts. The present work utilizes a sensitive label-free MRM method to quantify seven milk proteins (α-lactalbumin, lactoferrin, secretory immunoglobulin A, immunoglobulin G, immunoglobulin M, α1-antitrypsin, and lysozyme) using their unique peptides while at the same time, quantifying their site-specific N-glycosylation relative to the protein abundance. The method is highly reproducible, has low limit of quantitation, and accounts for differences in glycosylation due to variations in protein amounts. The method described here expands our knowledge about human milk proteins and provides vital details that could be used in monitoring the health of the infant and even the mother. PMID:27796459

Determination of itopride in human plasma by liquid chromatography coupled to tandem mass spectrometric detection: application to a bioequivalence study.

PubMed

Lee, Heon-Woo; Seo, Ji-Hyung; Choi, Seung-Ki; Lee, Kyung-Tae

2007-01-30

A simple method using a one-step liquid-liquid extraction (LLE) with butyl acetate followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of itopride in human plasma, using sulpiride as an internal standard (IS). Acquisition was performed in multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 359.5>166.1 for itopride and m/z 342.3>111.6 for IS, respectively. Analytes were chromatographed on an YMC C18 reverse-phase chromatographic column by isocratic elution with 1 mM ammonium acetate buffer-methanol (20: 80, v/v; pH 4.0 adjusted with acetic acid). Results were linear (r2=0.9999) over the studied range (0.5-1000 ng mL(-1)) with a total analysis time per run of 2 min for LC-MS/MS. The developed method was validated and successfully applied to bioequivalence studies of itopride hydrochloride in healthy male volunteers.

Characterization of nucleosides and nucleobases in natural Cordyceps by HILIC-ESI/TOF/MS and HILIC-ESI/MS.

PubMed

Zhao, Heng-Qiang; Wang, Xiao; Li, Hong-Mei; Yang, Bin; Yang, Hong-Jun; Huang, Luqi

2013-08-15

A method combining hydrophilic interaction chromatography (HILIC) and electrospray ionization mass spectrometry (ESI-MS) was developed for the characterization and determination of natural Cordyceps. Separation was achieved on a Waters Xbridge Amide column with gradient elution. Identification of 15 target nucleosides and nucleobases was based on retention time, UV spectra and mass measurements of the protonated molecules ([M+H]⁺) and main fragment ions (ESI-TOF/MS). Eight non-target compounds were tentatively identified by ESI-TOF/MS. The 15 target compounds were quantified by HILIC-ESI-MS/MS using time-programmed selective ion monitoring or multiple reaction monitoring in positive-ion mode under optimized mass conditions. This technique showed good linearity, repeatability and recovery. This approach was also successfully implemented in the analysis of nucleosides and nucleobases in 12 batches of natural Cordyceps samples that were collected from different regions in China. The developed HILIC-ESI-MS method exhibited clear advantages in identifying and determining highly polar bioactive components in Cordyceps, as well as their quality control.

Analytical methods for multiresidue determination of sulfonamides and trimethoprim in meat and ground water samples by CE-MS and CE-MS/MS.

PubMed

Soto-Chinchilla, Jorge J; García-Campaña, Ana M; Gámiz-Gracia, Laura

2007-11-01

This paper presents two methods based on CZE-MS detection and CZE-MS/MS detection developed for the multiresidue determination of ten sulfonamides (sulfapyridine, sulfadoxin, sulfamethazine, sulfadimethoxine, sulfameter, sulfamerazine, sulfachlorpyridazine, sulfadiazine, sulfamethoxazole, and sulfamethizole) and a potentiator, trimethoprim (TMP), whose contents are regulated by the EU Council Regulation no. 2377/90 in animal edible tissues. Experimental designs were employed to optimize the electrospray conditions. MS/MS experiments using an IT as analyzer operating in multiple reaction monitoring (MRM) mode were carried out to achieve the minimum number of points according to the 2002/657/EC European Decision for unambiguous identification. The proposed procedures have been compared in terms of the performance characteristics and trueness. The limits of detection and quantification were in all cases lower than the maximum residue limits legislated for these compounds and the recoveries were satisfactory, being possible the application for their monitoring in foodstuff of animal origin and in environmental samples, allowing the determination of sulfonamides and TMP residues in meat and in superficial water in the low microg/L range.

Identification and Relative Quantification of Bioactive Peptides Sequentially Released during Simulated Gastrointestinal Digestion of Commercial Kefir.

PubMed

Liu, Yufang; Pischetsrieder, Monika

2017-03-08

Health-promoting effects of kefir may be partially caused by bioactive peptides. To evaluate their formation or degradation during gastrointestinal digestion, we monitored changes of the peptide profile in a model of (1) oral, (2) gastric, and (3) small intestinal digestion of kefir. Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses revealed clearly different profiles between digests 2/3 and kefir/digest 1. Subsequent ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry identified 92 peptides in total (25, 25, 43, and 30, partly overlapping in kefir and digests 1, 2, and 3, respectively), including 16 peptides with ascribed bioactivity. Relative quantification in scheduled multiple reaction monitoring mode showed that many bioactive peptides were released by simulated digestion. Most prominently, the concentration of angiotensin-converting enzyme inhibitor β-casein 203-209 increased approximately 10 000-fold after combined oral, gastric, and intestinal digestion. Thus, physiological digestive processes may promote bioactive peptide formation from proteins and oligopeptides in kefir. Furthermore, bioactive peptides present in certain compartments of the gastrointestinal tract may exert local physiological effects.

40 CFR 264.98 - Detection monitoring program.

Code of Federal Regulations, 2010 CFR

2010-07-01

... conductance, total organic carbon, or total organic halogen), waste constituents, or reaction products that... reaction products in the unsaturated zone beneath the waste management area; (3) The detectability of indicator parameters, waste constituents, and reaction products in ground water; and (4) The concentrations...

[Metabolites and metabolic pathways of mesaconitine in rat liver microsomal investigated by using UPLC-MS/MS method in vitro].

PubMed

Bi, Yun-Feng; Liu, Shu; Zhang, Rui-Xing; Song, Feng-Rui; Liu, Zhi-Qiang

2013-12-01

Mesaconitine was incubated with rat liver microsomes in vitro. The metabolites of mesaconitine in rat liver microsomes were identified by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method with high resolution power. A typical reaction mixture of 100 mol L-1 Tris-HCI buffer (pH 7.4) containing 0.5 gL-1 microsomal protein and 50 micro molL-1 mesaconitine was prepared. The above reaction mixture was divided into six groups, and the volume of each group was 200 micro L. The incubation mixture was pre-incubated at 37 degrees C for 2 min and the reactions were initiated by adding NADPH generating system. After 90 min incubation at 37 degrees C, 200 micro L of acetonitrile was added to each group to stop the reaction. The metabolites of mesaconitine were investigated by UPLC-MS/MS method. Mesaconitine and 6 metabolites M1-M6 were found in the incubation system. The structures were characterized according to the data from MS/MS spectra and literatures. The metabolic reactions of mesaconitine in rat liver microsomes included the demethylation, deacetylation, dehydrogenation and hydroxylation. The major metabolic pathways of mesaconitine in rat liver microsomes were determined by UPLC-MS/MS on multiple reaction monitoring (MRM) mode combined with specific inhibitors of cytochrome P450 (CYP) isoforms, including alpha-naphthoflavone (CYP1A2), quinine (CYP2D), diethyldithiocarbamate (CYP2E1), ketoconazole (CYP3A) and sulfaphenazole (CYP2C), separately. Mesaconitine was mainly metabolized by CYP3A. CYP2C and CYP2D were also more important CYP isoforms for the metabolism reactions of mesaconitine, but CYP1A2 and CYP2E1 haven't any contribution to MA metabolism in rat liver microsomes.

Microbial Monitoring of Common Opportunistic Pathogens by Comparing Multiple Real-Time PCR Platforms for Potential Space Applications

NASA Technical Reports Server (NTRS)

Oubre, Cherie M.; Birmele, Michele N.; Castro, Victoria A.; Venkateswaran, Kasthuri J.; Vaishampayan, Parag A.; Jones, Kathy U.; Singhal, Adesh; Johnston, Angela S.; Roman, Monserrate C.; Ozbolt, Tamra A.;

Photoneutron cross sections for 59Co : Systematic uncertainties of data from various experiments

NASA Astrophysics Data System (ADS)

Varlamov, V. V.; Davydov, A. I.; Ishkhanov, B. S.

2017-09-01

Data on partial photoneutron reaction cross sections (γ ,1n), (γ ,2n), and (γ ,3n) for 59Co obtained in two experiments carried out at Livermore (USA) were analyzed. The sources of radiation in both experiments were the monoenergetic photon beams from the annihilation in flight of relativistic positrons. The total yield was sorted by the neutron multiplicity, taking into account the difference in the neutron energy spectra for different multiplicity. The two quoted studies differ in the method of determining the neutron. Significant systematic disagreements between the results of the two experiments exist. They are considered to be caused by large systematic uncertainties in partial cross sections, since they do not satisfy physical criteria for reliability of the data. To obtain reliable cross sections of partial and total photoneutron reactions a new method combining experimental data and theoretical evaluation was used. It is based on the experimental neutron yield cross section which is rather independent of neutron multiplicity and the transitional neutron multiplicity functions of the combined photonucleon reaction model (CPNRM). The model transitional multiplicity functions were used for the decomposition of the neutron yield cross section into the contributions of partial reactions. The results of the new evaluation noticeably differ from the partial cross sections obtained in the two experimental studies are under discussion.

Different enzyme kinetic models.

PubMed

Seibert, Eleanore; Tracy, Timothy S

2014-01-01

As described in Chapter 2 , a large number of enzymatic reactions can be adequately described by Michaelis-Menten kinetics. The Michaelis-Menten equation represents a rectangular hyperbola, with a y-asymptote at the V max value. In many cases, more complex kinetic models are required to explain the observed data. Atypical kinetic profiles are believed to arise from the simultaneous binding of multiple molecules within the active site of the enzyme (Tracy and Hummel, Drug Metab Rev 36:231-242, 2004). Several cytochromes P450 have large active sites that enable binding of multiple molecules (Wester et al. J Biol Chem 279:35630-35637, 2004; Yano et al. J Biol Chem 279:38091-38094, 2004). Thus, atypical kinetics are not uncommon in in vitro drug metabolism studies. This chapter covers enzyme kinetic reactions in which a single enzyme has multiple binding sites for substrates and/or inhibitors as well as reactions catalyzed by multiple enzymes.

Mapping the Complete Reaction Path of a Complex Photochemical Reaction.

PubMed

Smith, Adam D; Warne, Emily M; Bellshaw, Darren; Horke, Daniel A; Tudorovskya, Maria; Springate, Emma; Jones, Alfred J H; Cacho, Cephise; Chapman, Richard T; Kirrander, Adam; Minns, Russell S

2018-05-04

We probe the dynamics of dissociating CS_{2} molecules across the entire reaction pathway upon excitation. Photoelectron spectroscopy measurements using laboratory-generated femtosecond extreme ultraviolet pulses monitor the competing dissociation, internal conversion, and intersystem crossing dynamics. Dissociation occurs either in the initially excited singlet manifold or, via intersystem crossing, in the triplet manifold. Both product channels are monitored and show that, despite being more rapid, the singlet dissociation is the minor product and that triplet state products dominate the final yield. We explain this by a consideration of accurate potential energy curves for both the singlet and triplet states. We propose that rapid internal conversion stabilizes the singlet population dynamically, allowing for singlet-triplet relaxation via intersystem crossing and the efficient formation of spin-forbidden dissociation products on longer timescales. The study demonstrates the importance of measuring the full reaction pathway for defining accurate reaction mechanisms.

Mapping the Complete Reaction Path of a Complex Photochemical Reaction

NASA Astrophysics Data System (ADS)

Smith, Adam D.; Warne, Emily M.; Bellshaw, Darren; Horke, Daniel A.; Tudorovskya, Maria; Springate, Emma; Jones, Alfred J. H.; Cacho, Cephise; Chapman, Richard T.; Kirrander, Adam; Minns, Russell S.

2018-05-01

We probe the dynamics of dissociating CS2 molecules across the entire reaction pathway upon excitation. Photoelectron spectroscopy measurements using laboratory-generated femtosecond extreme ultraviolet pulses monitor the competing dissociation, internal conversion, and intersystem crossing dynamics. Dissociation occurs either in the initially excited singlet manifold or, via intersystem crossing, in the triplet manifold. Both product channels are monitored and show that, despite being more rapid, the singlet dissociation is the minor product and that triplet state products dominate the final yield. We explain this by a consideration of accurate potential energy curves for both the singlet and triplet states. We propose that rapid internal conversion stabilizes the singlet population dynamically, allowing for singlet-triplet relaxation via intersystem crossing and the efficient formation of spin-forbidden dissociation products on longer timescales. The study demonstrates the importance of measuring the full reaction pathway for defining accurate reaction mechanisms.

Beyond the Western front: Targeted proteomics and organelle abundance profiling

DOE PAGES

Parsons, Harriet T.; Heazlewood, Joshua L.

2015-05-05

The application of westerns or immunoblotting techniques for assessing the composition, dynamics, and purity of protein extracts from plant material has become common practice. While the approach is reproducible, can be readily applied and is generally considered robust, the field of plant science suffers from a lack of antibody variety against plant proteins. The development of approaches that employ mass spectrometry to enable both relative and absolute quantification of many hundreds of proteins in a single sample from a single analysis provides a mechanism to overcome the expensive impediment in having to develop antibodies in plant science. Here, we considermore » it an opportune moment to consider and better develop the adoption of multiple reaction monitoring (MRM)-based analyses in plant biochemistry.« less

Novel liquid chromatography-electrospray ionization mass spectrometry method for the quantification in human urine of microbial aromatic acid metabolites derived from dietary polyphenols.

PubMed

Gonthier, Marie-Paule; Rios, Laurent Y; Verny, Marie- Anne; Rémésy, Christian; Scalbert, Augustin

2003-06-15

An HPLC-ESI-MS-MS method was developed to quantify in human urine fourteen aromatic acids known as metabolites of dietary polyphenols. These metabolites were determined simultaneously in a single 20-min chromatographic analysis with multiple reaction monitoring detection. The inter- and intra-day precisions, calculated from quality control samples were 8.8 and 5.3%, respectively, and the mean accuracy was 2.3%. The method was tested on urine samples collected from one healthy volunteer who consumed a polyphenol-rich diet for 3 days. Increased levels of several aromatic acid metabolites were observed, demonstrating that the method can be used to detect changes in the excretion of microbial metabolites induced by the consumption of polyphenol-containing foods in humans.

Metabolite fingerprinting of Camptotheca acuminata and the HPLC-ESI-MS/MS analysis of camptothecin and related alkaloids.

PubMed

Montoro, Paola; Maldini, Mariateresa; Piacente, Sonia; Macchia, Mario; Pizza, Cosimo

2010-01-20

The major phytochemical constituents, namely, alkaloids, flavonoids and ellagic acid derivatives, of leaves of Camptotheca acuminata were identified using high performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ESI-MS) in extracts of plants cultivated in Italy and collected at different growth stages. Alkaloids related to camptothecin were identified and quantified by HPLC coupled with ESI-tandem mass spectrometry (MS/MS) employing, respectively, an ion trap and a triple quadrupole mass analyser. The fragmentation patterns of alkaloids related to camptothecin were analysed and a specific Multiple Reaction Monitoring HPLC-MS/MS method was developed for the quantitative determination of these constituents. The described method provides high sensitivity and specificity for the characterisation and quantitative determination of the alkaloids in C. acuminata.

Targeted Quantification of Isoforms of a Thylakoid-Bound Protein: MRM Method Development.

PubMed

Bru-Martínez, Roque; Martínez-Márquez, Ascensión; Morante-Carriel, Jaime; Sellés-Marchart, Susana; Martínez-Esteso, María José; Pineda-Lucas, José Luis; Luque, Ignacio

2018-01-01

Targeted mass spectrometric methods such as selected/multiple reaction monitoring (SRM/MRM) have found intense application in protein detection and quantification which competes with classical immunoaffinity techniques. It provides a universal procedure to develop a fast, highly specific, sensitive, accurate, and cheap methodology for targeted detection and quantification of proteins based on the direct analysis of their surrogate peptides typically generated by tryptic digestion. This methodology can be advantageously applied in the field of plant proteomics and particularly for non-model species since immunoreagents are scarcely available. Here, we describe the issues to take into consideration in order to develop a MRM method to detect and quantify isoforms of the thylakoid-bound protein polyphenol oxidase from the non-model and database underrepresented species Eriobotrya japonica Lindl.

Beyond the Western front: Targeted proteomics and organelle abundance profiling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parsons, Harriet T.; Heazlewood, Joshua L.

The application of westerns or immunoblotting techniques for assessing the composition, dynamics, and purity of protein extracts from plant material has become common practice. While the approach is reproducible, can be readily applied and is generally considered robust, the field of plant science suffers from a lack of antibody variety against plant proteins. The development of approaches that employ mass spectrometry to enable both relative and absolute quantification of many hundreds of proteins in a single sample from a single analysis provides a mechanism to overcome the expensive impediment in having to develop antibodies in plant science. Here, we considermore » it an opportune moment to consider and better develop the adoption of multiple reaction monitoring (MRM)-based analyses in plant biochemistry.« less

LC-MS Data Processing with MAVEN: A Metabolomic Analysis and Visualization Engine

PubMed Central

Clasquin, Michelle F.; Melamud, Eugene; Rabinowitz, Joshua D.

2014-01-01

MAVEN is an open-source software program for interactive processing of LC-MS-based metabolomics data. MAVEN enables rapid and reliable metabolite quantitation from multiple reaction monitoring data or high-resolution full-scan mass spectrometry data. It automatically detects and reports peak intensities for isotope-labeled metabolites. Menu-driven, click-based navigation allows visualization of raw and analyzed data. Here we provide a User Guide for MAVEN. Step-by-step instructions are provided for data import, peak alignment across samples, identification of metabolites that differ strongly between biological conditions, quantitation and visualization of isotope-labeling patterns, and export of tables of metabolite-specific peak intensities. Together, these instructions describe a workflow that allows efficient processing of raw LC-MS data into a form ready for biological analysis. PMID:22389014

An MRM-based workflow for absolute quantitation of lysine-acetylated metabolic enzymes in mouse liver.

PubMed

Xu, Leilei; Wang, Fang; Xu, Ying; Wang, Yi; Zhang, Cuiping; Qin, Xue; Yu, Hongxiu; Yang, Pengyuan

2015-12-07

As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.

Quantification of Free Phenytoin by Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS).

PubMed

Peat, Judy; Frazee, Clint; Garg, Uttam

2016-01-01

Phenytoin (diphenylhydantoin) is an anticonvulsant drug that has been used for decades for the treatment of many types of seizures. The drug is highly protein bound and measurement of free-active form of the drug is warranted particularly in patients with conditions that can affect drug protein binding. Here, we describe a LC/MS/MS method for the measurement of free phenytoin. Free drug is separated by ultrafiltration of serum or plasma. Ultrafiltrate is treated with acetonitrile containing internal standard phenytoin d-10 to precipitate proteins. The mixture is centrifuged and supernatant is injected onto LC-MS-MS, and analyzed using multiple reaction monitoring. This method is linear from 0.1 to 4.0 μg/mL and does not demonstrate any significant ion suppression or enhancement.

[Rapid screening the alkaloids of poppy shell in hot pot condiment, beef noodle soup and seasoning by direct analysis in real time-tandem mass spectrometry].

PubMed

Zhang, Baile; Gao, Lihong; Xie, Yingshuang; Zhou, Wei; Chen, Xiaofeng; Lei, Chunni; Zhang, Huan

2017-07-08

A direct analysis in real time tandem mass spectrometry (DART-MS/MS) method was established for quickly screening five illegally added alkaloids of poppy shell from the hot pot condiment, beef noodle soup and seasoning. The samples were extracted and purified by acetonitrile, and then injected under the conditions of ionization temperature of 300℃, grid electrode voltage of 150 V and sampling rate of 0.8 mm/s using DART in the positive ion mode. The determination was conducted by tandem mass spectrometry in positive ESI mode under multiple reaction monitoring (MRM) mode. The method is simple and rapid, and can meet the requirement of rapid screening and analysis of large quantities of samples.

LC-MS data processing with MAVEN: a metabolomic analysis and visualization engine.

PubMed

Clasquin, Michelle F; Melamud, Eugene; Rabinowitz, Joshua D

2012-03-01

MAVEN is an open-source software program for interactive processing of LC-MS-based metabolomics data. MAVEN enables rapid and reliable metabolite quantitation from multiple reaction monitoring data or high-resolution full-scan mass spectrometry data. It automatically detects and reports peak intensities for isotope-labeled metabolites. Menu-driven, click-based navigation allows visualization of raw and analyzed data. Here we provide a User Guide for MAVEN. Step-by-step instructions are provided for data import, peak alignment across samples, identification of metabolites that differ strongly between biological conditions, quantitation and visualization of isotope-labeling patterns, and export of tables of metabolite-specific peak intensities. Together, these instructions describe a workflow that allows efficient processing of raw LC-MS data into a form ready for biological analysis.

A new dimethyl labeling-based SID-MRM-MS method and its application to three proteases involved in insulin maturation.

PubMed

Cheng, Dongwan; Zheng, Li; Hou, Junjie; Wang, Jifeng; Xue, Peng; Yang, Fuquan; Xu, Tao

2015-01-01

The absolute quantification of target proteins in proteomics involves stable isotope dilution coupled with multiple reactions monitoring mass spectrometry (SID-MRM-MS). The successful preparation of stable isotope-labeled internal standard peptides is an important prerequisite for the SID-MRM absolute quantification methods. Dimethyl labeling has been widely used in relative quantitative proteomics and it is fast, simple, reliable, cost-effective, and applicable to any protein sample, making it an ideal candidate method for the preparation of stable isotope-labeled internal standards. MRM mass spectrometry is of high sensitivity, specificity, and throughput characteristics and can quantify multiple proteins simultaneously, including low-abundance proteins in precious samples such as pancreatic islets. In this study, a new method for the absolute quantification of three proteases involved in insulin maturation, namely PC1/3, PC2 and CPE, was developed by coupling a stable isotope dimethyl labeling strategy for internal standard peptide preparation with SID-MRM-MS quantitative technology. This method offers a new and effective approach for deep understanding of the functional status of pancreatic β cells and pathogenesis in diabetes.

Continuous monitoring of dissolved gases with membrane inlet mass spectrometry to fingerprint river biochemical activity

NASA Astrophysics Data System (ADS)

Vautier, Camille; Chatton, Eliot; Abbott, Benjamin; Harjung, Astrid; Labasque, Thierry; Guillou, Aurélie; Pannard, Alexandrine; Piscart, Christophe; Laverman, Anniet; Kolbe, Tamara; Massé, Stéphanie; de Dreuzy, Jean-Raynald; Thomas, Zahra; Aquilina, Luc; Pinay, Gilles

2017-04-01

Water quality in rivers results from biogeochemical processes in contributing hydrological compartments (soils, aquifers, hyporheic and riparian zones) and biochemical activity in the river network itself. Consequently, chemical fluxes fluctuate on multiple spatial and temporal scales, leading eventually to complex concentration signals in rivers. We characterized these fluctuations with innovative continuous monitoring of dissolved gases, to quantify transport and reaction processes occurring in different hydrological compartments. We performed stream-scale experiments in two headwater streams in Brittany, France. Factorial injections of inorganic nitrogen (NH4NO3), inorganic phosphate (P2O5) and multiple sources of labile carbon (acetate, tryptophan) were implemented in the two streams. We used a new field application of membrane inlet mass spectrometry to continuously monitor dissolved gases for multiple day-night periods (Chatton et al., 2016). Quantified gases included He, O2, N2, CO2, CH4, N2O, and 15N of dissolved N2 and N2O. We calibrated and assessed the methodology with well-established complementary techniques including gas chromatography and high-frequency water quality sensors. Wet chemistry and radon analysis complemented the study. The analyses provided several methodological and ecological insights and demonstrated that high frequency variations linked to background noise can be efficiently determined and filtered to derive effective fluxes. From a more fundamental point of view, the tested stream segments were fully characterized with extensive sampling of riverbeds and laboratory experiments, allowing scaling of point-level microbial and invertebrate diversity and activity on in-stream processing. This innovative technology allows fully-controlled in-situ experiments providing rich information with a high signal to noise ratio. We present the integrated nutrient demand and uptake and discuss limiting processes and elements at the reach and catchment scales. Eliot Chatton, Thierry Labasque, Jérôme de La Bernardie, Nicolas Guihéneuf, Olivier Bour, Luc Aquilina. 2016. Field Continuous Measurement of Dissolved Gases with a CF-MIMS: Applications to the Physics and Biogeochemistry of Groundwater Flow. Environ. Sci. Technol.

Perspective: Chemical reactions in ionic liquids monitored through the gas (vacuum)/liquid interface.

PubMed

Maier, F; Niedermaier, I; Steinrück, H-P

2017-05-07

This perspective analyzes the potential of X-ray photoelectron spectroscopy under ultrahigh vacuum (UHV) conditions to follow chemical reactions in ionic liquids in situ. Traditionally, only reactions occurring on solid surfaces were investigated by X-ray photoelectron spectroscopy (XPS) in situ. This was due to the high vapor pressures of common liquids or solvents, which are not compatible with the required UHV conditions. It was only recently realized that the situation is very different when studying reactions in Ionic Liquids (ILs), which have an inherently low vapor pressure, and first studies have been performed within the last years. Compared to classical spectroscopy techniques used to monitor chemical reactions, the advantage of XPS is that through the analysis of their core levels all relevant elements can be quantified and their chemical state can be analyzed under well-defined (ultraclean) conditions. In this perspective, we cover six very different reactions which occur in the IL, with the IL, or at an IL/support interface, demonstrating the outstanding potential of in situ XPS to gain insights into liquid phase reactions in the near-surface region.

Developing Inventory and Monitoring Programs Based on Multiple Objectives

Treesearch

Daniel L. Schmoldt; David L. Peterson; David G. Silsbee

1995-01-01

Resource inventory and monitoring (I&M) programs in national parks combine multiple objectives in order to create a plan of action over a finite time horizon. Because all program activities are constrained by time and money, it is critical to plan I&M activities that make the best use of available agency resources. However, multiple objectives complicate a...

Protein isoform-specific validation defines multiple chloride intracellular channel and tropomyosin isoforms as serological biomarkers of ovarian cancer

PubMed Central

Tang, Hsin-Yao; Beer, Lynn A.; Tanyi, Janos L.; Zhang, Rugang; Liu, Qin; Speicher, David W.

2013-01-01

New serological biomarkers for early detection and clinical management of ovarian cancer are urgently needed, and many candidates have been reported. A major challenge frequently encountered when validating candidates in patients is establishing quantitative assays that distinguish between highly homologous proteins. The current study tested whether multiple members of two recently discovered ovarian cancer biomarker protein families, chloride intracellular channel (CLIC) proteins and tropomyosins (TPM), were detectable in ovarian cancer patient sera. A multiplexed, label-free multiple reaction monitoring (MRM) assay was established to target peptides specific to all detected CLIC and TPM family members, and their serum levels were quantitated for ovarian cancer patients and non-cancer controls. In addition to CLIC1 and TPM1, which were the proteins initially discovered in a xenograft mouse model, CLIC4, TPM2, TPM3, and TPM4 were present in ovarian cancer patient sera at significantly elevated levels compared with controls. Some of the additional biomarkers identified in this homolog-centric verification and validation approach may be superior to the previously identified biomarkers at discriminating between ovarian cancer and non-cancer patients. This demonstrates the importance of considering all potential protein homologs and using quantitative assays for cancer biomarker validation with well-defined isoform specificity. PMID:23792823

Thin layer chromatography coupled with surface-enhanced Raman scattering as a facile method for on-site quantitative monitoring of chemical reactions.

PubMed

Zhang, Zong-Mian; Liu, Jing-Fu; Liu, Rui; Sun, Jie-Fang; Wei, Guo-Hua

2014-08-05

By coupling surface-enhanced Raman spectroscopy (SERS) with thin layer chromatography (TLC), a facile and powerful method was developed for on-site monitoring the process of chemical reactions. Samples were preseparated on a TLC plate following a common TLC procedure, and then determined by SERS after fabricating a large-area, uniform SERS substrate on the TLC plate by spraying gold nanoparticles (AuNPs). Reproducible and strong SERS signals were obtained with substrates prepared by spraying 42-nm AuNPs at a density of 5.54 × 10(10) N/cm(2) on the TLC plate. The capacity of this TLC-SERS method was evaluated by monitoring a typical Suzuki coupling reaction of phenylboronic acid and 2-bromopyridine as a model. Results showed that this proposed method is able to identify reaction product that is invisible to the naked eye, and distinguish the reactant 2-bromopyridine and product 2-phenylpyridine, which showed almost the same retention factors (R(f)). Under the optimized conditions, the peak area of the characteristic Raman band (755 cm(-1)) of the product 2-phenylpyridine showed a good linear correlation with concentration in the range of 2-200 mg/L (R(2) = 0.9741), the estimated detection limit (1 mg/L 2-phenylpyridine) is much lower than the concentration of the chemicals in the common organic synthesis reaction system, and the product yield determined by the proposed TLC-SERS method agreed very well with that by UPLC-MS/MS. In addition, a new byproduct in the reaction system was found and identified through continuous Raman detection from the point of sample to the solvent front. This facile TLC-SERS method is quick, easy to handle, low-cost, sensitive, and can be exploited in on-site monitoring the processes of chemical reactions, as well as environmental and biological processes.

Life Span Differences in Electrophysiological Correlates of Monitoring Gains and Losses during Probabilistic Reinforcement Learning

ERIC Educational Resources Information Center

Hammerer, Dorothea; Li, Shu-Chen; Muller, Viktor; Lindenberger, Ulman

2011-01-01

By recording the feedback-related negativity (FRN) in response to gains and losses, we investigated the contribution of outcome monitoring mechanisms to age-associated differences in probabilistic reinforcement learning. Specifically, we assessed the difference of the monitoring reactions to gains and losses to investigate the monitoring of…

Flow chemistry: intelligent processing of gas-liquid transformations using a tube-in-tube reactor.

PubMed

Brzozowski, Martin; O'Brien, Matthew; Ley, Steven V; Polyzos, Anastasios

2015-02-17

CONSPECTUS: The previous decade has witnessed the expeditious uptake of flow chemistry techniques in modern synthesis laboratories, and flow-based chemistry is poised to significantly impact our approach to chemical preparation. The advantages of moving from classical batch synthesis to flow mode, in order to address the limitations of traditional approaches, particularly within the context of organic synthesis are now well established. Flow chemistry methodology has led to measurable improvements in safety and reduced energy consumption and has enabled the expansion of available reaction conditions. Contributions from our own laboratories have focused on the establishment of flow chemistry methods to address challenges associated with the assembly of complex targets through the development of multistep methods employing supported reagents and in-line monitoring of reaction intermediates to ensure the delivery of high quality target compounds. Recently, flow chemistry approaches have addressed the challenges associated with reactions utilizing reactive gases in classical batch synthesis. The small volumes of microreactors ameliorate the hazards of high-pressure gas reactions and enable improved mixing with the liquid phase. Established strategies for gas-liquid reactions in flow have relied on plug-flow (or segmented flow) regimes in which the gas plugs are introduced to a liquid stream and dissolution of gas relies on interfacial contact of the gas bubble with the liquid phase. This approach confers limited control over gas concentration within the liquid phase and is unsuitable for multistep methods requiring heterogeneous catalysis or solid supported reagents. We have identified the use of a gas-permeable fluoropolymer, Teflon AF-2400, as a simple method of achieving efficient gas-liquid contact to afford homogeneous solutions of reactive gases in flow. The membrane permits the transport of a wide range of gases with significant control of the stoichiometry of reactive gas in a given reaction mixture. We have developed a tube-in-tube reactor device consisting of a pair of concentric capillaries in which pressurized gas permeates through an inner Teflon AF-2400 tube and reacts with dissolved substrate within a liquid phase that flows within a second gas impermeable tube. This Account examines our efforts toward the development of a simple, unified methodology for the processing of gaseous reagents in flow by way of development of a tube-in-tube reactor device and applications to key C-C, C-N, and C-O bond forming and hydrogenation reactions. We further describe the application to multistep reactions using solid-supported reagents and extend the technology to processes utilizing multiple gas reagents. A key feature of our work is the development of computer-aided imaging techniques to allow automated in-line monitoring of gas concentration and stoichiometry in real time. We anticipate that this Account will illustrate the convenience and benefits of membrane tube-in-tube reactor technology to improve and concomitantly broaden the scope of gas/liquid/solid reactions in organic synthesis.

pH-sensitive pHluorins as a molecular sensor for in situ monitoring of enzyme-catalyzed prodrug activation.

PubMed

Liu, Hui; Cao, Xiaodan; Wang, Ping; Ma, Xingyuan

2017-07-01

This work examines the feasibility of using a pH-sensitive fluorescent protein as a molecular reporter for enzyme-catalyzed prodrug activation reaction. Specifically, a ratiometric pHluorins was examined for detection of the activity of horseradish peroxidase (HRP) for the activation of indole-3-acetic acid. The pHluorins and HRP were conjugated chemically, forming a biocatalyst with a self-reporting function. Results showed that the characteristic fluorescence intensity ratio of the conjugate shifted from 1.47 to 1.40 corresponding to the progress of the prodrug activation reaction. The effectiveness of applying the conjugate for inhibition of the growth of Bcap-37 cells was also demonstrated simultaneously with reaction monitoring. The results reveal a very promising approach to realizing in situ monitoring of enzyme activities based on pH shifting for enzyme-based prodrug therapy applications. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Raman spectroscopic approach to monitor the in vitro cyclization of creatine → creatinine

NASA Astrophysics Data System (ADS)

Gangopadhyay, Debraj; Sharma, Poornima; Singh, Sachin Kumar; Singh, Pushkar; Tarcea, Nicolae; Deckert, Volker; Popp, Jürgen; Singh, Ranjan K.

2015-01-01

The creatine → creatinine cyclization, an important metabolic phenomenon has been initiated in vitro at acidic pH and studied through Raman spectroscopic and DFT approach. The equilibrium composition of neutral, zwitterionic and protonated microspecies of creatine has been monitored with time as the reaction proceeds. Time series Raman spectra show clear signature of creatinine formation at pH 3 after ∼240 min at room temperature and reaction is faster at higher temperature. The spectra at pH 1 and pH 5 do not show such signature up to 270 min implying faster reaction rate at pH 3.

The effect of milling frequency on a mechanochemical organic reaction monitored by in situ Raman spectroscopy

PubMed Central

Julien, Patrick A; Malvestiti, Ivani

2017-01-01

We provide the first in situ and real-time study of the effect of milling frequency on the course of a mechanochemical organic reaction conducted using a vibratory shaker (mixer) ball mill. The use of in situ Raman spectroscopy for real-time monitoring of the mechanochemical synthesis of a 2,3-diphenylquinoxaline derivative revealed a pronounced dependence of chemical reactivity on small variations in milling frequency. In particular, in situ measurements revealed the establishment of two different regimes of reaction kinetics at different frequencies, providing tentative insight into processes of mechanical activation in organic mechanochemical synthesis. PMID:29114323

Metal oxide nanoparticle mediated enhanced Raman scattering and its use in direct monitoring of interfacial chemical reactions.

PubMed

Li, Li; Hutter, Tanya; Finnemore, Alexander S; Huang, Fu Min; Baumberg, Jeremy J; Elliott, Stephen R; Steiner, Ullrich; Mahajan, Sumeet

2012-08-08

Metal oxide nanoparticles (MONPs) have widespread usage across many disciplines, but monitoring molecular processes at their surfaces in situ has not been possible. Here we demonstrate that MONPs give highly enhanced (×10(4)) Raman scattering signals from molecules at the interface permitting direct monitoring of their reactions, when placed on top of flat metallic surfaces. Experiments with different metal oxide materials and molecules indicate that the enhancement is generic and operates at the single nanoparticle level. Simulations confirm that the amplification is principally electromagnetic and is a result of optical modulation of the underlying plasmonic metallic surface by MONPs, which act as scattering antennae and couple light into the confined region sandwiched by the underlying surface. Because of additional functionalities of metal oxides as magnetic, photoelectrochemical and catalytic materials, enhanced Raman scattering mediated by MONPs opens up significant opportunities in fundamental science, allowing direct tracking and understanding of application-specific transformations at such interfaces. We show a first example by monitoring the MONP-assisted photocatalytic decomposition reaction of an organic dye by individual nanoparticles.

Age-Related Differences in Multiple Task Monitoring

PubMed Central

Todorov, Ivo; Del Missier, Fabio; Mäntylä, Timo

2014-01-01

Coordinating multiple tasks with narrow deadlines is particularly challenging for older adults because of age related decline in cognitive control functions. We tested the hypothesis that multiple task performance reflects age- and gender-related differences in executive functioning and spatial ability. Young and older adults completed a multitasking session with four monitoring tasks as well as separate tasks measuring executive functioning and spatial ability. For both age groups, men exceeded women in multitasking, measured as monitoring accuracy. Individual differences in executive functioning and spatial ability were independent predictors of young adults' monitoring accuracy, but only spatial ability was related to sex differences. For older adults, age and executive functioning, but not spatial ability, predicted multitasking performance. These results suggest that executive functions contribute to multiple task performance across the adult life span and that reliance on spatial skills for coordinating deadlines is modulated by age. PMID:25215609

Age-related differences in multiple task monitoring.

PubMed

Todorov, Ivo; Del Missier, Fabio; Mäntylä, Timo

2014-01-01

Coordinating multiple tasks with narrow deadlines is particularly challenging for older adults because of age related decline in cognitive control functions. We tested the hypothesis that multiple task performance reflects age- and gender-related differences in executive functioning and spatial ability. Young and older adults completed a multitasking session with four monitoring tasks as well as separate tasks measuring executive functioning and spatial ability. For both age groups, men exceeded women in multitasking, measured as monitoring accuracy. Individual differences in executive functioning and spatial ability were independent predictors of young adults' monitoring accuracy, but only spatial ability was related to sex differences. For older adults, age and executive functioning, but not spatial ability, predicted multitasking performance. These results suggest that executive functions contribute to multiple task performance across the adult life span and that reliance on spatial skills for coordinating deadlines is modulated by age.

Characterisation of a proposed internet synthesis of N,N-dimethyltryptamine using liquid chromatography/electrospray ionisation tandem mass spectrometry.

PubMed

Martins, Cláudia P B; Freeman, Sally; Alder, John F; Brandt, Simon D

2009-08-14

The psychoactive properties of N,N-dimethyltryptamine (DMT) are known to induce altered states of consciousness in humans. These properties attract great interest from clinical, neuroscientific, clandestine and forensic communities. The Breath of Hope Synthesis was reported on an internet website as a convenient two-step methodology for the preparation of DMT. The analytical characterisation of the first stage was the subject of previous publications by the authors and involved the thermal decarboxylation of tryptophan and the formation of tryptamine. The present study reports on the characterisation of the second step of this procedure which was based on the methylation of tryptamine. This employed methyl iodide and benzyltriethylammonium chloride/sodium hydroxide as a phase transfer catalyst. The reaction product was characterised by liquid chromatography/electrospray ionisation tandem mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry. Quantitative evaluation was carried out in positive multiple reaction monitoring mode (MRM), which included synthesis of the identified reaction products. MRM screening of the product did not lead to the detection of DMT. Instead, 11.1% tryptamine starting material, 21.0% N,N,N-trimethyltryptammonium iodide (TMT) and 47.4% 1-N-methyl-TMT were detected. A 0.5% trace of the monomethylated N-methyltryptamine was also detected. This study demonstrated the impact on product purity of doubtful synthetic methodologies discussed on the internet.

Liquid microjunction surface sampling probe fluid dynamics: Characterization and application of an analyte plug formation operational mode

DOE Office of Scientific and Technical Information (OSTI.GOV)

ElNaggar, Mariam S.; Van Berkel, Gary J.

2011-08-10

The recently discovered sample plug formation and injection operational mode of a continuous flow, coaxial tube geometry, liquid microjunction surface sampling probe (LMJ-SSP) (J. Am. Soc. Mass Spectrom, 2011) was further characterized and applied for concentration and mixing of analyte extracted from multiple areas on a surface and for nanoliter-scale chemical reactions of sampled material. A transparent LMJ-SSP was constructed and colored analytes were used so that the surface sampling process, plug formation, and the chemical reactions could be visually monitored at the sampling end of the probe before being analyzed by mass spectrometry of the injected sample plug. Injectionmore » plug peak widths were consistent for plug hold times as long as the 8 minute maximum attempted (RSD below 1.5%). Furthermore, integrated injection peak signals were not significantly different for the range of hold times investigated. The ability to extract and completely mix individual samples within a fixed volume at the sampling end of the probe was demonstrated and a linear mass spectral response to the number of equivalent analyte spots sampled was observed. Lastly, using the color and mass changing chemical reduction of the redox dye 2,6-dichlorophenol-indophenol with ascorbic acid, the ability to sample, concentrate, and efficiently run reactions within the same plug volume within the probe was demonstrated.« less

Autonomous Multi-Sensor Coordination: The Science Goal Monitor

NASA Technical Reports Server (NTRS)

Koratkar, Anuradha; Grosvenor, Sandy; Jung, John; Hess, Melissa; Jones, Jeremy

2004-01-01

Many dramatic earth phenomena are dynamic and coupled. In order to fully understand them, we need to obtain timely coordinated multi-sensor observations from widely dispersed instruments. Such a dynamic observing system must include the ability to Schedule flexibly and react autonomously to sciencehser driven events; Understand higher-level goals of a sciencehser defined campaign; Coordinate various space-based and ground-based resources/sensors effectively and efficiently to achieve goals. In order to capture transient events, such a 'sensor web' system must have an automated reactive capability built into its scientific operations. To do this, we must overcome a number of challenges inherent in infusing autonomy. The Science Goal Monitor (SGM) is a prototype software tool being developed to explore the nature of automation necessary to enable dynamic observing. The tools being developed in SGM improve our ability to autonomously monitor multiple independent sensors and coordinate reactions to better observe dynamic phenomena. The SGM system enables users to specify what to look for and how to react in descriptive rather than technical terms. The system monitors streams of data to identify occurrences of the key events previously specified by the scientisther. When an event occurs, the system autonomously coordinates the execution of the users' desired reactions between different sensors. The information can be used to rapidly respond to a variety of fast temporal events. Investigators will no longer have to rely on after-the-fact data analysis to determine what happened. Our paper describes a series of prototype demonstrations that we have developed using SGM and NASA's Earth Observing-1 (EO-1) satellite and Earth Observing Systems' Aqua/Terra spacecrafts' MODIS instrument. Our demonstrations show the promise of coordinating data from different sources, analyzing the data for a relevant event, autonomously updating and rapidly obtaining a follow-on relevant image. SGM was used to investigate forest fires, floods and volcanic eruptions. We are now identifying new Earth science scenarios that will have more complex SGM reasoning. By developing and testing a prototype in an operational environment, we are also establishing and gathering metrics to gauge the success of automating science campaigns.

Microprocessor Based Real-Time Monitoring of Multiple ECG Signals

PubMed Central

Nasipuri, M.; Basu, D.K.; Dattagupta, R.; Kundu, M.; Banerjee, S.

1987-01-01

A microprocessor based system capable of realtime monitoring of multiple ECG signals has been described. The system consists of a number of microprocessors connected in a hierarchical fashion and capable of working concurrently on ECG data collected from different channels. The system can monitor different arrhythmic abnormalities for at least 36 patients even for a heart rate of 500 beats/min.

A data fusion approach for track monitoring from multiple in-service trains

NASA Astrophysics Data System (ADS)

Lederman, George; Chen, Siheng; Garrett, James H.; Kovačević, Jelena; Noh, Hae Young; Bielak, Jacobo

2017-10-01

We present a data fusion approach for enabling data-driven rail-infrastructure monitoring from multiple in-service trains. A number of researchers have proposed using vibration data collected from in-service trains as a low-cost method to monitor track geometry. The majority of this work has focused on developing novel features to extract information about the tracks from data produced by individual sensors on individual trains. We extend this work by presenting a technique to combine extracted features from multiple passes over the tracks from multiple sensors aboard multiple vehicles. There are a number of challenges in combining multiple data sources, like different relative position coordinates depending on the location of the sensor within the train. Furthermore, as the number of sensors increases, the likelihood that some will malfunction also increases. We use a two-step approach that first minimizes position offset errors through data alignment, then fuses the data with a novel adaptive Kalman filter that weights data according to its estimated reliability. We show the efficacy of this approach both through simulations and on a data-set collected from two instrumented trains operating over a one-year period. Combining data from numerous in-service trains allows for more continuous and more reliable data-driven monitoring than analyzing data from any one train alone; as the number of instrumented trains increases, the proposed fusion approach could facilitate track monitoring of entire rail-networks.

Oxygen Mass Flow Rate Generated for Monitoring Hydrogen Peroxide Stability

NASA Technical Reports Server (NTRS)

Ross, H. Richard

2002-01-01

Recent interest in propellants with non-toxic reaction products has led to a resurgence of interest in hydrogen peroxide for various propellant applications. Because peroxide is sensitive to contaminants, material interactions, stability and storage issues, monitoring decomposition rates is important. Stennis Space Center (SSC) uses thermocouples to monitor bulk fluid temperature (heat evolution) to determine reaction rates. Unfortunately, large temperature rises are required to offset the heat lost into the surrounding fluid. Also, tank penetration to accomodate a thermocouple can entail modification of a tank or line and act as a source of contamination. The paper evaluates a method for monitoring oxygen evolution as a means to determine peroxide stability. Oxygen generation is not only directly related to peroxide decomposition, but occurs immediately. Measuring peroxide temperature to monitor peroxide stability has significant limitations. The bulk decomposition of 1% / week in a large volume tank can produce in excess of 30 cc / min. This oxygen flow rate corresponds to an equivalent temperature rise of approximately 14 millidegrees C, which is difficult to measure reliably. Thus, if heat transfer were included, there would be no temperature rise. Temperature changes from the surrounding environment and heat lost to the peroxide will also mask potential problems. The use of oxygen flow measurements provides an ultra sensitive technique for monitoring reaction events and will provide an earlier indication of an abnormal decomposition when compared to measuring temperature rise.

CHROMATOGRAPHIC SEPARATION AND IDENTIFICATION OF PRODUCTS FROM THE REACTION OF DIMETHYLARSINIC ACID WITH HYDROGEN SULFIDE

EPA Science Inventory

The reaction of dimethylarsinic acid (DMAV) with hydrogen sulfide (H2S) is of biological significance and may be implicated in the overall toxicity and carcinogenicity of arsenic. The course of the reaction in aqueous phase was monitored and an initial product, dimethylthioarsin...

Online monitoring of a photocatalytic reaction by real-time high resolution FlowNMR spectroscopy.

PubMed

Hall, Andrew M R; Broomfield-Tagg, Rachael; Camilleri, Matthew; Carbery, David R; Codina, Anna; Whittaker, David T E; Coombes, Steven; Lowe, John P; Hintermair, Ulrich

2017-12-19

We demonstrate how FlowNMR spectroscopy can readily be applied to investigate photochemical reactions that require sustained input of light and air to yield mechanistic insight under realistic conditions. The Eosin Y mediated photo-oxidation of N-allylbenzylamine is shown to produce imines as primary reaction products from which undesired aldehydes form after longer reaction times. Facile variation of reaction conditions during the reaction in flow allows for probe experiments that give information about the mode of action of the photocatalyst.

Computational multiple steady states for enzymatic esterification of ethanol and oleic acid in an isothermal CSTR.

PubMed

Ho, Pang-Yen; Chuang, Guo-Syong; Chao, An-Chong; Li, Hsing-Ya

2005-05-01

The capacity of complex biochemical reaction networks (consisting of 11 coupled non-linear ordinary differential equations) to show multiple steady states, was investigated. The system involved esterification of ethanol and oleic acid by lipase in an isothermal continuous stirred tank reactor (CSTR). The Deficiency One Algorithm and the Subnetwork Analysis were applied to determine the steady state multiplicity. A set of rate constants and two corresponding steady states are computed. The phenomena of bistability, hysteresis and bifurcation are discussed. Moreover, the capacity of steady state multiplicity is extended to the family of the studied reaction networks.

40 CFR 75.82 - Monitoring of Hg mass emissions and heat input at common and multiple stacks.

Code of Federal Regulations, 2010 CFR

2010-07-01

... heat input at common and multiple stacks. 75.82 Section 75.82 Protection of Environment ENVIRONMENTAL... Provisions § 75.82 Monitoring of Hg mass emissions and heat input at common and multiple stacks. (a) Unit... systems and perform the Hg emission testing described under § 75.81(b). If reporting of the unit heat...

Fusion of microlitre water-in-oil droplets for simple, fast and green chemical assays.

PubMed

Chiu, S-H; Urban, P L

2015-08-07

A simple format for microscale chemical assays is proposed. It does not require the use of test tubes, microchips or microtiter plates. Microlitre-range (ca. 0.7-5.0 μL) aqueous droplets are generated by a commercial micropipette in a non-polar matrix inside a Petri dish. When two droplets are pipetted nearby, they spontaneously coalesce within seconds, priming a chemical reaction. Detection of the reaction product is accomplished by colorimetry, spectrophotometry, or fluorimetry using simple light-emitting diode (LED) arrays as the sources of monochromatic light, while chemiluminescence detection of the analytes present in single droplets is conducted in the dark. A smartphone camera is used as the detector. The limits of detection obtained for the developed in-droplet assays are estimated to be: 1.4 nmol (potassium permanganate by colorimetry), 1.4 pmol (fluorescein by fluorimetry), and 580 fmol (sodium hypochlorite by chemiluminescence detection). The format has successfully been used to monitor the progress of chemical and biochemical reactions over time with sub-second resolution. A semi-quantitative analysis of ascorbic acid using Tillman's reagent is presented. A few tens of individual droplets can be scanned in parallel. Rapid switching of the LED light sources with different wavelengths enables a spectral analysis of multiple droplets. Very little solid waste is produced. The assay matrix is readily recycled, thus the volume of liquid waste produced each time is also very small (typically, 1-10 μL per analysis). Various water-immiscible translucent liquids can be used as the reaction matrix: including silicone oil, 1-octanol as well as soybean cooking oil.

Analysis of Protein Adduction Kinetics by Quantitative Mass Spectrometry. Competing Adduction Reactions of Glutathione-S-Transferase P1-1 with Electrophiles

PubMed Central

Orton, Christopher R.; Liebler, Daniel C.

2007-01-01

Defining the mechanisms and consequences of protein adduction is crucial to understanding the toxicity of reactive electrophiles. Application of tandem mass spectrometry and data analysis algorithms enables detection and mapping of chemical adducts at the level of amino acid sequence. Nevertheless, detection of adducts does not indicate relative reactivity of different sites. Here we describe a method to measure the kinetics of competing adduction reactions at different sites on the same protein. Adducts are formed by electrophiles at Cys14 and Cys47 on the metabolic enzyme glutathione-S-transferase P1-1 and modification is accompanied by a loss of enzymatic activity. Relative quantitation of protein adducts was done by tagging N-termini of peptide digests with isotopically labeled phenyl isocyanate and tracking the ratio of light-tagged peptide adducts to heavy-tagged reference samples in liquid chromatography-tandem mass spectrometry analyses using a multiple reaction monitoring method. This approach was used to measure rate constants for adduction at both positions with two different model electrophiles, N-iodoacetyl-N-biotinylhexylenediamine and 1-biotinamido-4-(4′-[maleimidoethyl-cyclohexane]-carboxamido)butane. The results indicate that Cys47 was approximately 2–3-fold more reactive toward both electrophiles than was Cys14. This result was consistent with the relative reactivity of these electrophiles in a complex proteome system and with previously reported trends in reactivity of these sites. Kinetic analyses of protein modification reactions provide a means of evaluating the selectivity of reactive mediators of chemical toxicity. PMID:17433278

Chemical methods and techniques to monitor early Maillard reaction in milk products; A review.

PubMed

Aalaei, Kataneh; Rayner, Marilyn; Sjöholm, Ingegerd

2018-01-23

Maillard reaction is an extensively studied, yet unresolved chemical reaction that occurs as a result of application of the heat and during the storage of foods. The formation of advanced glycation end products (AGEs) has been the focus of several investigations recently. These molecules which are formed at the advanced stage of the Maillard reaction, are suspected to be involved in autoimmune diseases in humans. Therefore, understanding to which extent this reaction occurs in foods, is of vital significance. Because of their composition, milk products are ideal media for this reaction, especially when application of heat and prolonged storage are considered. Thus, in this work several chemical approaches to monitor this reaction in an early stage are reviewed. This is mostly done regarding available lysine blockage which takes place in the very beginning of the reaction. The most popular methods and their applications to various products are reviewed. The methods including their modifications are described in detail and their findings are discussed. The present paper provides an insight into the history of the most frequently-used methods and provides an overview on the indicators of the Maillard reaction in the early stage with its focus on milk products and especially milk powders.

Anti-Peptide Monoclonal Antibodies Generated for Immuno-Multiple Reaction Monitoring-Mass Spectrometry Assays Have a High Probability of Supporting Western blot and ELISA*

PubMed Central

Schoenherr, Regine M.; Saul, Richard G.; Whiteaker, Jeffrey R.; Yan, Ping; Whiteley, Gordon R.; Paulovich, Amanda G.

2015-01-01

Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium's (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first determination of the success rate (92%) for generating mAbs for immuno-MRM using a recombinant B cell cloning approach, which is considerably faster than the traditional hybridoma approach. PMID:25512614

Optimization by infusion of multiple reaction monitoring transitions for sensitive quantification of peptides by liquid chromatography/mass spectrometry.

PubMed

Alghanem, Bandar; Nikitin, Frédéric; Stricker, Thomas; Duchoslav, Eva; Luban, Jeremy; Strambio-De-Castillia, Caterina; Muller, Markus; Lisacek, Frédérique; Varesio, Emmanuel; Hopfgartner, Gérard

2017-05-15

In peptide quantification by liquid chromatography/mass spectrometry (LC/MS), the optimization of multiple reaction monitoring (MRM) parameters is essential for sensitive detection. We have compared different approaches to build MRM assays, based either on flow injection analysis (FIA) of isotopically labelled peptides, or on the knowledge and the prediction of the best settings for MRM transitions and collision energies (CE). In this context, we introduce MRMOptimizer, an open-source software tool that processes spectra and assists the user in selecting transitions in the FIA workflow. MS/MS spectral libraries with CE voltages from 10 to 70 V are automatically acquired in FIA mode for isotopically labelled peptides. Then MRMOptimizer determines the optimal MRM settings for each peptide. To assess the quantitative performance of our approach, 155 peptides, representing 84 proteins, were analysed by LC/MRM-MS and the peak areas were compared between: (A) the MRMOptimizer-based workflow, (B1) the SRMAtlas transitions set used 'as-is'; (B2) the same SRMAtlas set with CE parameters optimized by Skyline. 51% of the three most intense transitions per peptide were shown to be common to both A and B1/B2 methods, and displayed similar sensitivity and peak area distributions. The peak areas obtained with MRMOptimizer for transitions sharing either the precursor ion charge state or the fragment ions with the SRMAtlas set at unique transitions were increased 1.8- to 2.3-fold. The gain in sensitivity using MRMOptimizer for transitions with different precursor ion charge state and fragment ions (8% of the total), reaches a ~ 11-fold increase. Isotopically labelled peptides can be used to optimize MRM transitions more efficiently in FIA than by searching databases. The MRMOptimizer software is MS independent and enables the post-acquisition selection of MRM parameters. Coefficients of variation for optimal CE values are lower than those obtained with the SRMAtlas approach (B2) and one additional peptide was detected. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Second generation multiple reaction monitoring assays for enhanced detection of ultra-low abundance Mycobacterium tuberculosis peptides in human serum.

PubMed

Mehaffy, Carolina; Dobos, Karen M; Nahid, Payam; Kruh-Garcia, Nicole A

2017-01-01

Mycobacterium tuberculosis (Mtb) is the causative agent of Tuberculosis (TB), the number one cause of death due to an infectious disease. TB diagnosis is performed by microscopy, culture or PCR amplification of bacterial DNA, all of which require patient sputum or the biopsy of infected tissue. Detection of mycobacterial products in serum, as biomarkers of diagnosis or disease status would provide an improvement over current methods. Due to the low-abundance of mycobacterial products in serum, we have explored exosome enrichment to improve sensitivity. Mtb resides intracellularly where its secreted proteins have been shown to be packaged into host exosomes and released into the bloodstream. Exosomes can be readily purified assuring an enrichment of mycobacterial analytes from the complex mix of host serum proteins. Multiple reaction monitoring assays were optimized for the enhanced detection of 41 Mtb peptides in exosomes purified from the serum of individuals with TB. Exosomes isolated from the serum of healthy individuals was used to create and validate a unique data analysis algorithm and identify filters to reduce the rate of false positives, attributed to host m / z interference. The final optimized method was tested in 40 exosome samples from TB positive patients. Our enhanced methods provide limit of detection and quantification averaging in the low femtomolar range for detection of mycobacterial products in serum. At least one mycobacterial peptide was identified in 92.5% of the TB positive patients. Four peptides from the Mtb proteins, Cfp2, Mpt32, Mpt64 and BfrB, show normalized total peak areas significantly higher in individuals with active TB as compared to healthy controls; three of the peptides from these proteins have not previously been associated with serum exosomes from individuals with active TB disease. Some of the detected peptides were significantly associated with specific geographical locations, highlighting potential markers that can be linked to the Mtb strains circulating within each given region. An enhanced MRM method to detect ultra-low abundance Mtb peptides in human serum exosomes is demonstrated, highlighting the potential of this methodology for TB diagnostic biomarker development.

Mass Spectrometry-based Assay for High Throughput and High Sensitivity Biomarker Verification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Xuejiang; Tang, Keqi

Searching for disease specific biomarkers has become a major undertaking in the biomedical research field as the effective diagnosis, prognosis and treatment of many complex human diseases are largely determined by the availability and the quality of the biomarkers. A successful biomarker as an indicator to a specific biological or pathological process is usually selected from a large group of candidates by a strict verification and validation process. To be clinically useful, the validated biomarkers must be detectable and quantifiable by the selected testing techniques in their related tissues or body fluids. Due to its easy accessibility, protein biomarkers wouldmore » ideally be identified in blood plasma or serum. However, most disease related protein biomarkers in blood exist at very low concentrations (<1ng/mL) and are “masked” by many none significant species at orders of magnitude higher concentrations. The extreme requirements of measurement sensitivity, dynamic range and specificity make the method development extremely challenging. The current clinical protein biomarker measurement primarily relies on antibody based immunoassays, such as ELISA. Although the technique is sensitive and highly specific, the development of high quality protein antibody is both expensive and time consuming. The limited capability of assay multiplexing also makes the measurement an extremely low throughput one rendering it impractical when hundreds to thousands potential biomarkers need to be quantitatively measured across multiple samples. Mass spectrometry (MS)-based assays have recently shown to be a viable alternative for high throughput and quantitative candidate protein biomarker verification. Among them, the triple quadrupole MS based assay is the most promising one. When it is coupled with liquid chromatography (LC) separation and electrospray ionization (ESI) source, a triple quadrupole mass spectrometer operating in a special selected reaction monitoring (SRM) mode, also known as multiple reaction monitoring (MRM), is capable of quantitatively measuring hundreds of candidate protein biomarkers from a relevant clinical sample in a single analysis. The specificity, reproducibility and sensitivity could be as good as ELISA. Furthermore, SRM MS can also quantify protein isoforms and post-translational modifications, for which traditional antibody-based immunoassays often don’t exist.« less

[Methods Used for Monitoring Cure Reactions in Real-time in an Autoclave

NASA Technical Reports Server (NTRS)

Cooper, John B.; Wise, Kent L.; Jensen, Brian J. (Technical Monitor)

2000-01-01

The goal of the research was to investigate methods for monitoring cure reactions in real-time in an autoclave. This is of particular importance to NASA Langley Research Center because polyimides were proposed for use in the High Speed Civil Transport (HSCT) program. Understanding the cure chemistry behind the polyimides would allow for intelligent processing of the composites made from their use. This work has led to two publications in peer-reviewed journals and a patent. The journal articles are listed as Appendix A which is on the instrument design of the research and Appendix B which is on the cure chemistry. Also, a patent has been awarded for the instrumental design developed under this grant which is given as Appendix C. There has been a significant amount of research directed at developing methods for monitoring cure reactions in real-time within the autoclave. The various research efforts can be categorized as methods providing either direct chemical bonding information or methods that provide indirect chemical bonding information. Methods falling into the latter category are fluorescence, dielectric loss, ultrasonic and similar type methods. Correlation of such measurements with the underlying chemistry is often quite difficult since these techniques do not allow monitoring of the curing chemistry which is ultimately responsible for material properties. Direct methods such as vibrational spectroscopy, however, can often be easily correlated with the underlying chemistry of a reaction. Such methods include Raman spectroscopy, mid-IR absorbance, and near-IR absorbance. With the recent advances in fiber-optics, these spectroscopic techniques can be applied to remote on-line monitoring.

Revealing time bunching effect in single-molecule enzyme conformational dynamics.

PubMed

Lu, H Peter

2011-04-21

In this perspective, we focus our discussion on how the single-molecule spectroscopy and statistical analysis are able to reveal enzyme hidden properties, taking the study of T4 lysozyme as an example. Protein conformational fluctuations and dynamics play a crucial role in biomolecular functions, such as in enzymatic reactions. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of a polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. Based on the single-molecule spectroscopic results, molecular dynamics simulation, a random walk model analysis, and a novel 2D statistical correlation analysis, we have revealed a time bunching effect in protein conformational motion dynamics that is critical to enzymatic functions. Bunching effect implies that conformational motion times tend to bunch in a finite and narrow time window. We show that convoluted multiple Poisson rate processes give rise to the bunching effect in the enzymatic reaction dynamics. Evidently, the bunching effect is likely common in protein conformational dynamics involving in conformation-gated protein functions. In this perspective, we will also discuss a new approach of 2D regional correlation analysis capable of analyzing fluctuation dynamics of complex multiple correlated and anti-correlated fluctuations under a non-correlated noise background. Using this new method, we are able to map out any defined segments along the fluctuation trajectories and determine whether they are correlated, anti-correlated, or non-correlated; after which, a cross correlation analysis can be applied for each specific segment to obtain a detailed fluctuation dynamics analysis.

Development of the gateway recycling cloning system for multiple linking of expression cassettes in a defined order, and direction on gateway compatible binary vectors.

PubMed

Kimura, Tetsuya; Nakao, Akihide; Murata, Sachiko; Kobayashi, Yasuyuki; Tanaka, Yuji; Shibahara, Kenta; Kawazu, Tetsu; Nakagawa, Tsuyoshi

2013-01-01

We developed the Gateway recycling cloning system, which allows multiple linking of expression cassettes by multiple rounds of the Gateway LR reaction. Employing this system, the recycling donor vector pRED419 was subjected to the first LR reaction with an attR1-attR2 type destination vector. Then conversion vector pCON was subjected to an LR reaction to restore the attR1-attR2 site on the destination vector for the next cloning cycle. By repetition of these two simple steps, we linked four expression cassettes of a reporter gene in Gateway binary vector pGWB1, introduced the constructs into tobacco BY-2 cells, and observed the expression of transgenes.

Single-molecule enzymology based on the principle of the Millikan oil drop experiment.

PubMed

Leiske, Danielle L; Chow, Andrea; Dettloff, Roger; Farinas, Javier

2014-03-01

The ability to monitor the progress of single-molecule enzyme reactions is often limited by the need to use fluorogenic substrates. A method based on the principle of the Millikan oil drop experiment was developed to monitor the change in charge of substrates bound to a nanoparticle and offers a means of detecting single-enzyme reactions without fluorescence detection. As a proof of principle of the ability to monitor reactions that result in a change in substrate charge, polymerization on a single DNA template was detected. A custom oligonucleotide was synthesized that allowed for the attachment of single DNA templates to gold nanoparticles with a single polymer tether. The nanoparticles were then tethered to the surface of a microfluidic channel where the positions of the nanoparticles, subjected to an oscillating electric field, were monitored using dark field microscopy. With short averaging times, the signal-to-noise level was low enough to discriminate changes in charge of less than 1.2%. Polymerization of a long DNA template demonstrated the ability to use the system to monitor single-molecule enzymatic activity. Finally, nanoparticle surfaces were modified with thiolated moieties to reduce and/or shield the number of unproductive charges and allow for improved sensitivity. Copyright © 2013 Elsevier Inc. All rights reserved.

Single-Molecule Enzymology Based On The Principle Of The Millikan Oil Drop Experiment

PubMed Central

Leiske, Danielle L.; Chow, Andrea; Dettloff, Roger; Farinas, Javier

2014-01-01

The ability to monitor the progress of single molecule enzyme reactions is often limited by the need to use fluorogenic substrates. A method based on the principle of the Millikan Oil Drop Experiment was developed to monitor the change in charge of substrates bound to a nanoparticle and offers a means of detecting single enzyme reactions without fluorescence detection. As a proof of principle of the ability to monitor reactions which result in a change in substrate charge, polymerization on a single DNA template was detected. A custom oligonucleotide was synthesized which allowed for the attachment of single DNA templates to gold nanoparticles with a single polymer tether. The nanoparticles were then tethered to the surface of a microfluidic channel where the positions of the nanoparticles, subjected to an oscillating electric field, were monitored using darkfield microscopy. With short averaging times, the signal-to-noise level was low enough to discriminate changes in charge of less than 1.2%. Polymerization of a long DNA template demonstrated the ability to use the system to monitor single molecule enzymatic activity. Finally, nanoparticle surfaces were modified with thiolated moieties in order to reduce and/or shield the number of unproductive charges and allow for improved sensitivity. PMID:24291542

No Evidence of Reaction Time Slowing in Autism Spectrum Disorder

ERIC Educational Resources Information Center

Ferraro, F. Richard

2016-01-01

A total of 32 studies comprising 238 simple reaction time and choice reaction time conditions were examined in individuals with autism spectrum disorder (n?=?964) and controls (n?=?1032). A Brinley plot/multiple regression analysis was performed on mean reaction times, regressing autism spectrum disorder performance onto the control performance as…

Light charged particle multiplicities in fusion and quasifission reactions

NASA Astrophysics Data System (ADS)

Kalandarov, Sh. A.; Adamian, G. G.; Antonenko, N. V.; Lacroix, D.; Wieleczko, J. P.

2018-01-01

The light charged particle evaporation from the compound nucleus and from the complex fragments in the reactions 32S+100Mo, 121Sb+27Al, 40Ar+164Dy, and 40Ar+ nat Ag is studied within the dinuclear system model. The possibility to distinguish the reaction products from different reaction mechanisms is discussed.

Simultaneous Determination of Multiple Classes of Hydrophilic and Lipophilic Components in Shuang-Huang-Lian Oral Liquid Formulations by UPLC-Triple Quadrupole Linear Ion Trap Mass Spectrometry.

PubMed

Liang, Jun; Sun, Hui-Min; Wang, Tian-Long

2017-11-24

The Shuang-Huang-Lian (SHL) oral liquid is a combined herbal prescription used in the treatment of acute upper respiratory tract infection, acute bronchitis and pneumonia. Multiple constituents are considered to be responsible for the therapeutic effects of SHL. However, the quantitation of the multi-components from multiple classes is still unsatisfactory because of the high complexity of constituents in SHL. In this study, an accurate, rapid, and specific UPLC-MS/MS method was established for simultaneous quantification of 18 compounds from multiple classes in SHL oral liquid formulations. Chromatographic separation was performed on a HSS T3 (1.8 μm, 2.1 mm × 100 mm) column, using a gradient mobile phase system of 0.1% formic acid in acetonitrile and 0.1% formic acid in water at a flow rate of 0.2 mL·min -1 ; the run time was 23 min. The MS was operated in negative electrospray ionization (ESI - ) for analysis of 18 compounds using multiple reaction monitoring (MRM) mode. UPLC-ESI - -MRM-MS/MS method showed good linear relationships ( R ² > 0.999), repeatability (RSD < 3%), precisions (RSD < 3%) and recovery (84.03-101.62%). The validated method was successfully used to determine multiple classes of hydrophilic and lipophilic components in the SHL oral liquids. Finally, principal component analysis (PCA) was used to classify and differentiate SHL oral liquid samples attributed to different manufacturers of China. The proposed UPLC-ESI - -MRM-MS/MS coupled with PCA has been elucidated to be a simple and reliable method for quality evaluation of SHL oral liquids.

When big brother is watching: goal orientation shapes reactions to electronic monitoring during online training.

PubMed

Watson, Aaron M; Foster Thompson, Lori; Rudolph, Jane V; Whelan, Thomas J; Behrend, Tara S; Gissel, Amanda L

2013-07-01

Web-based training is frequently used by organizations as a convenient and low-cost way to teach employees new knowledge and skills. As web-based training is typically unproctored, employees may be held accountable to the organization by computer software that monitors their behaviors. The current study examines how the introduction of electronic performance monitoring may provoke negative emotional reactions and decrease learning among certain types of e-learners. Through motivated action theory and trait activation theory, we examine the role of performance goal orientation when e-learners are exposed to asynchronous and synchronous monitoring. We show that some e-learners are more susceptible than others to evaluation apprehension when they perceive their activities are being monitored electronically. Specifically, e-learners higher in avoid performance goal orientation exhibited increased evaluation apprehension if they believed asynchronous monitoring was present, and they showed decreased skill attainment as a result. E-learners higher on prove performance goal orientation showed greater evaluation apprehension if they believed real-time monitoring was occurring, resulting in decreased skill attainment. PsycINFO Database Record (c) 2013 APA, all rights reserved.

Biodiesel transesterification kinetics monitored by pH measurement.

PubMed

Clark, William M; Medeiros, Nicholas J; Boyd, Donal J; Snell, Jared R

2013-05-01

Quantification of a pH change that was observed over the course of the transesterification reaction that converts vegetable oil to biodiesel may provide a simple method to monitor the reaction. Transesterification of canola oil at 6:1 methanol to oil ratio with 0.5 wt.% KOH as catalyst was studied at 25, 35, and 45 °C. Reaction conversion was correlated to pH measurements and the results were shown to be in agreement with an independent measure of conversion using an enzymatic assay for glycerol. Rate constants obtained from these measurements are consistent with those in the literature. The measured pH change appears to be related to dilution of OH(-) ions as the oil is converted to products rather than to depletion of OH(-) due to reaction. Copyright © 2013 Elsevier Ltd. All rights reserved.

Real-time monitoring of enzyme-free strand displacement cascades by colorimetric assays

NASA Astrophysics Data System (ADS)

Duan, Ruixue; Wang, Boya; Hong, Fan; Zhang, Tianchi; Jia, Yongmei; Huang, Jiayu; Hakeem, Abdul; Liu, Nannan; Lou, Xiaoding; Xia, Fan

2015-03-01

The enzyme-free toehold-mediated strand displacement reaction has shown potential for building programmable DNA circuits, biosensors, molecular machines and chemical reaction networks. Here we report a simple colorimetric method using gold nanoparticles as signal generators for the real-time detection of the product of the strand displacement cascade. During the process the assembled gold nanoparticles can be separated, resulting in a color change of the solution. This assay can also be applied in complex mixtures, fetal bovine serum, and to detect single-base mismatches. These results suggest that this method could be of general utility to monitor more complex enzyme-free strand displacement reaction-based programmable systems or for further low-cost diagnostic applications.The enzyme-free toehold-mediated strand displacement reaction has shown potential for building programmable DNA circuits, biosensors, molecular machines and chemical reaction networks. Here we report a simple colorimetric method using gold nanoparticles as signal generators for the real-time detection of the product of the strand displacement cascade. During the process the assembled gold nanoparticles can be separated, resulting in a color change of the solution. This assay can also be applied in complex mixtures, fetal bovine serum, and to detect single-base mismatches. These results suggest that this method could be of general utility to monitor more complex enzyme-free strand displacement reaction-based programmable systems or for further low-cost diagnostic applications. Electronic supplementary information (ESI) available: Experimental procedures and analytical data are provided. See DOI: 10.1039/c5nr00697j

In situ monitoring of atomic layer controlled pore reduction in alumina tubular membranes using sequential surface reactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berland, B.S.; Gartland, I.P.; Ott, A.W.

1998-12-01

The pore diameter in alumina tubular membranes with an initial diameter of 50 {angstrom} was systematically reduced using the atomic layer controlled deposition of Al{sub 2}O{sub 3}. The Al{sub 2}O{sub 3} was deposited using sequential exposures of Al(CH{sub 3}){sub 3} (trimethylaluminum, TMA) and H{sub 2}O in an ABAB... binary reaction sequence. The pore diameter reduction was monitored using in situ N{sub 2} and Ar conductance measurements. The conductance, C = Q/{Delta}P, was measured using a mass flow controller to define a constant gas throughput, Q, and a pair of capacitance manometers to monitor the transmembrane pressure drop, {Delta}P. Conductance measurementsmore » were periodically obtained at 298 K as a function of AB binary reaction cycles. These conductance measurements were consistent with a pore diameter reduction from 50 {angstrom} to {approximately}5--10 {angstrom} at a rate of {approximately}2.5 {angstrom} for each AB cycle. Conductance measurements were also performed during the Al{sub 2}O{sub 3} deposition at 500 K after each half-reaction in the binary reaction sequence. These in situ conductance measurements demonstrate that the pore diameters in mesoporous membranes can be reduced to molecular dimensions with atomic layer control using sequential surface reactions. Poe diameters can be tailored for specific applications by varying the number of AB cycles and changing the nature of the terminating surface functional groups.« less

Pollution monitoring system. [photographic laboratory by-products

NASA Technical Reports Server (NTRS)

Goodding, R. A.

1973-01-01

An investigation was undertaken to identify those photographic laboratory by-products which can produce harmful reactions if released untreated. After identification of these by-products, specific monitoring systems for each of the offending ions were investigated and recommendations for implementation are presented. Appropriate monitoring systems are discussed.

Implementation of Multiple Host Nodes in Wireless Sensing Node Network System for Landslide Monitoring

NASA Astrophysics Data System (ADS)

Abas, Faizulsalihin bin; Takayama, Shigeru

2015-02-01

This paper proposes multiple host nodes in Wireless Sensing Node Network System (WSNNS) for landslide monitoring. As landslide disasters damage monitoring system easily, one major demand in landslide monitoring is the flexibility and robustness of the system to evaluate the current situation in the monitored area. For various reasons WSNNS can provide an important contribution to reach that aim. In this system, acceleration sensors and GPS are deployed in sensing nodes. Location information by GPS, enable the system to estimate network topology and enable the system to perceive the location in emergency by monitoring the node mode. Acceleration sensors deployment, capacitate this system to detect slow mass movement that can lead to landslide occurrence. Once deployed, sensing nodes self-organize into an autonomous wireless ad hoc network. The measurement parameter data from sensing nodes is transmitted to Host System via host node and "Cloud" System. The implementation of multiple host nodes in Local Sensing Node Network System (LSNNS), improve risk- management of the WSNNS for real-time monitoring of landslide disaster.

Use of multiple competitors for quantification of human immunodeficiency virus type 1 RNA in plasma.

PubMed

Vener, T; Nygren, M; Andersson, A; Uhlén, M; Albert, J; Lundeberg, J

1998-07-01

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma has rapidly become an important tool in basic HIV research and in the clinical care of infected individuals. Here, a quantitative HIV assay based on competitive reverse transcription-PCR with multiple competitors was developed. Four RNA competitors containing identical PCR primer binding sequences as the viral HIV-1 RNA target were constructed. One of the PCR primers was fluorescently labeled, which facilitated discrimination between the viral RNA and competitor amplicons by fragment analysis with conventional automated sequencers. The coamplification of known amounts of the RNA competitors provided the means to establish internal calibration curves for the individual reactions resulting in exclusion of tube-to-tube variations. Calibration curves were created from the peak areas, which were proportional to the starting amount of each competitor. The fluorescence detection format was expanded to provide a dynamic range of more than 5 log units. This quantitative assay allowed for reproducible analysis of samples containing as few as 40 viral copies of HIV-1 RNA per reaction. The within- and between-run coefficients of variation were <24% (range, 10 to 24) and <36% (range, 27 to 36), respectively. The high reproducibility (standard deviation, <0.13 log) of the overall procedure for quantification of HIV-1 RNA in plasma, including sample preparation, amplification, and detection variations, allowed reliable detection of a 0.5-log change in RNA viral load. The assay could be a useful tool for monitoring HIV-1 disease progression and antiviral treatment and can easily be adapted to the quantification of other pathogens.

Comprehensive analysis of phospholipids and glycolipids in the opportunistic pathogen Enterococcus faecalis

PubMed Central

Gao, Iris H.; Nair, Zeus J.; Kumar, Jaspal K.; Gao, Liang; Kline, Kimberly A.; Wenk, Markus R.

2017-01-01

Enterococcus faecalis is a Gram-positive, opportunistic, pathogenic bacterium that causes a significant number of antibiotic-resistant infections in hospitalized patients. The development of antibiotic resistance in hospital-associated pathogens is a formidable public health threat. In E. faecalis and other Gram-positive pathogens, correlations exist between lipid composition and antibiotic resistance. Resistance to the last-resort antibiotic daptomycin is accompanied by a decrease in phosphatidylglycerol (PG) levels, whereas multiple peptide resistance factor (MprF) converts anionic PG into cationic lysyl-PG via a trans-esterification reaction, providing resistance to cationic antimicrobial peptides. Unlike previous studies that relied on thin layer chromatography and spectrophotometry, we have performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) directly on lipids extracted from E. faecalis, and quantified the phospholipids through multiple reaction monitoring (MRM). In the daptomycin-sensitive E. faecalis strain OG1RF, we have identified 17 PGs, 8 lysyl-PGs (LPGs), 23 cardiolipins (CL), 3 glycerophospho-diglucosyl-diacylglycerols (GPDGDAG), 5 diglucosyl-diacylglycerols (DGDAG), 3 diacylglycerols (DAGs), and 4 triacylglycerols (TAGs). We have quantified PG and shown that PG levels vary during growth of E. faecalis in vitro. We also show that two daptomycin-resistant (DapR) strains of E. faecalis have substantially lower levels of PG and LPG levels. Since LPG levels in these strains are lower, daptomycin resistance is likely due to the reduction in PG. This lipidome map is the first comprehensive analysis of membrane phospholipids and glycolipids in the important human pathogen E. faecalis, for which antimicrobial resistance and altered lipid homeostasis have been intimately linked. PMID:28423018

Real-time monitoring of enzyme-free strand displacement cascades by colorimetric assays.

PubMed

Duan, Ruixue; Wang, Boya; Hong, Fan; Zhang, Tianchi; Jia, Yongmei; Huang, Jiayu; Hakeem, Abdul; Liu, Nannan; Lou, Xiaoding; Xia, Fan

2015-03-19

The enzyme-free toehold-mediated strand displacement reaction has shown potential for building programmable DNA circuits, biosensors, molecular machines and chemical reaction networks. Here we report a simple colorimetric method using gold nanoparticles as signal generators for the real-time detection of the product of the strand displacement cascade. During the process the assembled gold nanoparticles can be separated, resulting in a color change of the solution. This assay can also be applied in complex mixtures, fetal bovine serum, and to detect single-base mismatches. These results suggest that this method could be of general utility to monitor more complex enzyme-free strand displacement reaction-based programmable systems or for further low-cost diagnostic applications.

[Irrational use of drugs as a source of drug - induced diseases].

PubMed

Woroń, Jarosław; Porebski, Grzegorz; Kostka-Trabka, Elzbieta; Goszcz, Aleksandra

2007-01-01

The irrational use of medication, by which we understand the administration of drugs for indications where their effectiveness has not been confirmed, the disregard of restrictions and warnings against their use, and the use of drug combinations which do not increase the therapeutic effect but to the contrary increase the risk of adverse drug reactions, is a serious problem encountered in paediatric pharmacotherapy. Each year the centres for monitoring of adverse drug reactions receive many reports, the analysis of which show that the reasons of occurrence of adverse drug reactions after drug administration, are specifically due to irrational use of medications. In order to prevent in an active way the occurrence, of adverse drug reactions following drug administration it is worthwhile to bring to attention the reasons for their occurrence which not infrequently bring about pathological effects. Our work which is based on reports received by the Regional Centre for Adverse Drug Reactions Monitoring in Krakow concerning the occurrence of adverse drug reactions in an attempt to bring to attention in our view important problems in current pharmacotherapy.

Comprehensive near infrared study of Jatropha oil esterification with ethanol for biodiesel production

NASA Astrophysics Data System (ADS)

Oliveira, Alianda Dantas de; Sá, Ananda Franco de; Pimentel, Maria Fernanda; Pacheco, José Geraldo A.; Pereira, Claudete Fernandes; Larrechi, Maria Soledad

2017-01-01

This work presents a comprehensive near infrared study for in-line monitoring of the esterification reaction of high acid oils, such as Jatropha curcas oil, using ethanol. Parallel reactions involved in the process were carried out to select a spectral region that characterizes the evolution of the esterification reaction. Using absorbance intensities at 5176 cm- 1, the conversion and kinetic behaviors of the esterification reaction were estimated. This method was applied to evaluate the influence of temperature and catalyst concentration on the estimates of initial reaction rate and ester conversion as responses to a 22 factorial experimental design. Employment of an alcohol/oil ratio of 16:1, catalyst concentration of 1.5% w/w, and temperatures at 65 °C or 75 °C, made it possible to reduce the initial acidity from 18% to 1.3% w/w, which is suitable for transesterification of high free fatty acid oils for biodiesel production. Using the proposed analytical method in the esterification reaction of raw materials with high free fatty acid content for biodiesel makes the monitoring process inexpensive, fast, simple, and practical.

A Focused Multiple Reaction Monitoring (MRM) Quantitative Method for Bioactive Grapevine Stilbenes by Ultra-High-Performance Liquid Chromatography Coupled to Triple-Quadrupole Mass Spectrometry (UHPLC-QqQ).

PubMed

Hurtado-Gaitán, Elías; Sellés-Marchart, Susana; Martínez-Márquez, Ascensión; Samper-Herrero, Antonio; Bru-Martínez, Roque

2017-03-07

Grapevine stilbenes are a family of polyphenols which derive from trans -resveratrol having antifungal and antimicrobial properties, thus being considered as phytoalexins. In addition to their diverse bioactive properties in animal models, they highlight a strong potential in human health maintenance and promotion. Due to this relevance, highly-specific qualitative and quantitative methods of analysis are necessary to accurately analyze stilbenes in different matrices derived from grapevine. Here, we developed a rapid, sensitive, and specific analysis method using ultra-high-performance liquid chromatography coupled to triple-quadrupole mass spectrometry (UHPLC-QqQ) in MRM mode to detect and quantify five grapevine stilbenes, trans -resveratrol, trans -piceid, trans -piceatannol, trans -pterostilbene, and trans -ε-viniferin, whose interest in relation to human health is continuously growing. The method was optimized to minimize in-source fragmentation of piceid and to avoid co-elution of cis -piceid and trans -resveratrol, as both are detected with resveratrol transitions. The applicability of the developed method of stilbene analysis was tested successfully in different complex matrices including cellular extracts of Vitis vinifera cell cultures, reaction media of biotransformation assays, and red wine.

From radioimmunoassay to mass spectrometry: a new method to quantify orexin-A (hypocretin-1) in cerebrospinal fluid

PubMed Central

Hirtz, Christophe; Vialaret, Jérôme; Gabelle, Audrey; Nowak, Nora; Dauvilliers, Yves; Lehmann, Sylvain

2016-01-01

I125 radioimmunoassay (RIA) is currently the standard technique for quantifying cerebrospinal fluid (CSF) orexin-A/hypocretin-1, a biomarker used to diagnose narcolepsy type 1. However, orexin-A RIA is liable to undergo cross-reactions with matrix constituents generating interference, high variability between batches, low precision and accuracy, and requires special radioactivity precautions. Here we developed the first quantitative mass spectrometry assay of orexin-A based on a multiple reaction monitoring (MRM) approach. This method was tested in keeping with the Clinical and Laboratory Standards Institute (CLSI) guidelines and its clinical relevance was confirmed by comparing patients with narcolepsy type 1 versus patients with other neurological conditions. The results obtained using MRM and RIA methods were highly correlated, and Bland–Altman analysis established their interchangeability. However, the MRM values had a wider distribution and were 2.5 time lower than the RIA findings. In conclusion, this method of assay provides a useful alternative to RIA to quantify orexin-A, and may well replace it not only in narcolepsy type 1, but also in the increasing number of pathologies in which the quantification of this analyte is relevant. PMID:27165941

Monitoring dynamic reactions of red blood cells to UHF electromagnetic waves radiation using a novel micro-imaging technology.

PubMed

Ruan, Ping; Yong, Junguang; Shen, Hongtao; Zheng, Xianrong

2012-12-01

Multiple state-of-the-art techniques, such as multi-dimensional micro-imaging, fast multi-channel micro-spetrophotometry, and dynamic micro-imaging analysis, were used to dynamically investigate various effects of cell under the 900 MHz electromagnetic radiation. Cell changes in shape, size, and parameters of Hb absorption spectrum under different power density electromagnetic waves radiation were presented in this article. Experimental results indicated that the isolated human red blood cells (RBCs) do not have obviously real-time responses to the ultra-low density (15 μW/cm(2), 31 μW/cm(2)) electromagnetic wave radiation when the radiation time is not more than 30 min; however, the cells do have significant reactions in shape, size, and the like, to the electromagnetic waves radiation with power densities of 1 mW/cm(2) and 5 mW/cm(2). The data also reveal the possible influences and statistical relationships among living human cell functions, radiation amount, and exposure time with high-frequency electromagnetic waves. The results of this study may be significant on protection of human being and other living organisms against possible radiation affections of the high-frequency electromagnetic waves.

Measurements of the thermal neutron cross-section and resonance integral for the 108Pd(n,γ)109Pd reaction

NASA Astrophysics Data System (ADS)

Hien, Nguyen Thi; Kim, Guinyun; Kim, Kwangsoo; Do, Nguyen Van; Khue, Pham Duc; Thanh, Kim Tien; Shin, Sung-Gyun; Cho, Moo-Hyun

2018-06-01

The thermal neutron capture cross-section (σ0) and resonance integral (I0) of the 108Pd(n,γ)109Pd reaction have been measured relative to that of the monitor reaction 197Au(n,γ)198Au. The measurements were carried out using the neutron activation with the cadmium ratio method. Both the samples and monitors were irradiated with and without cadmium cover of 0.5 mm thickness. The induced activities of the reaction products were measured with a well calibrated HPGe γ-ray detector. In order to improve the accuracy of the results, the necessary corrections for the counting losses were made. The thermal neutron capture cross-section and resonance integral of the 108Pd(n,γ)109Pd reaction were determined to be σ0,Pd = 8.68 ± 0.41 barn and I0,Pd = 245.6 ± 24.8 barn, respectively. The obtained results are compared with literature values and discussed.

Multiple-step preparation and physicochemical characterization of crystalline α-germanium hydrogenphosphate

NASA Astrophysics Data System (ADS)

Romano, Ricardo; Ruiz, Ana I.; Alves, Oswaldo L.

2004-04-01

The reaction between germanium oxide and phosphoric acid has previously been described and led to impure germanium hydrogenphosphate samples with low crystallinity. A new multiple-step route involving the same reaction under refluxing and soft hydrothermal conditions is described for the preparation of pure and crystalline α-GeP. The physicochemical characterization of the samples allows accompaniment of the reaction evolution as well as determining short- and long-range structural organization. The phase purity of the α-GeP sample was confirmed by applying Rietveld's profile analysis, which also determined the cell parameters of its crystals.

Catalytic bismetallative multicomponent coupling reactions: scope, applications, and mechanisms

PubMed Central

Cho, Hee Yeon

2014-01-01

Catalytic reactions have played an indispensable role in organic chemistry for the last several decades. In particular, catalytic multicomponent reactions have attracted a lot of attention due to their efficiency and expediency towards complex molecule synthesis. The presence of bismetallic reagents (e.g. B–B, Si–Si, B–Si, Si–Sn, etc.) in this process renders the products enriched with various functional groups and multiple stereocenters. For this reason, catalytic bismetallative coupling is considered an effective method to generate the functional and stereochemical complexity of simple hydrocarbon substrates. This review highlights key developments of transition-metal catalyzed bismetallative reactions involving multiple π components. Specifically, it will highlight the scope, synthetic applications, and proposed mechanistic pathways of this process. PMID:24736839

Biological water quality monitoring using chemiluminescent and bioluminescent techniques

NASA Technical Reports Server (NTRS)

Thomas, R. R.

1978-01-01

Automated chemiluminescence and bioluminescence sensors were developed for the continuous monitoring of microbial levels in water supplies. The optimal chemical procedures were determined for the chemiluminescence system to achieve maximum sensitivity. By using hydrogen peroxide, reaction rate differentiation, ethylene diamine tetraacetic acid (EDTA), and carbon monoxide pretreatments, factors which cause interference were eliminated and specificity of the reaction for living and dead bacteria was greatly increased. By employing existing technology with some modifications, a sensitive and specific bioluminescent system was developed.

Parallel Reaction Monitoring: A Targeted Experiment Performed Using High Resolution and High Mass Accuracy Mass Spectrometry

PubMed Central

Rauniyar, Navin

2015-01-01

The parallel reaction monitoring (PRM) assay has emerged as an alternative method of targeted quantification. The PRM assay is performed in a high resolution and high mass accuracy mode on a mass spectrometer. This review presents the features that make PRM a highly specific and selective method for targeted quantification using quadrupole-Orbitrap hybrid instruments. In addition, this review discusses the label-based and label-free methods of quantification that can be performed with the targeted approach. PMID:26633379

Numerical Problem Solving Using Mathcad in Undergraduate Reaction Engineering

ERIC Educational Resources Information Center

Parulekar, Satish J.

2006-01-01

Experience in using a user-friendly software, Mathcad, in the undergraduate chemical reaction engineering course is discussed. Example problems considered for illustration deal with simultaneous solution of linear algebraic equations (kinetic parameter estimation), nonlinear algebraic equations (equilibrium calculations for multiple reactions and…

Monitoring Enzymatic Reactions in Real Time Using Venturi Easy Ambient Sonic-Spray Ionization Mass Spectrometry

PubMed Central

2016-01-01

We developed a technique to monitor spatially confined surface reactions with mass spectrometry under ambient conditions, without the need for voltage or organic solvents. Fused-silica capillaries immersed in an aqueous solution, positioned in close proximity to each other and the functionalized surface, created a laminar flow junction with a resulting reaction volume of ∼5 pL. The setup was operated with a syringe pump, delivering reagents to the surface through a fused-silica capillary. The other fused-silica capillary was connected to a Venturi easy ambient sonic-spray ionization source, sampling the resulting analytes at a slightly higher flow rate compared to the feeding capillary. The combined effects of the inflow and outflow maintains a chemical microenvironment, where the rate of advective transport overcomes diffusion. We show proof-of-concept where acetylcholinesterase was immobilized on an organosiloxane polymer through electrostatic interactions. The hydrolysis of acetylcholine by acetylcholinesterase into choline was monitored in real-time for a range of acetylcholine concentrations, fused-silica capillary geometries, and operating flow rates. Higher reaction rates and conversion yields were observed with increasing acetylcholine concentrations, as would be expected. PMID:27249533

Correlation between y-type ions observed in ion trap and triple quadrupole mass spectrometers.

PubMed

Sherwood, Carly A; Eastham, Ashley; Lee, Lik Wee; Risler, Jenni; Vitek, Olga; Martin, Daniel B

2009-09-01

Multiple reaction monitoring mass spectrometry (MRM-MS) is a technique for high-sensitivity targeted analysis. In proteomics, MRM-MS can be used to monitor and quantify a peptide based on the production of expected fragment peaks from the selected peptide precursor ion. The choice of which fragment ions to monitor in order to achieve maximum sensitivity in MRM-MS can potentially be guided by existing MS/MS spectra. However, because the majority of discovery experiments are performed on ion trap platforms, there is concern in the field regarding the generalizability of these spectra to MRM-MS on a triple quadrupole instrument. In light of this concern, many operators perform an optimization step to determine the most intense fragments for a target peptide on a triple quadrupole mass spectrometer. We have addressed this issue by targeting, on a triple quadrupole, the top six y-ion peaks from ion trap-derived consensus library spectra for 258 doubly charged peptides from three different sample sets and quantifying the observed elution curves. This analysis revealed a strong correlation between the y-ion peak rank order and relative intensity across platforms. This suggests that y-type ions obtained from ion trap-based library spectra are well-suited for generating MRM-MS assays for triple quadrupoles and that optimization is not required for each target peptide.

A method to measure the ozone penetration factor in residences under infiltration conditions: application in a multifamily apartment unit.

PubMed

Zhao, H; Stephens, B

2016-08-01

Recent experiments have demonstrated that outdoor ozone reacts with materials inside residential building enclosures, potentially reducing indoor exposures to ozone or altering ozone reaction byproducts. However, test methods to measure ozone penetration factors in residences (P) remain limited. We developed a method to measure ozone penetration factors in residences under infiltration conditions and applied it in an unoccupied apartment unit. Twenty-four repeated measurements were made, and results were explored to (i) evaluate the accuracy and repeatability of the new procedure using multiple solution methods, (ii) compare results from 'interference-free' and conventional UV absorbance ozone monitors, and (iii) compare results against those from a previously published test method requiring artificial depressurization. The mean (±s.d.) estimate of P was 0.54 ± 0.10 across a wide range of conditions using the new method with an interference-free monitor; the conventional monitor was unable to yield meaningful results due to relatively high limits of detection. Estimates of P were not clearly influenced by any indoor or outdoor environmental conditions or changes in indoor decay rate constants. This work represents the first known measurements of ozone penetration factors in a residential building operating under natural infiltration conditions and provides a new method for widespread application in buildings. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Purification of CFTR for mass spectrometry analysis: identification of palmitoylation and other post-translational modifications

PubMed Central

McClure, Michelle; DeLucas, Lawrence J.; Wilson, Landon; Ray, Marjorie; Rowe, Steven M.; Wu, Xiaoyun; Dai, Qun; Hong, Jeong S.; Sorscher, Eric J.; Kappes, John C.; Barnes, Stephen

2012-01-01

Post-translational modifications (PTMs) play a crucial role during biogenesis of many transmembrane proteins. Previously, it had not been possible to evaluate PTMs in cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial ion channel responsible for cystic fibrosis, because of difficulty obtaining sufficient amounts of purified protein. We recently used an inducible overexpression strategy to generate recombinant CFTR protein at levels suitable for purification and detailed analysis. Using liquid chromatography (LC) tandem and multiple reaction ion monitoring (MRM) mass spectrometry, we identified specific sites of PTMs, including palmitoylation, phosphorylation, methylation and possible ubiquitination. Many of these covalent CFTR modifications have not been described previously, but are likely to influence key and clinically important molecular processes including protein maturation, gating and the mechanisms underlying certain mutations associated with disease. PMID:22119790

A standardized kit for automated quantitative assessment of candidate protein biomarkers in human plasma.

PubMed

Percy, Andrew J; Mohammed, Yassene; Yang, Juncong; Borchers, Christoph H

2015-12-01

An increasingly popular mass spectrometry-based quantitative approach for health-related research in the biomedical field involves the use of stable isotope-labeled standards (SIS) and multiple/selected reaction monitoring (MRM/SRM). To improve inter-laboratory precision and enable more widespread use of this 'absolute' quantitative technique in disease-biomarker assessment studies, methods must be standardized. Results/methodology: Using this MRM-with-SIS-peptide approach, we developed an automated method (encompassing sample preparation, processing and analysis) for quantifying 76 candidate protein markers (spanning >4 orders of magnitude in concentration) in neat human plasma. The assembled biomarker assessment kit - the 'BAK-76' - contains the essential materials (SIS mixes), methods (for acquisition and analysis), and tools (Qualis-SIS software) for performing biomarker discovery or verification studies in a rapid and standardized manner.

Multiple reaction monitoring (MRM) of plasma proteins in cardiovascular proteomics.

PubMed

Dardé, Verónica M; Barderas, Maria G; Vivanco, Fernando

2013-01-01

Different methodologies have been used through years to discover new potential biomarkers related with cardiovascular risk. The conventional proteomic strategy involves a discovery phase that requires the use of mass spectrometry (MS) and a validation phase, usually on an alternative platform such as immunoassays that can be further implemented in clinical practice. This approach is suitable for a single biomarker, but when large panels of biomarkers must be validated, the process becomes inefficient and costly. Therefore, it is essential to find an alternative methodology to perform the biomarker discovery, validation, and -quantification. The skills provided by quantitative MS turn it into an extremely attractive alternative to antibody-based technologies. Although it has been traditionally used for quantification of small molecules in clinical chemistry, MRM is now emerging as an alternative to traditional immunoassays for candidate protein biomarker validation.

Recent Advances in Clinical Glycoproteomics of Immunoglobulins (Igs).

PubMed

Plomp, Rosina; Bondt, Albert; de Haan, Noortje; Rombouts, Yoann; Wuhrer, Manfred

2016-07-01

Antibody glycosylation analysis has seen methodological progress resulting in new findings with regard to antibody glycan structure and function in recent years. For example, antigen-specific IgG glycosylation analysis is now applicable for clinical samples because of the increased sensitivity of measurements, and this has led to new insights in the relationship between IgG glycosylation and various diseases. Furthermore, many new methods have been developed for the purification and analysis of IgG Fc glycopeptides, notably multiple reaction monitoring for high-throughput quantitative glycosylation analysis. In addition, new protocols for IgG Fab glycosylation analysis were established revealing autoimmune disease-associated changes. Functional analysis has shown that glycosylation of IgA and IgE is involved in transport across the intestinal epithelium and receptor binding, respectively. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Development of an UPLC-MS/MS micromethod for quantitation of cinitapride in plasma and its application in a pharmacokinetic interaction trial.

PubMed

Marcelín-Jiménez, Gabriel; Contreras, Leticia; Esquivel, Javier; Ávila, Óscar; Batista, Dany; Ángeles, Alionka P; García-González, Alberto

2017-03-01

Cinitapride (CIN) is a benzamide-derived molecule used for the treatment of gastroesophageal reflux and dyspepsia. Its pharmacokinetics are controversial due to the use of supratherapeutic doses and the lack of sensitive methodology. Therefore, a sensitive and accurate micromethod was developed for its quantitation in human plasma. CIN was extracted from 300 µl of heparinized plasma by liquid-liquid extraction using cisapride as internal standard, and analyzed with an ultra performance liquid chromatograph employing positive multiple-reaction monitoring-MS. The method proved to be rapid, accurate and stable within a range between 50 and 2000 pg/ml and was successfully validated and applied in a pharmacokinetic interaction trial, where it was demonstrated that oral co-administration of simethicone does not modify the bioavailability of CIN.

Quantitation of iothalamate in urine and plasma using liquid chromatography electrospray tandem mass spectrometry (HPLC-ESI-MS/MS).

PubMed

Molinaro, Ross J; Ritchie, James C

2010-01-01

The following chapter describes a method to measure iothalamate in plasma and urine samples using high performance liquid chromatography combined with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Methanol and water are spiked with the internal standard (IS) iohexol. Iothalamate is isolated from plasma after IS spiked methanol extraction and from urine by IS spiked water addition and quick-spin filtration. The plasma extractions are dried under a stream of nitrogen. The residue is reconstituted in ammonium acetate-formic acid-water. The reconstituted plasma and filtered urine are injected into the HPLC-ESI-MS/MS. Iothalamate and iohexol show similar retention times in plasma and urine. Quantification of iothalamate in the samples is made by multiple reaction monitoring using the hydrogen adduct mass transitions, from a five-point calibration curve.

Direct analysis of [6,6-(2)H2]glucose and [U-(13)C6]glucose dry blood spot enrichments by LC-MS/MS.

PubMed

Coelho, Margarida; Mendes, Vera M; Lima, Inês S; Martins, Fátima O; Fernandes, Ana B; Macedo, M Paula; Jones, John G; Manadas, Bruno

2016-06-01

A liquid chromatography tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring (MRM) in a triple-quadrupole scan mode was developed and comprehensively validated for the determination of [6,6-(2)H2]glucose and [U-(13)C6]glucose enrichments from dried blood spots (DBS) without prior derivatization. The method is demonstrated with dried blood spots obtained from rats administered with a primed-constant infusion of [U-(13)C6]glucose and an oral glucose load enriched with [6,6-(2)H2]glucose. The sensitivity is sufficient for analysis of the equivalent to <5μL of blood and the overall method was accurate and precise for the determination of DBS isotopic enrichments. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantification of short chain amines in aqueous matrices using liquid chromatography electrospray ionization tandem mass spectrometry.

PubMed

Viidanoja, Jyrki

2017-01-13

A new liquid chromatography-electrospray ionization-tandem Mass Spectrometry (LC-ESI-MS/MS) method was developed for the determination of more than 20 C 1 -C 6 alkyl and alkanolamines in aqueous matrices. The method employs Hydrophilic Interaction Liquid Chromatography Multiple Reaction Monitoring (HILIC-MRM) with a ZIC-pHILIC column and four stable isotope labeled amines as internal standards for signal normalization and quantification of the amines. The method was validated using a refinery process water sample that was obtained from a cooling cycle of crude oil distillation. The averaged within run precision, between run precision and accuracy were generally within 2-10%, 1-9% and 80-120%, respectively, depending on the analyte and concentration level. Selected aqueous process samples were analyzed with the method. Copyright © 2016 Elsevier B.V. All rights reserved.

Site-specific quantitative analysis of cardiac mitochondrial protein phosphorylation.

PubMed

Lam, Maggie P Y; Lau, Edward; Scruggs, Sarah B; Wang, Ding; Kim, Tae-Young; Liem, David A; Zhang, Jun; Ryan, Christopher M; Faull, Kym F; Ping, Peipei

2013-04-09

We report the development of a multiple-reaction monitoring (MRM) strategy specifically tailored to the detection and quantification of mitochondrial protein phosphorylation. We recently derived 68 MRM transitions specific to protein modifications in the respiratory chain, voltage-dependent anion channel, and adenine nucleotide translocase. Here, we have now expanded the total number of MRM transitions to 176 to cover proteins from the tricarboxylic acid cycle, pyruvate dehydrogenase complex, and branched-chain alpha-keto acid dehydrogenase complex. We utilized the transition set to analyze endogenous protein phosphorylation in human heart, mouse heart, and mouse liver. The data demonstrate the potential utility of the MRM workflow for studying the functional details of mitochondrial phosphorylation signaling. This article is part of a Special Issue entitled: From protein structures to clinical applications. Copyright © 2012 Elsevier B.V. All rights reserved.

Serial measurement of serum cytokines, cytokine receptors and neopterin in leprosy patients with reversal reactions.

PubMed

Faber, W R; Iyer, A M; Fajardo, T T; Dekker, T; Villahermosa, L G; Abalos, R M; Das, P K

2004-09-01

Serum levels of cytokines (IL-4, IL-5, IFN-gamma, TNF-alpha), cytokine receptors (TNFR I and II) and one monokine (neopterin) were estimated in seven leprosy patients to establish disease associated markers for reversal reactions (RR). Sera were collected at diagnosis of leprosy, at the onset of reversal reaction and at different time points during and at the end of prednisone treatment of reactions. It was expected that the serum cytokine and monokine profile before and at different time points during reactions would provide guidelines for the diagnosis and monitoring of reversal reactions in leprosy. The cytokines and cytokine receptors were measured by ELISA, whereas a radioimmunoassay was used for neopterin measurement. Six of the seven patients showed increased levels of neopterin either at the onset of RR or 1 month thereafter, and levels declined on prednisone treatment to that seen at the time of diagnosis without reactions. No consistent disease associated cytokine profile was observed in these patients. Interestingly, serum TNF-alpha levels were increased in the same patients even after completion of prednisone treatment, indicating ongoing immune activity. In conclusion, this study demonstrates that despite cytokines levels in leprosy serum being inconsistent in relation to reversal reactions, serum neopterin measurement appears to be an useful biomarker in monitoring RR patients during corticosteroid therapy.

Monitoring Time-Dependent Formation of Oligomers and Brown Carbon in Reactions of Glycolaldehyde, Methylglyoxal, and Amines

NASA Astrophysics Data System (ADS)

Espelien, B.; Galloway, M. M.; De Haan, D. O.

2012-12-01

Authors: Brenna Espelien, Melissa Galloway, and David De Haan The brown carbon components of atmospheric aerosol exhibit strong UV absorbance with a featureless 'tail' that extends into the visible range. Recent work has shown that brown carbon (or HULIS) is formed at least in part by aqueous-phase chemical reactions in the atmosphere. Reactions between aldehydes (such as glycolaldehyde and methylglyoxal) and amines create brown products that have similar light-absorbing spectra as HULIS extracted from atmospheric aerosol. However, the structures of these products have not been well-characterized. Bulk-phase reactions were monitored using LCMS and UV-Vis spectroscopy over a period of 2-3 weeks to see what products formed, whether oligomerization is occurring, and how this correlates with the development of absorbance peaks in the visible range. UV-Vis data shows that these reactions generally take several days to reach maximum absorbance in the visible range. For the glycolaldehyde/glycine reaction, the appearance of a strong absorber at about 400 nm correlated with the appearance of high-mass products at m/z 227, 363, 393, and 431. Additional reactions between aldehydes and amines that quickly produce brown products are being studied. We suggest that imine oligomers are major products of these reactions.

Monitoring of selected non-ionic surfactants in river water by liquid chromatography-tandem mass spectrometry.

PubMed

Zembrzuska, Joanna; Budnik, Irena; Lukaszewski, Zenon

2016-03-15

Alcohol ethoxylates (AEs) are a significant component of the non-ionic surfactant (NS) flux discharged into surface water. Due to the polydispersity of the majority of NS, they are easily recognizable by their 'fingerprints', i.e. a series of mass peaks which differ by m/z = 44, namely the m/z of a single oxyethylene subunit. Dodecanol ethoxylates (C12EOx) represent AEs from both renewable and petrochemical sources. Therefore, C12Ex are suitable fingerprints of NS in the aquatic environment. The aim of this work was to develop an LC-MS/MS method suitable for AE monitoring in river water. River water samples taken from the River Warta in Poznan (Poland) were extracted with ethyl acetate, evaporated, reconstituted in the mobile phase and processed by the LC - Multistage MS procedure (LC-MS/MS) using optimum multiple reaction monitoring (MRM). The method of multiple standard additions was used for the evaluation of each AE fingerprint concentration. The concentration of C12EOx having 2-9 oxyethylene subunits was determined. Standards for higher C12EOx are not yet available. The developed method offers an LOD of between 1 and 9 ng L(-1), and is suitable for the monitoring of NS fingerprints in river water. The range of C12EO2-9 concentrations determined in the River Warta varied within two orders of magnitude in all cases. The lowest determined concentration was 17 ± 1 ng L(-1), while the highest was 2.6 ± 0.14 μg L(-1). The total concentration of C12EO2-C12EO9 homologues varied between 1.4 and 11.2 μg L(-1). A relatively high concentration of short-chained homologues (2-5 oxyethylene subunits) was observed in the investigated river water. This provides evidence of a biodegradation pathway involving the gradual shortening of the AE oxyethylene chain. Distinct evidence was also obtained of unregulated NS discharges into the river. Copyright © 2016 Elsevier Ltd. All rights reserved.

DEMONSTRATION OF LOW COST, LOW BURDEN EXPOSURE MONITORING STRATEGIES FOR USE IN LONGITUDINAL COHORT STUDIES

EPA Science Inventory

A large longitudinal cohort study designed to evaluate the association between children's exposures to environmental agents and health outcomes presents many challenges for exposure monitoring. Exposure of the child must be measured for multiple chemicals through multiple path...

In-line monitoring of cocrystallization process and quantification of carbamazepine-nicotinamide cocrystal using Raman spectroscopy and chemometric tools.

PubMed

Soares, Frederico L F; Carneiro, Renato L

2017-06-05

A cocrystallization process may involve several molecular species, which are generally solid under ambient conditions. Thus, accurate monitoring of different components that might appear during the reaction is necessary, as well as quantification of the final product. This work reports for the first time the synthesis of carbamazepine-nicotinamide cocrystal in aqueous media with a full conversion. The reactions were monitored by Raman spectroscopy coupled with Multivariate Curve Resolution - Alternating Least Squares, and the quantification of the final product among its coformers was performed using Raman spectroscopy and Partial Least Squares regression. The slurry reaction was made in four different conditions: room temperature, 40°C, 60°C and 80°C. The slurry reaction at 80°C enabled a full conversion of initial substrates into the cocrystal form, using water as solvent for a greener method. The employment of MCR-ALS coupled with Raman spectroscopy enabled to observe the main steps of the reactions, such as drug dissolution, nucleation and crystallization of the cocrystal. The PLS models gave mean errors of cross validation around 2.0 (% wt/wt), and errors of validation between 2.5 and 8.2 (% wt/wt) for all components. These were good results since the spectra of cocrystals and the physical mixture of the coformers present some similar peaks. Copyright © 2017 Elsevier B.V. All rights reserved.

Hybrid hard- and soft-modeling of spectrophotometric data for monitoring of ciprofloxacin and its main photodegradation products at different pH values

NASA Astrophysics Data System (ADS)

Razuc, Mariela; Garrido, Mariano; Caro, Yamile S.; Teglia, Carla M.; Goicoechea, Héctor C.; Fernández Band, Beatriz S.

2013-04-01

A simple and fast on line spectrophotometric method combined with a hybrid hard-soft modeling multivariate curve resolution (HS-MCR) was proposed for the monitoring of photodegradation reaction of ciprofloxacin under UV radiation. The studied conditions attempt to emulate the effect of sunlight on these antibiotics that could be eventually present in the environment. The continuous flow system made it possible to study the ciprofloxacin degradation at different pH values almost at real time, avoiding errors that could arise from typical batch monitoring of the reaction. On the base of a concentration profiles obtained by previous pure soft-modeling approach, reaction pathways have been proposed for the parent compound and its photoproducts at different pH values. These kinetic models were used as a constraint in the HS-MCR analysis. The kinetic profiles and the corresponding pure response profile (UV-Vis spectra) of ciprofloxacin and its main degradation products were recovered after the application of HS-MCR analysis to the spectra recorded throughout the reaction. The observed behavior showed a good agreement with the photodegradation studies reported in the bibliography. Accordingly, the photodegradation reaction was studied by high performance liquid chromatography coupled with UV-Vis diode array detector (HPLC-DAD). The spectra recorded during the chromatographic analysis present a good correlation with the ones recovered by UV-Vis/HS-MCR method.

In-line monitoring of cocrystallization process and quantification of carbamazepine-nicotinamide cocrystal using Raman spectroscopy and chemometric tools

NASA Astrophysics Data System (ADS)

Soares, Frederico L. F.; Carneiro, Renato L.

2017-06-01

A cocrystallization process may involve several molecular species, which are generally solid under ambient conditions. Thus, accurate monitoring of different components that might appear during the reaction is necessary, as well as quantification of the final product. This work reports for the first time the synthesis of carbamazepine-nicotinamide cocrystal in aqueous media with a full conversion. The reactions were monitored by Raman spectroscopy coupled with Multivariate Curve Resolution - Alternating Least Squares, and the quantification of the final product among its coformers was performed using Raman spectroscopy and Partial Least Squares regression. The slurry reaction was made in four different conditions: room temperature, 40 °C, 60 °C and 80 °C. The slurry reaction at 80 °C enabled a full conversion of initial substrates into the cocrystal form, using water as solvent for a greener method. The employment of MCR-ALS coupled with Raman spectroscopy enabled to observe the main steps of the reactions, such as drug dissolution, nucleation and crystallization of the cocrystal. The PLS models gave mean errors of cross validation around 2.0 (% wt/wt), and errors of validation between 2.5 and 8.2 (% wt/wt) for all components. These were good results since the spectra of cocrystals and the physical mixture of the coformers present some similar peaks.

Flexibility, Diversity, and Cooperativity: Pillars of Enzyme Catalysis

PubMed Central

Hammes, Gordon G.; Benkovic, Stephen J.; Hammes-Schiffer, Sharon

2011-01-01

This brief review discusses our current understanding of the molecular basis of enzyme catalysis. A historical development is presented, beginning with steady state kinetics and progressing through modern fast reaction methods, NMR, and single molecule fluorescence techniques. Experimental results are summarized for ribonuclease, aspartate aminotransferase, and especially dihydrofolate reductase (DHFR). Multiple intermediates, multiple conformations, and cooperative conformational changes are shown to be an essential part of virtually all enzyme mechanisms. In the case of DHFR, theoretical investigations have provided detailed information about the movement of atoms within the enzyme-substrate complex as the reaction proceeds along the collective reaction coordinate for hydride transfer. A general mechanism is presented for enzyme catalysis that includes multiple intermediates and a complex, multidimensional standard free energy surface. Protein flexibility, diverse protein conformations, and cooperative conformational changes are important features of this model. PMID:22029278

XML Encoding of Features Describing Rule-Based Modeling of Reaction Networks with Multi-Component Molecular Complexes

PubMed Central

Blinov, Michael L.; Moraru, Ion I.

2011-01-01

Multi-state molecules and multi-component complexes are commonly involved in cellular signaling. Accounting for molecules that have multiple potential states, such as a protein that may be phosphorylated on multiple residues, and molecules that combine to form heterogeneous complexes located among multiple compartments, generates an effect of combinatorial complexity. Models involving relatively few signaling molecules can include thousands of distinct chemical species. Several software tools (StochSim, BioNetGen) are already available to deal with combinatorial complexity. Such tools need information standards if models are to be shared, jointly evaluated and developed. Here we discuss XML conventions that can be adopted for modeling biochemical reaction networks described by user-specified reaction rules. These could form a basis for possible future extensions of the Systems Biology Markup Language (SBML). PMID:21464833

Measurement of Walking Ground Reactions in Real-Life Environments: A Systematic Review of Techniques and Technologies.

PubMed

Shahabpoor, Erfan; Pavic, Aleksandar

2017-09-12

Monitoring natural human gait in real-life environments is essential in many applications, including quantification of disease progression, monitoring the effects of treatment, and monitoring alteration of performance biomarkers in professional sports. Nevertheless, developing reliable and practical techniques and technologies necessary for continuous real-life monitoring of gait is still an open challenge. A systematic review of English-language articles from scientific databases including Scopus, ScienceDirect, Pubmed, IEEE Xplore, EBSCO and MEDLINE were carried out to analyse the 'accuracy' and 'practicality' of the current techniques and technologies for quantitative measurement of the tri-axial walking ground reactions outside the laboratory environment, and to highlight their strengths and shortcomings. In total, 679 relevant abstracts were identified, 54 full-text papers were included in the paper and the quantitative results of 17 papers were used for meta-analysis and comparison. Three classes of methods were reviewed: (1) methods based on measured kinematic data; (2) methods based on measured plantar pressure; and (3) methods based on direct measurement of ground reactions. It was found that all three classes of methods have competitive accuracy levels with methods based on direct measurement of the ground reactions showing highest accuracy while being least practical for long-term real-life measurement. On the other hand, methods that estimate ground reactions using measured body kinematics show highest practicality of the three classes of methods reviewed. Among the most prominent technical and technological challenges are: (1) reducing the size and price of tri-axial load-cells; (2) improving the accuracy of orientation measurement using IMUs; (3) minimizing the number and optimizing the location of required IMUs for kinematic measurement; (4) increasing the durability of pressure insole sensors, and (5) enhancing the robustness and versatility of the ground reactions estimation methods to include pathological gaits and natural variability of gait in real-life physical environment.

Measurement of Walking Ground Reactions in Real-Life Environments: A Systematic Review of Techniques and Technologies

PubMed Central

Shahabpoor, Erfan; Pavic, Aleksandar

2017-01-01

Monitoring natural human gait in real-life environments is essential in many applications, including quantification of disease progression, monitoring the effects of treatment, and monitoring alteration of performance biomarkers in professional sports. Nevertheless, developing reliable and practical techniques and technologies necessary for continuous real-life monitoring of gait is still an open challenge. A systematic review of English-language articles from scientific databases including Scopus, ScienceDirect, Pubmed, IEEE Xplore, EBSCO and MEDLINE were carried out to analyse the ‘accuracy’ and ‘practicality’ of the current techniques and technologies for quantitative measurement of the tri-axial walking ground reactions outside the laboratory environment, and to highlight their strengths and shortcomings. In total, 679 relevant abstracts were identified, 54 full-text papers were included in the paper and the quantitative results of 17 papers were used for meta-analysis and comparison. Three classes of methods were reviewed: (1) methods based on measured kinematic data; (2) methods based on measured plantar pressure; and (3) methods based on direct measurement of ground reactions. It was found that all three classes of methods have competitive accuracy levels with methods based on direct measurement of the ground reactions showing highest accuracy while being least practical for long-term real-life measurement. On the other hand, methods that estimate ground reactions using measured body kinematics show highest practicality of the three classes of methods reviewed. Among the most prominent technical and technological challenges are: (1) reducing the size and price of tri-axial load-cells; (2) improving the accuracy of orientation measurement using IMUs; (3) minimizing the number and optimizing the location of required IMUs for kinematic measurement; (4) increasing the durability of pressure insole sensors, and (5) enhancing the robustness and versatility of the ground reactions estimation methods to include pathological gaits and natural variability of gait in real-life physical environment. PMID:28895909

Multiple sclerosis affects the frequency content in the vertical ground reaction forces during walking.

PubMed

Wurdeman, Shane R; Huisinga, Jessie M; Filipi, Mary; Stergiou, Nicholas

2011-02-01

Multiple sclerosis is a progressive neurological disease that results in a high incident of gait disturbance. Exploring the frequency content of the ground reaction forces generated during walking may provide additional insights to gait in patients with multiple sclerosis that could lead to specific tools for differential diagnosis. The purpose of this study was to investigate differences in the frequency content of these forces in an effort to contribute to improved clinical management of this disease. Eighteen patients and eighteen healthy controls walked across a 10 meter long walkway. The anterior-posterior and vertical ground reaction forces generated during the stance phase of gait were evaluated in the frequency domain using fast Fourier transformation. T-tests were utilized for comparison of median frequency, the 99.5% frequency, and the frequency bandwidth between patients and healthy controls and also for comparisons between patients with mild and moderate severity. Patients with multiple sclerosis had significantly lower 99.5% frequency (P=0.006) and median frequency (P<0.001) in the vertical ground reaction force. No differences were found in the anterior-posterior reaction force frequency content. There were no differences between patients with mild and moderate severity. The lower frequency content suggests lesser vertical oscillation of the center of gravity. Lack of differences between severities may suggest presence of differences prior to currently established diagnosis timelines. Analysis of the frequency content may potentially serve to provide earlier diagnostic assessment of this debilitating disease. Copyright © 2010 Elsevier Ltd. All rights reserved.

Chuckwalla Valley multiple-well monitoring site, Chuckwalla Valley, Riverside County

USGS Publications Warehouse

Everett, Rhett

2013-01-01

The U.S. Geological Survey (USGS), in cooperation with the Bureau of Land Management, is evaluating the geohydrology and water availability of the Chuckwalla Valley, California. As part of this evaluation, the USGS installed the Chuckwalla Valley multiple-well monitoring site (CWV1) in the southeastern portion of the Chuckwalla Basin. Data collected at this site provide information about the geology, hydrology, geophysics, and geochemistry of the local aquifer system, thus enhancing the understanding of the geohydrologic framework of the Chuckwalla Valley. This report presents construction information for the CWV1 multiple-well monitoring site and initial geohydrologic data collected from the site.

Orthogonal tandem catalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lohr, Tracy L.; Marks, Tobin J.

2015-05-20

Tandem catalysis is a growing field that is beginning to yield important scientific and technological advances toward new and more efficient catalytic processes. 'One-pot' tandem reactions, where multiple catalysts and reagents, combined in a single reaction vessel undergo a sequence of precisely staged catalytic steps, are highly attractive from the standpoint of reducing both waste and time. Orthogonal tandem catalysis is a subset of one-pot reactions in which more than one catalyst is used to promote two or more mechanistically distinct reaction steps. This Perspective summarizes and analyses some of the recent developments and successes in orthogonal tandem catalysis, withmore » particular focus on recent strategies to address catalyst incompatibility. We also highlight the concept of thermodynamic leveraging by coupling multiple catalyst cycles to effect challenging transformations not observed in single-step processes, and to encourage application of this technique to energetically unfavourable or demanding reactions.« less

A sensitive UPLC-MS/MS method for simultaneous determination of eleven bioactive components of Tong-Xie-Yao-Fang decoction in rat biological matrices.

PubMed

Li, Tian-xue; Hu, Lang; Zhang, Meng-meng; Sun, Jian; Qiu, Yue; Rui, Jun-qian; Yang, Xing-hao

2014-01-01

There is a growing concern for the sensitive quantification of multiple components using advanced data acquisition method in herbal medicines (HMs). An improved and rugged UPLC-MS/MS method has been developed and validated for sensitive and rapid determination of multiply analytes from Tong-Xie-Yao-Fang (TXYF) decoction in three biological matrices (plasma/brain tissue/urine) using geniposide and formononetin as internal standards. After solid-phase extraction, chromatographic separation was performed on a C18 column using gradient elution. Quantifier and qualifier transitions were monitored using novel Triggered Dynamic multiple reaction monitoring (TdMRM) in the positive ionization mode. A significant peak symmetry and sensitivity improvement in the TdMRM mode was achieved as compared to conventional MRM. The reproducibility (RSD%) was ≤7.9% by applying TdMRM transition while the values were 6.8-20.6% for MRM. Excellent linear calibration curves were obtained under TdMRM transitions over the tested concentration ranges. Intra- and inter-day precisions (RSD%) were ≤14.2% and accuracies (RE%) ranged from -9.6% to 10.6%. The validation data of specificity, carryover, recovery, matrix effect and stability were within the required limits. The method was effectively applied to simultaneously detect and quantify 1 lactone, 2 monoterpene glucosides, 1 alkaloid, 5 flavonoids and 2 chromones in plasma, brain tissue and urine after oral administration of TXYF decoction. In conclusion, this new and reliable method is beneficial for quantification and confirmation assays of multiply components in complex biological samples. Copyright © 2013 Elsevier B.V. All rights reserved.

Resolution and Assignment of Differential Ion Mobility Spectra of Sarcosine and Isomers.

PubMed

Berthias, Francis; Maatoug, Belkis; Glish, Gary L; Moussa, Fathi; Maitre, Philippe

2018-04-01

Due to their central role in biochemical processes, fast separation and identification of amino acids (AA) is of importance in many areas of the biomedical field including the diagnosis and monitoring of inborn errors of metabolism and biomarker discovery. Due to the large number of AA together with their isomers and isobars, common methods of AA analysis are tedious and time-consuming because they include a chromatographic separation step requiring pre- or post-column derivatization. Here, we propose a rapid method of separation and identification of sarcosine, a biomarker candidate of prostate cancer, from isomers using differential ion mobility spectrometry (DIMS) interfaced with a tandem mass spectrometer (MS/MS) instrument. Baseline separation of protonated sarcosine from α- and β-alanine isomers can be easily achieved. Identification of DIMS peak is performed using an isomer-specific activation mode where DIMS- and mass-selected ions are irradiated at selected wavenumbers allowing for the specific fragmentation via an infrared multiple photon dissociation (IRMPD) process. Two orthogonal methods to MS/MS are thus added, where the MS/MS(IRMPD) is nothing but an isomer-specific multiple reaction monitoring (MRM) method. The identification relies on the comparison of DIMS-MS/MS(IRMPD) chromatograms recorded at different wavenumbers. Based on the comparison of IR spectra of the three isomers, it is shown that specific depletion of the two protonated α- and β-alanine can be achieved, thus allowing for clear identification of the sarcosine peak. It is also demonstrated that DIMS-MS/MS(IRMPD) spectra in the carboxylic C=O stretching region allow for the resolution of overlapping DIMS peaks. Graphical Abstract ᅟ.

A targeted strategy to identify untargeted metabolites from in vitro to in vivo: Rapid and sensitive metabolites profiling of licorice in rats using ultra-high performance liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry.

PubMed

Huang, Meilin; Cheng, Zhongzhe; Wang, Lu; Feng, Yulin; Huang, Jiangeng; Du, Zhifeng; Jiang, Hongliang

2018-05-29

It is challenging to conduct in vivo metabolic study for traditional Chinese medicines (TCMs) because of complex components, unpredictable metabolic pathways and low metabolite concentrations. Herein, we proposed a sensitive strategy to characterize TCM metabolites in vivo at an orally clinical dose using ultra-high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry (UHPLC-QTRAP-MS). Firstly, the metabolism of individual compounds in rat liver microsomes was studied to obtain the metabolic pathways and fragmentation patterns. The untargeted metabolites in vitro were detected by multiple ion monitoring-enhanced product ion (EPI) and neutral loss-EPI scans. Subsequently, a sensitive multiple reaction monitoring-EPI method was developed according to the in vitro results and predicted metabolites to profile the in vivo metabolites. Licorice as a model herb was used to evaluate and validate our strategy. A clinical dose of licorice water extract was orally administered to rats, then a total of 45 metabolites in urine, 21 metabolites in feces and 35 metabolites in plasma were detected. Among them, 18 minor metabolites have not been reported previously and 6 minor metabolites were first detected in vivo. Several isomeric metabolites were well separated and differentiated in our strategy. These results suggested that this new strategy could be widely used for the detection and characterization of in vivo metabolites of TCMs. Copyright © 2018. Published by Elsevier B.V.

Air-Track: a real-world floating environment for active sensing in head-fixed mice.

PubMed

Nashaat, Mostafa A; Oraby, Hatem; Sachdev, Robert N S; Winter, York; Larkum, Matthew E

2016-10-01

Natural behavior occurs in multiple sensory and motor modalities and in particular is dependent on sensory feedback that constantly adjusts behavior. To investigate the underlying neuronal correlates of natural behavior, it is useful to have access to state-of-the-art recording equipment (e.g., 2-photon imaging, patch recordings, etc.) that frequently requires head fixation. This limitation has been addressed with various approaches such as virtual reality/air ball or treadmill systems. However, achieving multimodal realistic behavior in these systems can be challenging. These systems are often also complex and expensive to implement. Here we present "Air-Track," an easy-to-build head-fixed behavioral environment that requires only minimal computational processing. The Air-Track is a lightweight physical maze floating on an air table that has all the properties of the "real" world, including multiple sensory modalities tightly coupled to motor actions. To test this system, we trained mice in Go/No-Go and two-alternative forced choice tasks in a plus maze. Mice chose lanes and discriminated apertures or textures by moving the Air-Track back and forth and rotating it around themselves. Mice rapidly adapted to moving the track and used visual, auditory, and tactile cues to guide them in performing the tasks. A custom-controlled camera system monitored animal location and generated data that could be used to calculate reaction times in the visual and somatosensory discrimination tasks. We conclude that the Air-Track system is ideal for eliciting natural behavior in concert with virtually any system for monitoring or manipulating brain activity. Copyright © 2016 the American Physiological Society.

Air-Track: a real-world floating environment for active sensing in head-fixed mice

PubMed Central

Oraby, Hatem; Sachdev, Robert N. S.; Winter, York

2016-01-01

Natural behavior occurs in multiple sensory and motor modalities and in particular is dependent on sensory feedback that constantly adjusts behavior. To investigate the underlying neuronal correlates of natural behavior, it is useful to have access to state-of-the-art recording equipment (e.g., 2-photon imaging, patch recordings, etc.) that frequently requires head fixation. This limitation has been addressed with various approaches such as virtual reality/air ball or treadmill systems. However, achieving multimodal realistic behavior in these systems can be challenging. These systems are often also complex and expensive to implement. Here we present “Air-Track,” an easy-to-build head-fixed behavioral environment that requires only minimal computational processing. The Air-Track is a lightweight physical maze floating on an air table that has all the properties of the “real” world, including multiple sensory modalities tightly coupled to motor actions. To test this system, we trained mice in Go/No-Go and two-alternative forced choice tasks in a plus maze. Mice chose lanes and discriminated apertures or textures by moving the Air-Track back and forth and rotating it around themselves. Mice rapidly adapted to moving the track and used visual, auditory, and tactile cues to guide them in performing the tasks. A custom-controlled camera system monitored animal location and generated data that could be used to calculate reaction times in the visual and somatosensory discrimination tasks. We conclude that the Air-Track system is ideal for eliciting natural behavior in concert with virtually any system for monitoring or manipulating brain activity. PMID:27486102

Differences between Drug-Induced and Contrast Media-Induced Adverse Reactions Based on Spontaneously Reported Adverse Drug Reactions.

PubMed

Ryu, JiHyeon; Lee, HeeYoung; Suh, JinUk; Yang, MyungSuk; Kang, WonKu; Kim, EunYoung

2015-01-01

We analyzed differences between spontaneously reported drug-induced (not including contrast media) and contrast media-induced adverse reactions. Adverse drug reactions reported by an in-hospital pharmacovigilance center (St. Mary's teaching hospital, Daejeon, Korea) from 2010-2012 were classified as drug-induced or contrast media-induced. Clinical patterns, frequency, causality, severity, Schumock and Thornton's preventability, and type A/B reactions were recorded. The trends among causality tools measuring drug and contrast-induced adverse reactions were analyzed. Of 1,335 reports, 636 drug-induced and contrast media-induced adverse reactions were identified. The prevalence of spontaneously reported adverse drug reaction-related admissions revealed a suspected adverse drug reaction-reporting rate of 20.9/100,000 (inpatient, 0.021%) and 3.9/100,000 (outpatients, 0.004%). The most common adverse drug reaction-associated drug classes included nervous system agents and anti-infectives. Dermatological and gastrointestinal adverse drug reactions were most frequently and similarly reported between drug and contrast media-induced adverse reactions. Compared to contrast media-induced adverse reactions, drug-induced adverse reactions were milder, more likely to be preventable (9.8% vs. 1.1%, p < 0.001), and more likely to be type A reactions (73.5% vs. 18.8%, p < 0.001). Females were over-represented among drug-induced adverse reactions (68.1%, p < 0.001) but not among contrast media-induced adverse reactions (56.6%, p = 0.066). Causality patterns differed between the two adverse reaction classes. The World Health Organization-Uppsala Monitoring Centre causality evaluation and Naranjo algorithm results significantly differed from those of the Korean algorithm version II (p < 0.001). We found differences in sex, preventability, severity, and type A/B reactions between spontaneously reported drug and contrast media-induced adverse reactions. The World Health Organization-Uppsala Monitoring Centre and Naranjo algorithm causality evaluation afforded similar results.

Differences between Drug-Induced and Contrast Media-Induced Adverse Reactions Based on Spontaneously Reported Adverse Drug Reactions

PubMed Central

Suh, JinUk; Yang, MyungSuk; Kang, WonKu; Kim, EunYoung

2015-01-01

Objective We analyzed differences between spontaneously reported drug-induced (not including contrast media) and contrast media-induced adverse reactions. Methods Adverse drug reactions reported by an in-hospital pharmacovigilance center (St. Mary’s teaching hospital, Daejeon, Korea) from 2010–2012 were classified as drug-induced or contrast media-induced. Clinical patterns, frequency, causality, severity, Schumock and Thornton’s preventability, and type A/B reactions were recorded. The trends among causality tools measuring drug and contrast-induced adverse reactions were analyzed. Results Of 1,335 reports, 636 drug-induced and contrast media-induced adverse reactions were identified. The prevalence of spontaneously reported adverse drug reaction-related admissions revealed a suspected adverse drug reaction-reporting rate of 20.9/100,000 (inpatient, 0.021%) and 3.9/100,000 (outpatients, 0.004%). The most common adverse drug reaction-associated drug classes included nervous system agents and anti-infectives. Dermatological and gastrointestinal adverse drug reactions were most frequently and similarly reported between drug and contrast media-induced adverse reactions. Compared to contrast media-induced adverse reactions, drug-induced adverse reactions were milder, more likely to be preventable (9.8% vs. 1.1%, p < 0.001), and more likely to be type A reactions (73.5% vs. 18.8%, p < 0.001). Females were over-represented among drug-induced adverse reactions (68.1%, p < 0.001) but not among contrast media-induced adverse reactions (56.6%, p = 0.066). Causality patterns differed between the two adverse reaction classes. The World Health Organization–Uppsala Monitoring Centre causality evaluation and Naranjo algorithm results significantly differed from those of the Korean algorithm version II (p < 0.001). Conclusions We found differences in sex, preventability, severity, and type A/B reactions between spontaneously reported drug and contrast media-induced adverse reactions. The World Health Organization–Uppsala Monitoring Centre and Naranjo algorithm causality evaluation afforded similar results. PMID:26544039

Calculation of individual isotope equilibrium constants for implementation in geochemical models

USGS Publications Warehouse

Thorstenson, Donald C.; Parkhurst, David L.

2002-01-01

Theory is derived from the work of Urey to calculate equilibrium constants commonly used in geochemical equilibrium and reaction-transport models for reactions of individual isotopic species. Urey showed that equilibrium constants of isotope exchange reactions for molecules that contain two or more atoms of the same element in equivalent positions are related to isotope fractionation factors by , where is n the number of atoms exchanged. This relation is extended to include species containing multiple isotopes, for example and , and to include the effects of nonideality. The equilibrium constants of the isotope exchange reactions provide a basis for calculating the individual isotope equilibrium constants for the geochemical modeling reactions. The temperature dependence of the individual isotope equilibrium constants can be calculated from the temperature dependence of the fractionation factors. Equilibrium constants are calculated for all species that can be formed from and selected species containing , in the molecules and the ion pairs with where the subscripts g, aq, l, and s refer to gas, aqueous, liquid, and solid, respectively. These equilibrium constants are used in the geochemical model PHREEQC to produce an equilibrium and reaction-transport model that includes these isotopic species. Methods are presented for calculation of the individual isotope equilibrium constants for the asymmetric bicarbonate ion. An example calculates the equilibrium of multiple isotopes among multiple species and phases.

Synchronized Survey Scan Approach Allows for Efficient Discrimination of Isomeric and Isobaric Compounds during LC-MS/MS Analyses

PubMed Central

Masike, Keabetswe

2018-01-01

Liquid chromatography-mass spectrometry- (LC-MS-) based multiple reaction monitoring (MRM) methods have been used to detect and quantify metabolites for years. These approaches rely on the monitoring of various fragmentation pathways of multiple precursors and the subsequent corresponding product ions. However, MRM methods are incapable of confidently discriminating between isomeric and isobaric molecules and, as such, the development of methods capable of overcoming this challenge has become imperative. Due to increasing scanning rates of recent MS instruments, it is now possible to operate MS instruments both in the static and dynamic modes. One such method is known as synchronized survey scan (SSS), which is capable of acquiring a product ion scan (PIS) during MRM analysis. The current study shows, for the first time, the use of SSS-based PIS approach as a feasible identification feature of MRM. To achieve the above, five positional isomers of dicaffeoylquinic acids (diCQAs) were studied with the aid of SSS-based PIS method. Here, the MRM transitions were automatically optimized using a 3,5-diCQA isomer by monitoring fragmentation transitions common to all five isomers. Using the mixture of these isomers, fragmentation spectra of the five isomers achieved with SSS-based PIS were used to identify each isomer based on previously published hierarchical fragmentation keys. The optimized method was also used to detect and distinguish between diCQA components found in Bidens pilosa and their isobaric counterparts found in Moringa oleifera plants. Thus, the method was shown to distinguish (by differences in fragmentation patterns) between diCQA and their isobars, caffeoylquinic acid (CQA) glycosides. In conclusion, SSS allowed the detection and discrimination of isomeric and isobaric compounds in a single chromatographic run by producing a PIS spectrum, triggered in the automatic MS/MS synchronized survey scan mode. PMID:29805830

Integration of multisensor hybrid reasoners to support personal autonomy in the smart home.

PubMed

Valero, Miguel Ángel; Bravo, José; Chamizo, Juan Manuel García; López-de-Ipiña, Diego

2014-09-17

The deployment of the Ambient Intelligence (AmI) paradigm requires designing and integrating user-centered smart environments to assist people in their daily life activities. This research paper details an integration and validation of multiple heterogeneous sensors with hybrid reasoners that support decision making in order to monitor personal and environmental data at a smart home in a private way. The results innovate on knowledge-based platforms, distributed sensors, connected objects, accessibility and authentication methods to promote independent living for elderly people. TALISMAN+, the AmI framework deployed, integrates four subsystems in the smart home: (i) a mobile biomedical telemonitoring platform to provide elderly patients with continuous disease management; (ii) an integration middleware that allows context capture from heterogeneous sensors to program environment's reaction; (iii) a vision system for intelligent monitoring of daily activities in the home; and (iv) an ontologies-based integrated reasoning platform to trigger local actions and manage private information in the smart home. The framework was integrated in two real running environments, the UPM Accessible Digital Home and MetalTIC house, and successfully validated by five experts in home care, elderly people and personal autonomy.

MRM-DIFF: data processing strategy for differential analysis in large scale MRM-based lipidomics studies.

PubMed

Tsugawa, Hiroshi; Ohta, Erika; Izumi, Yoshihiro; Ogiwara, Atsushi; Yukihira, Daichi; Bamba, Takeshi; Fukusaki, Eiichiro; Arita, Masanori

2014-01-01

Based on theoretically calculated comprehensive lipid libraries, in lipidomics as many as 1000 multiple reaction monitoring (MRM) transitions can be monitored for each single run. On the other hand, lipid analysis from each MRM chromatogram requires tremendous manual efforts to identify and quantify lipid species. Isotopic peaks differing by up to a few atomic masses further complicate analysis. To accelerate the identification and quantification process we developed novel software, MRM-DIFF, for the differential analysis of large-scale MRM assays. It supports a correlation optimized warping (COW) algorithm to align MRM chromatograms and utilizes quality control (QC) sample datasets to automatically adjust the alignment parameters. Moreover, user-defined reference libraries that include the molecular formula, retention time, and MRM transition can be used to identify target lipids and to correct peak abundances by considering isotopic peaks. Here, we demonstrate the software pipeline and introduce key points for MRM-based lipidomics research to reduce the mis-identification and overestimation of lipid profiles. The MRM-DIFF program, example data set and the tutorials are downloadable at the "Standalone software" section of the PRIMe (Platform for RIKEN Metabolomics, http://prime.psc.riken.jp/) database website.

MRM-DIFF: data processing strategy for differential analysis in large scale MRM-based lipidomics studies

PubMed Central

Tsugawa, Hiroshi; Ohta, Erika; Izumi, Yoshihiro; Ogiwara, Atsushi; Yukihira, Daichi; Bamba, Takeshi; Fukusaki, Eiichiro; Arita, Masanori

2015-01-01

Based on theoretically calculated comprehensive lipid libraries, in lipidomics as many as 1000 multiple reaction monitoring (MRM) transitions can be monitored for each single run. On the other hand, lipid analysis from each MRM chromatogram requires tremendous manual efforts to identify and quantify lipid species. Isotopic peaks differing by up to a few atomic masses further complicate analysis. To accelerate the identification and quantification process we developed novel software, MRM-DIFF, for the differential analysis of large-scale MRM assays. It supports a correlation optimized warping (COW) algorithm to align MRM chromatograms and utilizes quality control (QC) sample datasets to automatically adjust the alignment parameters. Moreover, user-defined reference libraries that include the molecular formula, retention time, and MRM transition can be used to identify target lipids and to correct peak abundances by considering isotopic peaks. Here, we demonstrate the software pipeline and introduce key points for MRM-based lipidomics research to reduce the mis-identification and overestimation of lipid profiles. The MRM-DIFF program, example data set and the tutorials are downloadable at the “Standalone software” section of the PRIMe (Platform for RIKEN Metabolomics, http://prime.psc.riken.jp/) database website. PMID:25688256

A Method for Comprehensive Glycosite-Mapping and Direct Quantitation of Serum Glycoproteins.

PubMed

Hong, Qiuting; Ruhaak, L Renee; Stroble, Carol; Parker, Evan; Huang, Jincui; Maverakis, Emanual; Lebrilla, Carlito B

2015-12-04

A comprehensive glycan map was constructed for the top eight abundant glycoproteins in plasma using both specific and nonspecific enzyme digestions followed by nano liquid chromatography (LC)-chip/quadrupole time-of-flight mass spectrometry (MS) analysis. Glycopeptides were identified using an in-house software tool, GPFinder. A sensitive and reproducible multiple reaction monitoring (MRM) technique on a triple quadrupole MS was developed and applied to quantify immunoglobulins G, A, M, and their site-specific glycans simultaneously and directly from human serum/plasma without protein enrichments. A total of 64 glycopeptides and 15 peptides were monitored for IgG, IgA, and IgM in a 20 min ultra high performance (UP)LC gradient. The absolute protein contents were quantified using peptide calibration curves. The glycopeptide ion abundances were normalized to the respective protein abundances to separate protein glycosylation from protein expression. This technique yields higher method reproducibility and less sample loss when compared with the quantitation method that involves protein enrichments. The absolute protein quantitation has a wide linear range (3-4 orders of magnitude) and low limit of quantitation (femtomole level). This rapid and robust quantitation technique, which provides quantitative information for both proteins and glycosylation, will further facilitate disease biomarker discoveries.

MRMPlus: an open source quality control and assessment tool for SRM/MRM assay development.

PubMed

Aiyetan, Paul; Thomas, Stefani N; Zhang, Zhen; Zhang, Hui

2015-12-12

Selected and multiple reaction monitoring involves monitoring a multiplexed assay of proteotypic peptides and associated transitions in mass spectrometry runs. To describe peptide and associated transitions as stable, quantifiable, and reproducible representatives of proteins of interest, experimental and analytical validation is required. However, inadequate and disparate analytical tools and validation methods predispose assay performance measures to errors and inconsistencies. Implemented as a freely available, open-source tool in the platform independent Java programing language, MRMPlus computes analytical measures as recommended recently by the Clinical Proteomics Tumor Analysis Consortium Assay Development Working Group for "Tier 2" assays - that is, non-clinical assays sufficient enough to measure changes due to both biological and experimental perturbations. Computed measures include; limit of detection, lower limit of quantification, linearity, carry-over, partial validation of specificity, and upper limit of quantification. MRMPlus streamlines assay development analytical workflow and therefore minimizes error predisposition. MRMPlus may also be used for performance estimation for targeted assays not described by the Assay Development Working Group. MRMPlus' source codes and compiled binaries can be freely downloaded from https://bitbucket.org/paiyetan/mrmplusgui and https://bitbucket.org/paiyetan/mrmplusgui/downloads respectively.

Liquid chromatography-tandem mass spectrometry method for the analysis of N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine, a biocidal disinfectant, in dairy products.

PubMed

Slimani, Kahina; Pirotais, Yvette; Maris, Pierre; Abjean, Jean-Pierre; Hurtaud-Pessel, Dominique

2018-10-01

A novel and reliable method to quantify residual levels of N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine in dairy products using ion-pairing reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and fully validated. Sample extraction was done with salting-out technique using acetonitrile and sodium chloride. For LC-MS/MS, the analyte was detected using positive electrospray ionization (ESI+) and two multiple reaction monitoring (MRM) transitions were monitored. The method was validated in the 5-150 µg kg -1 range using total error approach. Thus, performance criteria of the method were evaluated. Relative standard deviations for trueness and precision were lower than 10%; with the exception of hard pressed cheese at 5 µg kg -1 for precision. The limit of quantification (LOQ) was around 5-7 µg kg -1 depending on the matrix of interest. The method was successfully applied to accurately quantify N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine in 146 various dairy products with a maximum contamination level of 225 µg kg -1 in cheese. Copyright © 2018 Elsevier Ltd. All rights reserved.

Ultrasound/microwave-assisted solid-liquid-solid dispersive extraction with high-performance liquid chromatography coupled to tandem mass spectrometry for the determination of neonicotinoid insecticides in Dendrobium officinale.

PubMed

Zheng, Shuilian; Wu, Huizhen; Li, Zuguang; Wang, Jianmei; Zhang, Hu; Qian, Mingrong

2015-01-01

A one-step ultrasound/microwave-assisted solid-liquid-solid dispersive extraction procedure was used for the simultaneous determination of eight neonicotinoids (dinotefuran, nitenpyram, thiamethoxam, clothianidin, imidacloprid, acetamiprid, thiacloprid, imidaclothiz) in dried Dendrobium officinale by liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry in multiple reaction monitoring mode. The samples were quickly extracted by acetonitrile and cleaned up by the mixed dispersing sorbents including primary secondary amine, C18 , and carbon-GCB. Parameters that could influence the ultrasound/microwave-assisted extraction efficiency such as microwave irradiation power, ultrasound irradiation power, temperature, and solvent were investigated. Recovery studies were performing well (70.4-113.7%) at three examined spiking levels (10, 50, and 100 μg/kg). Meanwhile, the limits of quantification for the neonicotinoids ranged from 0.87 to 1.92 μg/kg. The method showed good linearity in the concentration range of 1-100 μg/L with correlation coefficients >0.99. This quick and useful analytical method could provide a basis for monitoring neonicotinoid insecticide residues in herbs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Integration of Multisensor Hybrid Reasoners to Support Personal Autonomy in the Smart Home

PubMed Central

Valero, Miguel Ángel; Bravo, José; Chamizo, Juan Manuel García; López-de-Ipiña, Diego

2014-01-01

The deployment of the Ambient Intelligence (AmI) paradigm requires designing and integrating user-centered smart environments to assist people in their daily life activities. This research paper details an integration and validation of multiple heterogeneous sensors with hybrid reasoners that support decision making in order to monitor personal and environmental data at a smart home in a private way. The results innovate on knowledge-based platforms, distributed sensors, connected objects, accessibility and authentication methods to promote independent living for elderly people. TALISMAN+, the AmI framework deployed, integrates four subsystems in the smart home: (i) a mobile biomedical telemonitoring platform to provide elderly patients with continuous disease management; (ii) an integration middleware that allows context capture from heterogeneous sensors to program environment's reaction; (iii) a vision system for intelligent monitoring of daily activities in the home; and (iv) an ontologies-based integrated reasoning platform to trigger local actions and manage private information in the smart home. The framework was integrated in two real running environments, the UPM Accessible Digital Home and MetalTIC house, and successfully validated by five experts in home care, elderly people and personal autonomy. PMID:25232910