Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants

Six ebolavirus species are reported to date, including human pathogens Bundibugyo virus (BDBV), Ebola virus (EBOV), Sudan virus (SUDV), and Taï Forest virus (TAFV); non-human pathogen Reston virus (RESTV); and the plausible Bombali virus (BOMV). Since there are differences in the disease severity caused by different species, species identification and viral burden quantification are critical for treating infected patients timely and effectively. Here we developed an immunoprecipitation-coupled mass spectrometry (IP-MS) assay for VP40 antigen detection and quantification. We carefully selected two regions of VP40, designated as peptide 8 and peptide12 from the protein sequence that showed minor variations among Ebolavirus species through MS analysis of tryptic peptides and antigenicity prediction based on available bioinformatic tools, and generated high-quality capture antibodies pan-specific for these variant peptides. We applied this assay to human plasma spiked with recombinant VP40 protein from EBOV, SUDV, and BDBV and virus-like particles (VLP), as well as EBOV infected NHP plasma. Sequence substitutions between EBOV and SUDV, the two species with highest lethality, produced affinity variations of 2.6-fold for p8 and 19-fold for p12. The proposed IP-MS assay differentiates four of the six known EBV species in one assay, through a combination of p8 and p12 data. The IP-MS assay limit of detection (LOD) using multiple reaction monitoring (MRM) as signal readout was determined to be 28 ng/mL and 7 ng/mL for EBOV and SUDV respectively, equivalent to ~1.625–6.5×105 Geq/mL, and comparable to the LOD of lateral flow immunoassays currently used for Ebola surveillance. The two peptides of the IP-MS assay were also identified by their tandem MS spectra using a miniature MALDI-TOF MS instrument, greatly increasing the feasibility of high specificity assay in a decentralized laboratory.

Download Full-text

Needle lost in the haystack: multiple reaction monitoring fails to detect Treponema pallidum candidate protein biomarkers in plasma and urine samples from individuals with syphilis

F1000Research ◽

10.12688/f1000research.13964.2 ◽

2018 ◽

Vol 7 ◽

pp. 336 ◽

Cited By ~ 2

Author(s):

Geert A. Van Raemdonck ◽

Kara K. Osbak ◽

Xaveer Van Ostade ◽

Chris R. Kenyon

Keyword(s):

Mass Spectrometer ◽

Limit Of Detection ◽

Multiple Reaction Monitoring ◽

Treponema Pallidum ◽

Protein Detection ◽

Reaction Monitoring ◽

Protein Biomarkers ◽

Diagnostic Strategies ◽

Plasma And Urine Samples ◽

Candidate Protein

Background:Current syphilis diagnostic strategies are lacking a sensitive manner of directly detectingTreponema pallidumantigens. A diagnostic test that could directly detectT. pallidumantigens in individuals with syphilis would be of considerable clinical utility, especially for the diagnosis of reinfections and for post-treatment serological follow-up.Methods:In this study, 11 candidateT. pallidumbiomarker proteins were chosen according to their physiochemical characteristics,T. pallidumspecificity and predicted abundance. Thirty isotopically labelled proteotypic surrogate peptides (hPTPs) were synthesized and incorporated into a scheduled multiple reaction monitoring assay. Protein extracts from undepleted/unenriched plasma (N = 18) and urine (N = 4) samples from 18 individuals with syphilis in various clinical stages were tryptically digested, spiked with the hPTP mixture and analysed with a triple quadruple mass spectrometer.Results:No endogenous PTPs corresponding to the eleven candidate biomarkers were detected in any samples analysed. To estimate the Limit of Detection (LOD) of a comparably sensitive mass spectrometer (LTQ-Orbitrap), two dilution series of rabbit cultured purifiedT. pallidumwere prepared in PBS. Polyclonal anti-T. pallidumantibodies coupled to magnetic Dynabeads were used to enrich one sample series; no LOD improvement was found compared to the unenriched series. The estimated LOD of MS instruments is 300T. pallidum/ml in PBS.Conclusions:Biomarker protein detection likely failed due to the low (femtomoles/liter) predicted concentration ofT. pallidumproteins. Alternative sample preparation strategies may improve the detectability ofT. pallidumproteins in biofluids.

Download Full-text

Quantification and Confirmation of Zearalenone Using a LC-MS/MS QTRAP System in Multiple Reaction Monitoring and Enhanced Product Ion Scan Modes.

10.21203/rs.3.rs-65392/v1 ◽

2020 ◽

Author(s):

Shencong lv ◽

Xiaoqiong Wu ◽

Guoying Zhu ◽

Jian Guan ◽

Yong Yan ◽

...

Keyword(s):

Time Perspective ◽

Ion Trap ◽

Corn Oil ◽

Limit Of Detection ◽

Multiple Reaction Monitoring ◽

Correlation Coefficients ◽

Sample Pretreatment ◽

Gradient Elution ◽

Reaction Monitoring ◽

Liquid Chromatography Tandem Mass

Abstract Background: A simple, rapid, and efficient liquid chromatography tandem mass spectrometry (LC–MS/MS) method, operated in electrospray ionization (ESI) and quadrupole linear ion trap modes, has been developed for the identification and structural characterization of zearalenone (ZEN) in corn oil. Methods: Samples (5 g) were extracted with acetonitrile/water (80:20, v/v). After centrifugation and dilution, the extracts were separated on a C18 analytical column by gradient elution (acetonitrile/water) and analyzed by UPLC–MS/MS. Enhanced product ion mode was used for qualitative analysis, while multiple reaction monitoring mode was used for quantitative analysis. Results: Calibration curve showed good linearity with correlation coefficients (r) higher than 0.995. Limit of detection was determined to be below 0.20 μg kg-1 for ZEN. The recovery for ZEN was in the acceptable range of 86.6% to 97.2%. 82.4 % of the samples were found to contain ZEN among the 51 samples.Conclusion: The sample pretreatment and LC–MS methods developed in this research, from a convenience and analysis time perspective, are simple, efficient, cheaper, and less time-consuming than existing methods.

Download Full-text

Screening of Danish traffic cases for synthetic cannabinoids in whole blood by LC-MS/MS

Scandinavian Journal of Forensic Science ◽

10.2478/sjfs-2013-0008 ◽

2013 ◽

Vol 19 (2) ◽

pp. 45-51 ◽

Cited By ~ 5

Author(s):

Niels Bjerre Holm ◽

Rebeca Sequera Pineda ◽

David Wederkinck Andersen ◽

Brian Schou Rasmussen ◽

Petur Weihe Dalsgaard ◽

...

Keyword(s):

Whole Blood ◽

Limit Of Detection ◽

Solid Phase ◽

Multiple Reaction Monitoring ◽

Synthetic Cannabinoids ◽

Screening Method ◽

Driving Under The Influence ◽

Reaction Monitoring ◽

Multiple Reaction Monitoring Mode ◽

Target Screening

ABSTRACT A target screening method for the detection of 13 synthetic cannabinoids in whole blood was developed and validated. Samples underwent automated solid-phase extraction, and sample extracts were analyzed by liquid chromatography-positive electrospray ionization-tandem mass spectrometry using two transitions in multiple reaction monitoring mode. The limit of detection was between 0.1-2.5 ng/mL for the compounds except HU-210, and extraction recovery ranged from 59 to 78%. The method was used to screen 393 Danish traffic cases from 2012, where the driver was suspected of driving under the influence of drugs. No synthetic cannabinoids were identified in these samples. Additionally, the method was applied to a clinical intoxication case, and the synthetic cannabinoid AM- 2201 was identified in serum. We conclude that the prevalence of driving under the influence of synthetic cannabinoids in Denmark is likely to be low, and that synthetic cannabinoids are most likely to be encountered in the clinical setting.

Download Full-text

BATL: Bayesian annotations for targeted lipidomics

10.1101/2021.03.18.435788 ◽

2021 ◽

Author(s):

Justin G Chitpin ◽

Graeme P Taylor ◽

Hongbin Xu ◽

Thao T Nguyen ◽

Roberto Ortega ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Multiple Reaction Monitoring ◽

Tandem Mass ◽

Reaction Monitoring ◽

Peak Shape ◽

Posterior Probabilities ◽

Bayes Classifier ◽

Bioinformatic Tools ◽

Ionization Tandem Mass Spectrometry

Motivation: Bioinformatic tools capable of annotating, rapidly and reproducibly, large targeted lipidomic datasets are limited. Specifically, few programs enable high-throughput peak assessment of liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) data acquired in either selected or multiple reaction monitoring (SRM and MRM) modes. Results: We present here Bayesian Annotations for Targeted Lipidomics (BATL), a Gaussian naive Bayes classifier for targeted lipidomics, that annotates peak identities according to eight features related to retention time, intensity, and peak shape. Lipid identification is achieved by modelling distributions of these eight input features across biological conditions and maximizing the joint posterior probabilities of all peak identities at a given transition. When applied to sphingolipid and glycerophosphocholine SRM datasets, we demonstrate over 95% of all peaks are rapidly and correctly identified. Availability: The BATL software is available on GitHub at https://github.com/lipidomic-uottawa/batl Contact: [email protected] and [email protected]

Download Full-text

Needle lost in the haystack: multiple reaction monitoring fails to detect Treponema pallidum candidate protein biomarkers in plasma and urine samples from individuals with syphilis

F1000Research ◽

10.12688/f1000research.13964.1 ◽

2018 ◽

Vol 7 ◽

pp. 336

Author(s):

Geert A. Van Raemdonck ◽

Kara K. Osbak ◽

Xaveer Van Ostade ◽

Chris R. Kenyon

Keyword(s):

Mass Spectrometer ◽

Limit Of Detection ◽

Multiple Reaction Monitoring ◽

Treponema Pallidum ◽

Protein Detection ◽

Reaction Monitoring ◽

Protein Biomarkers ◽

Diagnostic Strategies ◽

Plasma And Urine Samples ◽

Candidate Protein

Download Full-text

146 QUANTIFICATION OF PEPTIDE GROWTH FACTORS IN CATTLE UTERINE FLUID BY MULTIPLE REACTION MONITORING-LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab146 ◽

2015 ◽

Vol 27 (1) ◽

pp. 165

Author(s):

E. Gómez ◽

F. J. Corrales ◽

M. I. Mora ◽

E. Correia ◽

S. Carrocera ◽

...

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Dynamic Range ◽

Limit Of Detection ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Tissue Growth ◽

Reaction Monitoring ◽

Kit Ligand ◽

Uterine Fluid

Multiple reaction monitoring (MRM) allows targeted quantitative proteomics with a wide dynamic range and limit of detection down to femtomoles. We used MRM to study uterine growth factors (GF) presumed to promote embryonic development. A validated experimental model was used to recover uterine fluid (UF) and analyse GF expression in the presence or absence of embryos. Briefly, Day-6 in vitro-produced embryos (n = 50) or vehicle (sham transfer) were transferred into the uteri of each oestrus-synchronized Holstein heifer (n = 14) during nonconsecutive cycles. Blood P4 concentrations were measured on Days 0 (oestrus), 6, and 8. On Day 8, UF was recovered from embryo and sham recipients. After retrieval, UF were centrifuged and supernatants stored at –145°C. Sham and embryo UF selected for MRM were from n = 10 animals (n = 20 samples). Uterine fluid, recovered after embryo transfer, contained on average n = 43.1 ± 5.2 total and n = 34.1 ± 3.7% viable embryos per recipient. For MRM, UF samples were concentrated, and protein was precipitated and resuspended in ammonium bicarbonate. Protein (20 μg) was reduced with DTT, trypsin-digested, and desalted. Proteotypic peptides for targeted GF were selected with MRM Pilot software (ABsciex, Farmingham, MA, USA), with 3 to 5 transitions programmed for each peptide. A control, unrelated synthetic peptide was spiked as an internal standard. The area of the larger transition for the control peptide was used to normalise the area values of each other peptide. The MRM experiments were performed on a 5500 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer (ABsciex) equipped with an Eksigent 1D+plus nanoLC chromatographic system. Data analysis was performed with Analyst 1.5.2 and MultiQuant 2.0.2 softwares (ABsciex). The area of most abundant transition for each analysed peptide was used for relative quantitation. Proteins studied were betacellulin, heparin-binding EGF-like growth factor, neuregulin, artemin, connective tissue growth factor, nerve growth factor, kit ligand, stanniocalcin-1 (STC1), early pregnancy factor (EPF), and hepatoma-derived growth factor (HDGF). Proteotypic peptides were identified in all samples for HDGF, kit ligand, STC1, and EPF (n = 1, n = 1, n = 1, and n = 3 peptides, respectively), which precluded the analysis of the remaining GF. No differences in relative abundance were detected between UF containing or not containing embryos for HDGF, kit ligand, STC1, and EPF (2.85 ± 0.6 v. 4.43 ± 0.6; 0.15 ± 0.02 v. 0.16 ± 0.02; 0.03 ± 0.00 v. 0.04 ± 0.00; and 1.20 ± 0.16 v. 1.09 ± 1.16, respectively). However, STC1 and Day 8 blood P4 were highly correlated (r = 0.71; P = 0.0004), suggesting P4 regulation of STC1. Multiple reaction monitoring-LC-MS/MS is a useful technique to identify some scarce GF in UF at different dynamic ranges. MICINN, project AGL2012-37772 and FEDER. E. C. was supported by MEC-FPU-AP2009-5265. The authors are members of the COST Action FA1201 Epiconcept: Epigenetics and Periconception environment.

Download Full-text

Use of Deuterated Internal Standards for Quantitation of T-2 and HT-2 Toxins in Human Blood by Tandem Mass Spectrometry

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/72.5.807 ◽

1989 ◽

Vol 72 (5) ◽

pp. 807-812 ◽

Cited By ~ 1

Author(s):

Robert J Pawlosky ◽

Chester J Mirocha ◽

Y Wen ◽

Hamed K Abbas

Keyword(s):

Mass Spectrometry ◽

Detection Limit ◽

Tandem Mass Spectrometry ◽

Human Blood ◽

Limit Of Detection ◽

Multiple Reaction Monitoring ◽

Tandem Mass ◽

Reaction Monitoring ◽

Internal Standards ◽

Selection Of

Abstract Deuterated acetyl derivatives (3-trideutero-acetyl-T-2 and 15-trideutero- HT-2) were prepared for use as internal standards for the quantitation of T-2 and HT-2 in blood by tandem mass spectrometry. The method used was multiple reaction monitoring (MRM), which essentially involves the selection of a parent ion for analysis followed by monitoring of the daughter ions generated by collision activated decomposition. The parent ions chosen for the trifluoroacetate derivative of T-2 and HT-2 were m/z+ 478 and 532, respectively. Both parents yield the same daughter ions, i.e., 180, 138, and 121. HT-2 and T-2 were added to blood extracts in amounts ranging from 1 to 20 ppb. The limit of detection is about 0.5 ppb with an effective detection limit of 1.0 ppb in a range of 1-20 ppb. The recovery is about 90%. This method can be used by veterinarians for purposes of diagnostics. It can be used for urine as well as blood.

Download Full-text