scholarly journals 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 ◽  
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

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.

scholarly journals 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 ◽  
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

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.

Pseudo-Multiple Reaction Monitoring (Pseudo-MRM) Mode on the “Brick” Mass Spectrometer, Using the Grid-SWIFT Waveform

2019 ◽  
Vol 91 (21) ◽  
pp. 13838-13846 ◽  
Author(s):  
Zuqiang Xu ◽  
Ting Jiang ◽  
Qian Xu ◽  
Yanbing Zhai ◽  
Dayu Li ◽  
...  
Keyword(s):  
Mass Spectrometer ◽  
Multiple Reaction Monitoring ◽  
Reaction Monitoring

scholarly journals Rapid Identification of New Delhi Metallo-β-Lactamase (NDM) Using Tryptic Peptides and LC-MS/MS

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Honghui Wang ◽  
Jeffrey R. Strich ◽  
Steven K. Drake ◽  
Yong Chen ◽  
Jung-Ho Youn ◽  
...  
Keyword(s):  
Protein Expression ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Multiple Reaction Monitoring ◽  
Protein Detection ◽  
Turnaround Time ◽  
Rapid Identification ◽  
New Delhi ◽  
Negative Results ◽  
Resistance Protein

ABSTRACT There is significant interest in the development of mass spectrometry (MS) methods for antimicrobial resistance protein detection, given the ability of these methods to confirm protein expression. In this work, we studied the performance of a liquid chromatography, tandem MS multiple-reaction monitoring (LC-MS/MS MRM) method for the direct detection of the New Delhi metallo-β-lactamase (NDM) carbapenemase in clinical isolates. Using a genoproteomic approach, we selected three unique peptides (SLGNLGDADTEHYAASAR, AFGAAFPK, and ASMIVMSHSAPDSR) specific to NDM that were efficiently ionized and spectrally well-defined. These three peptides were used to build an assay with turnaround time of 90 min. In a blind set, the assay detected 21/24 blaNDM-containing isolates and 76/76 isolates with negative results, corresponding to a sensitivity value of 87.5% (95% confidence interval [CI], 67.6% to 97.3%) and a specificity value of 100% (95% CI, 95.3% to 100%). One of the missed identifications was determined by protein fractionation to be due to low (∼0.1 fm/μg) NDM protein expression (below the assay limit of detection). Parallel disk diffusion susceptibility testing demonstrated this isolate to be meropenem susceptible, consistent with low NDM expression. Total proteomic analysis of the other two missed identifications did not detect NDM peptides but detected other proteins expressed from the blaNDM-containing plasmids, confirming that the plasmids were not lost. The measurement of relative NDM concentrations over the entire isolate test set demonstrated variability spanning 4 orders of magnitude, further confirming the remarkable range that may be seen in levels of NDM expression. This report highlights the sensitivity of LC-MS/MS to variations in NDM protein expression, with implications for how this technology may be used.

scholarly journals Carbon Nanotube-Based Electrochemical Biosensor for Label-Free Protein Detection

Biosensors ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 144 ◽  
Author(s):  
Jesslyn Janssen ◽  
Mike Lambeta ◽  
Paul White ◽  
Ahmad Byagowi
Keyword(s):  
Limit Of Detection ◽  
Protein Detection ◽  
Protein Quantification ◽  
Clinical Diagnostics ◽  
Biological Research ◽  
Label Free ◽  
Protein Biomarkers ◽  
Biosensor System ◽  
Elisa Technique ◽  
Standard Protein

There is a growing need for biosensors that are capable of efficiently and rapidly quantifying protein biomarkers, both in the biological research and clinical setting. While accurate methods for protein quantification exist, the current assays involve sophisticated techniques, take long to administer and often require highly trained personnel for execution and analysis. Herein, we explore the development of a label-free biosensor for the detection and quantification of a standard protein. The developed biosensors comprise carbon nanotubes (CNTs), a specific antibody and cellulose filtration paper. The change in electrical resistance of the CNT-based biosensor system was used to sense a standard protein, bovine serum albumin (BSA) as a proof-of-concept. The developed biosensors were found to have a limit of detection of 2.89 ng/mL, which is comparable to the performance of the typical ELISA method for BSA quantification. Additionally, the newly developed method takes no longer than 10 min to perform, greatly reducing the time of analysis compared to the traditional ELISA technique. Overall, we present a versatile, affordable, simplified and rapid biosensor device capable of providing great benefit to both biological research and clinical diagnostics.

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

2011 ◽  
Vol 83 (6) ◽  
pp. 2162-2171 ◽  
Author(s):  
Mikhail E. Belov ◽  
Satendra Prasad ◽  
David C. Prior ◽  
William F. Danielson ◽  
Karl Weitz ◽  
...  
Keyword(s):  
Mass Spectrometer ◽  
Multiple Reaction Monitoring ◽  
Quadrupole Mass Spectrometer ◽  
Reaction Monitoring ◽  
Quadrupole Mass ◽  
Tandem Quadrupole Mass Spectrometer ◽  
Tandem Quadrupole

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

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.

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