Dynamic Range Of Quantification Research Articles

Assessment of current mass spectrometric workflows for the quantification of low abundant proteins and phosphorylation sites.

The data described here provide a systematic performance evaluation of popular data-dependent (DDA) and independent (DIA) mass spectrometric (MS) workflows currently used in quantitative proteomics. We assessed the limits of identification, quantification and detection for each method by analyzing a dilution series of 20 unmodified and 10 phosphorylated synthetic heavy labeled reference peptides, respectively, covering six orders of magnitude in peptide concentration with and without a complex human cell digest background. We found that all methods performed very similarly in the absence of background proteins, however, when analyzing whole cell lysates, targeted methods were at least 5–10 times more sensitive than directed or DDA methods. In particular, higher stage fragmentation (MS3) of the neutral loss peak using a linear ion trap increased dynamic quantification range of some phosphopeptides up to 100-fold. We illustrate the power of this targeted MS3 approach for phosphopeptide monitoring by successfully quantifying 9 phosphorylation sites of the kinetochore and spindle assembly checkpoint component Mad1 over different cell cycle states from non-enriched pull-down samples. The data are associated to the research article ‘Evaluation of data-dependent and data-independent mass spectrometric workflows for sensitive quantification of proteins and phosphorylation sites׳ (Bauer et al., 2014) [1]. The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000964.

Carbon dots as a fluorescent probe for label-free detection of physiological potassium level in human serum and red blood cells

A unique photoluminescence carbon dots (CDs) with larger size were prepared by microwave-assisted method. Complex functional groups on the surface of the CDs facilitate the nanoparticles to form affinity with some metal ions. Taking advantage of the effective fluorescence quenching effect of K+, a highly sensitive CD-based fluorescence analytical system for label-free detection of K+ with limit of detection (LOD) 1.0×10−12M was established. The concentrations of potassium ion in biological samples such as human serum are usually found at millimolar levels or even higher. The proposed method begins with a substantial dilution of the sample to place the K+ concentration in the dynamic range for quantification, which covers 3 orders of magnitude. This offers some advantages: the detection of K+ only needs very small quantities of biological samples, and the dilution of samples such as serum may effectively eliminate the potential interferences that often originate from the background matrix. The determined potassium levels were satisfactory and closely comparable with the results given by the hospital, indicating that this fluorescent probe is applicable to detection of physiological potassium level with high accuracy. Compared with other relative biosensors requiring modified design, bio-molecular modification or/and sophisticated instruments, this CD-based sensor is very simple, cost-effective and easy detection, suggesting great potential applications for successively monitoring physiological potassium level and the change in biological system.

Quantitative Proteomic Approach for MicroRNA Target Prediction Based on 18O/16O Labeling.

MOTIVATIONAmong many large-scale proteomic quantification methods, 18O/16O labeling requires neither specific amino acid in peptides nor label incorporation through several cell cycles, as in metabolic labeling; it does not cause significant elution time shifts between heavy- and light-labeled peptides, and its dynamic range of quantification is larger than that of tandem mass spectrometry-based quantification methods. These properties offer 18O/16O labeling the maximum flexibility in application. However, 18O/16O labeling introduces large quantification variations due to varying labeling efficiency. There lacks a processing pipeline that warrants the reliable identification of differentially expressed proteins (DEPs). This motivates us to develop a quantitative proteomic approach based on 18O/16O labeling and apply it on Kaposi sarcoma-associated herpesvirus (KSHV) microRNA (miR) target prediction. KSHV is a human pathogenic γ-herpesvirus strongly associated with the development of B-cell proliferative disorders, including primary effusion lymphoma. Recent studies suggest that miRs have evolved a highly complex network of interactions with the cellular and viral transcriptomes, and relatively few KSHV miR targets have been characterized at the functional level. While the new miR target prediction method, photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP), allows the identification of thousands of miR targets, the link between miRs and their targets still cannot be determined. We propose to apply the developed proteomic approach to establish such links.METHODWe integrate several 18O/16O data processing algorithms that we published recently and identify the messenger RNAs of downregulated proteins as potential targets in KSHV miR-transfected human embryonic kidney 293T cells. Various statistical tests are employed for picking DEPs, and we select the best test by examining the enrichment of PAR-CLIP-reported targets with seed match to the miRs of interest among top ranked DEPs returned by statistical tests. Subsequently, the list of DEPs picked by the selected statistical test is filtered with the criteria that they must have downregulated gene expressions, must have reported as targets by an miR target prediction algorithm SVMcrio, and must have reported as targets by PAR-CLIP.RESULTWe test the developed approach in the problem of finding targets of KSHV miR-K1. The RNAs of three DEPs are identified as miR-K1 targets, among which RAB23 and HNRNPU are novel. Results from both Western blotting and Luciferase reporter assays confirm the novel targets. These results show that the developed quantitative approach based on 18O/16O labeling can be combined with genomic, PAR-CLIP, and target prediction algorithms for the confident identification of KSHV miR targets. The developed approach could also be applied in other applications.

Comprehensive Lipidome Analysis by Shotgun Lipidomics on a Hybrid Quadrupole-Orbitrap-Linear Ion Trap Mass Spectrometer

Here we report on the application of a novel shotgun lipidomics platform featuring an Orbitrap Fusion mass spectrometer equipped with an automated nanoelectrospray ion source. To assess the performance of the platform for in-depth lipidome analysis, we evaluated various instrument parameters, including its high resolution power unsurpassed by any other contemporary Orbitrap instrumentation, its dynamic quantification range and its efficacy for in-depth structural characterization of molecular lipid species by quadrupole-based higher-energy collisional dissociation (HCD), and ion trap-based resonant-excitation collision-induced dissociation (CID). This evaluation demonstrated that FTMS analysis with a resolution setting of 450,000 allows distinguishing isotopes from different lipid species and features a linear dynamic quantification range of at least four orders of magnitude. Evaluation of fragmentation analysis demonstrated that combined use of HCD and CID yields complementary fragment ions of molecular lipid species. To support global lipidome analysis, we designed a method, termed MS(ALL), featuring high resolution FTMS analysis for lipid quantification, and FTMS(2) analysis using both HCD and CID and ITMS(3) analysis utilizing dual CID for in-depth structural characterization of molecular glycerophospholipid species. The performance of the MS(ALL) method was benchmarked in a comparative analysis of mouse cerebellum and hippocampus. This analysis demonstrated extensive lipidome quantification covering 311 lipid species encompassing 20 lipid classes, and identification of 202 distinct molecular glycerophospholipid species when applying a novel high confidence filtering strategy. The work presented here validates the performance of the Orbitrap Fusion mass spectrometer for in-depth lipidome analysis.

Investigation of the Role of Androstenedione-19-oic Acid in the Presence of 19-Norandrostenedione in Intact Male Horse Plasma Using Liquid Chromatography–Tandem Mass Spectrometry

A liquid chromatography–tandem mass spectrometry method was developed to confirm the presence of androstenedione-19-oic acid in intact male equine plasma and to show the source of 19-norandrostenedione in equine plasma. Androstenedione-19-oic acid was recovered from acidified plasma by liquid–liquid extraction using methyl tert-butyl ether and separated on an Ace 5 C8 column. A triple quadrupole mass spectrometer was used to detect the analytes in negative electrospray ionization mode. Limits of detection, quantification, and confirmation of the method were 0.1, 0.5, and 1.0 ng/mL, respectively. The linear dynamic range of quantification was 0.5–50 ng/mL. The presence of androstenedione-19-oic acid was confirmed in all plasma samples obtained from intact male horses but not those from gelded and female horses; the average concentration was 3.1 ± 1.6 ng/mL, suggesting androstenedione-19-oic acid is an endogenous compound only in intact male horse plasma samples. The conversion of androstenedione-19-oic acid to 19-norandrostenedione in equine plasma was demonstrated by spiking androstenedione-19-oic acid into blank plasma and monitoring the generation of 19-norandrostenedione and its increase in concentration during storage. Results indicated that androstenedione-19-oic acid was readily converted into 19-norandrostenedione; the higher the storage temperature, the faster the conversion. The conversion was not affected by the types of plasma samples collected from gelded and female horses or by anticoagulants used in blood collection to harvest plasma. Compared with other matrices such as water, methanol, and phosphate-buffered saline, the conversion of androstenedione-19-oic to 19-norandrostenedione in equine plasma was faster, suggesting that there is an unknown factor(s) in equine plasma that enhances the conversion.

Doping Control Analysis of 16 Non-Steroidal Anti-Inflammatory Drugs in Equine Plasma Using Liquid Chromatography-Tandem Mass Spectrometry

Non-steroidal anti-inflammatory drugs (NSAIDs) are classified as Class 4 agents by the Association of Racing Commissioners International and are banned in racehorses during competition in Pennsylvania (PA). To control the abuse of these agents in racehorses competing in PA, a forensic method for screening and confirmation of the presence of these agents is needed. Equine plasma (0.5 mL) was acidified with 75 μL 1M H3PO4 to increase recovery of the analytes by liquid-liquid extraction using methyl tert-butyl ether (MTBE). Extracted analytes were separated by reversed-phase liquid chromatography using a C8 column under gradient condition. All 16 analytes were detected, quantified and confirmed using a triple quadrupole tandem mass spectrometry with selected reaction monitoring (SRM) in both negative and positive electrospray ionization modes. The limit of detection, quantification and confirmation of the analytes were 1.0 - 5.0 ng/mL, 1.0 - 5.0 ng/mL and 1.0 - 20 ng/mL, respectively. The linear dynamic range of quantification was 5.0 - 200 ng/mL. The method is routinely used in anti-doping analysis to control the abuse of NSAIDs in racehorses competing in PA.

Error estimation in environmental DNA targets quantification due to PCR efficiencies differences between real samples and standards

IntroductionNowadays, as in the past, our knowledge of microbiology islimited because of the capacity of available technologytools. In recent decades, molecular techniques are playinga central role in the understanding of the microbial world. Inthis sense, a complete scientific evaluation of biologicalsamples is not possible without using molecular biology.The most influential technique has probably been the poly-merasechainreaction(PCR),whichallowsustoworkbeyondclassicalmicrobiology.WithPCR,insomecases,theneedforculture may be avoided. Nevertheless, until the developmentof real-time PCR, our evaluation of the microbial complexityhas been merely qualitative.Real-time PCR is an evolution of the conventional end-point PCR wherein the amplification of a target gene and itsfluorescence detection occurs simultaneously during eachcycle. Different strategies based on the use of nonspecificDNA intercalating dyes or specific fluorescent probes existfor linking target amplification to fluorescence detection.Regardless of the fluorescence emission mechanisms in-volved, real-time PCR enables both detection and quantifica-tion of a genetic target by a continuous fluorescencemonitoring of DNA amplification (Higuchi et al. 1993).An advantage in real-time PCR isthat target quantificationsensitivity is independent of the copy number within thesample (Freeman et al. 1999). Moreover, the key aspect thatdifferentiates real-time PCR from previous semi-quantitativemethodologies is that target DNA concentration can be deter-mined from the fractional cycle at which a threshold amountof target DNA is produced (i.e., measured signal exceeds abackground level), set at a point where amplicon DNA justbecomesdetectablebutisstillwithintheexponentialphaseoftheamplification(Higuchietal.1993;Rasmussen2001).Thisquantificationcycle(Cq)(orcyclethreshold,Ct)areinverselyproportionaltotheamountoftargetnucleicacidinthesample(i.e., the lower the Cq value, the greater the amount of targetnucleic acid in the sample). This approach ensures that inter-fering factors associated with later stages of the amplificationare minimized and considerably improves the measurementprecision (Rasmussen 2001).One of the most important points is that real-time PCRallows quantitative measurements. In order to estimate atarget concentration in environmental samples, absolutequantification can be performed using a standard curveconstructed by amplifying known amounts of target DNA(Rutledge and Cote 2003). Nowadays, this approach is themost commonly used in environmental microbiology.Real-time PCR has many advantages over conventionalendpoint PCR including speed, broad dynamic range ofquantification, reduced risk of contamination, and goodsensitivity. However, despite all these objective strengths,real-time PCR sensitivity is not better than conventionalPCR if a protocol validation is not performed (Bastien etal. 2008).

Quantification of superparamagnetic iron oxide using inversion recovery balanced steady-state free precession

Cellular and molecular MRI trafficking studies using superparamagnetic iron oxide (SPIO) have greatly improved non-invasive investigations of disease progression and drug efficacy, but thus far, these studies have largely been restricted to qualitative assessment of hypo- or hyperintense areas near SPIO. In this work, SPIO quantification using inversion recovery balanced steady-state free precession (IR-bSSFP) was demonstrated at 3T by extracting R2 values from a monoexponential model (P. Schmitt et al., 2004). A low flip angle was shown to reduce the apparent recovery rate of the IR-bSSFP time course, thus extending the dynamic range of quantification. However, low flip angle acquisitions preclude the use of traditional methods for combining RF phase-cycled images to reduce banding artifacts arising from off-resonance due to B0 inhomogeneity. To achieve R2 quantification of SPIO, we present a new algorithm applicable to low flip angle IR-bSSFP acquisitions that is specifically designed to identify on-resonance acquisitions. We demonstrate in this work, using both theoretical and empirical methods, that the smallest estimated R2 from multiple RF phase-cycled acquisitions correspond well to the on-resonance time course. Using this novel minimum R2 algorithm, homogeneous R2 maps and linear R2 calibration curves were created up to 100μg(Fe)/mL with 20° flip angles, despite substantial B0 inhomogeneity. In addition, we have shown this technique to be feasible for pre-clinical research: the minimum R2 algorithm was resistant to off-resonance in a single slice mouse R2 map, whereas maximum intensity projection resulted in banding artifacts and overestimated R2 values. With the application of recent advances in accelerated acquisitions, IR-bSSFP has the potential to quantify SPIO in vivo, thus providing important information for oncology, immunology, and regenerative medicine MRI studies.

A Real-Time Quantitative PCR Assay for Quantification of c-Myc DNA in Patients who Suffers from Leukemia

The MYC cancer gene contains instructions for the production of the c-Myc protein. The c-Myc protein is known as a transcription factor or a regulator of other genes. It is a protein that binds DNA at specific sites and instructs genes whether or not they should be transcribed into messages for cells to make additional or other new proteins. Quantitative real-time PCR (qRT-PCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology, archaeometry and diagnostics and has become the method of choice for the quantification of cDNA and nDNA. Therefore, we used Polymerase chain reaction (PCR)-based assays can target either DNA (the genome) or cDNA, namely used for research both DNA. We optimized a method for monitoring quantitative real-time PCR (qRT-PCR) of c-Myc cancer gene in patients with leukemia. We describe qRT-PCR a series of protocols that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible. In addition, our aim is to also quantify extracted DNA and determine its purity and the validation of extracted DNA from patients with leukemia including active Myc gene family. We also believe these protocols will be accessible to the researchers to provide them reliable data in this protocol. These analytical methods are essential for accurate gene quantification. With reference to, advantages of qRT-PCR are a large dynamic range of quantification, no requirement for post-PCR sample handling and the need for very small amounts of starting material. The specificity, reproducibility and detection limit of the assay was examined. The assay was used to monitor c-myc DNA levels in patients with leukemia.

Validated UHPLC–MS-MS Method for Rapid Analysis of Capsaicin and Dihydrocapsaicin in Equine Plasma for Doping Control

A method involving ultra high-performance liquid chromatography-tandem mass spectrometry was developed and validated for the analysis of capsaicin and dihydrocapsaicin in equine plasma. The analytes were recovered from plasma by liquid-liquid extraction using methyl tert-butyl ether and separated on a sub-2 micron column. The mobile phase was composed of 2 mM ammonium formate and methanol. A triple quadrupole mass spectrometer was used to detect the analytes in positive electrospray ionization mode with selected reaction monitoring. The limits of detection, quantification and confirmation for both analytes were 0.5, 1.0 and 2.5 pg/mL, respectively. The linear dynamic range of quantification was 1.0-1,000 pg/mL. During storage, both analytes in equine plasma were unstable at room temperature but stable at -20 and -70°C. The retention time and product ion ratios were employed as the criteria for confirmation of the presence of the analytes in plasma. The total analysis time was 2 min. The method is fast, selectively sensitive, reproducible, reliable and fully validated.

Upgraded Cobas TaqMan version 2: hidden consequences at cohort level

The viral load quantification constitutes a cornerstone of antiretroviral therapy management. After the switch from the Cobas Ampliprep Amplicor HIV‐1 Monitor Test, v. 1,5 (CAP/CA) (Roche Diagnostics, Mannheim, Germany) to CTM2 in August 2009 at the HIV department of the General Hospital in Vienna, multiple internal reports accumulated concerning an increase of detectable HIV‐1 viral loads in patients with previous long‐term virological suppression. In order to evaluate these observations and their clinical consequences, a retrospective analysis of the number of elevated VL measurements in formerly virological suppressed patients during the first year of CTM2 use was performed. Furthermore, we monitored for consecutive numbers of repeated VL measurements, genotypic testing and for changes of antiretroviral therapy (ART). We recruited 373 of 2078 patients meeting the chosen inclusion criteria (Initiation of ART prior to August 6, 2008;≥1 VL measurement in the pre‐CTM2 period from August 6, 2008 to August 5, 2009, all VL measurements below the limit of quantification as defined by applied nucleic acid quantification assay (CAP/CA with<50 copies/mL);≥1 VL measurement during the CTM2 period from August 6, 2009 to August 5, 2010). 221 (59.2%) remained with an undetectable HIV‐1 viral load after implementation of CTM2, whereas 152 (40.8%) became detectable. The newly detected viremia showed a clear increase at the lower end of the dynamic range of quantification by CTM2. Among our 152 patients, 111 (73.0%) had viral loads ranging from 20–200 copies/mL, 6 (4.0%) between 201–400 copies/mL, while 35 (23.0%) patients showed viral loads measurements above>400 copies/mL. Of these newly detectable patients, 132 had a VL repeat and 72 became undetectable, the remaining 60 patients remained detectable. Remarkably, it was striking to find that in the group of patients who when switching to CTM2 reached at once viral loads exceeding 400 copies/mL, 48.3% became undetectable after viral load repeat using again CTM2, suggesting a high test variability at low detection limit but also beyond. Three genotypic resistance testings were performed and 16 patients underwent subsequent ART changes. In summary, the transition to CTM2 was followed by a dramatic increase of detectable viral loads in patients with stable ART and prior virological suppression, which at least in part could not be reproduced in repeat measurements.

Simultaneous detection of diagnostic biomarkers of alkaptonuria, ornithine carbamoyltransferase deficiency, and neuroblastoma disease by high-performance liquid chromatography/tandem mass spectrometry

BackgroundUrinary homovanillic acid (HVA)/vanillylmandelic acid (VMA), orotic acid (OA), and homogentisic acid (HGA) are diagnostic biomarkers of neuroblastoma, ornithine carbamoyl transferase deficiency (OCTD), and alkaptonuria (AKU), respectively. In this study, a high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous quantification of HVA, VMA, OA, and HGA in urine. MethodsAfter sample preparation, which involved only the dilution procedure, samples were quantified by LC–MS/MS. Full-scan MS/MS mode enabled the urinary markers to be quantified with a high degree of specificity and sensitivity. Rather than using a separate enzymatic method to normalize the concentration of creatinine in urine, we quantified the level of creatinine in urine in one LC–MS run. ResultsThe limits of detection were 10μg/l for HGA, 25μg/l for HVA/VMA, and 50μg/l for OA with a single-to-noise ratio of 3; the limits of quantification were 50μg/l for HVA and HGA, 100μg/l for VMA, and 250μg/l for OA. The linear dynamic range for quantification of the analytes covered 2 to 3 orders of magnitude, depending on the analyte. The relative standard deviation of the developed LC–MS/MS method was less than 4% for the intra-day validation and 10% for the inter-day validation. ConclusionsThe results show that our LC–MS/MS technique is a highly sensitive and rapid method for screening for biomarkers that are diagnostic of three metabolic diseases.

Shotgun Lipidomics by Sequential Precursor Ion Fragmentation on a Hybrid Quadrupole Time-of-Flight Mass Spectrometer.

Shotgun lipidomics has evolved into a myriad of multi-dimensional strategies for molecular lipid characterization, including bioinformatics tools for mass spectrum interpretation and quantitative measurements to study systems-lipidomics in complex biological extracts. Taking advantage of spectral mass accuracy, scan speed and sensitivity of improved quadrupole linked time-of-flight mass analyzers, we developed a bias-free global lipid profiling acquisition technique of sequential precursor ion fragmentation called MS/MSALL. This generic information-independent tandem mass spectrometry (MS) technique consists of a Q1 stepped mass isolation window through a set mass range in small increments, fragmenting and recording all product ions and neutral losses. Through the accurate MS and MS/MS information, the molecular lipid species are resolved, including distinction of isobaric and isomeric species, and composed into more precise lipidomic outputs. The method demonstrates good reproducibility and at least 3 orders of dynamic quantification range for isomeric ceramides in human plasma. More than 400 molecular lipids in human plasma were uncovered and quantified in less than 12 min, including acquisitions in both positive and negative polarity modes. We anticipate that the performance of sequential precursor ion fragmentation both in quality and throughput will lead to the uncovering of new avenues throughout the biomedical research community, enhance biomarker discovery and provide novel information target discovery programs as it will prospectively shed new insight into affected metabolic and signaling pathways.

18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry

Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an (18)O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The (18)O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope ((18)O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope ((18)O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light ((16)O)/heavy ((18)O) peak area ratios. The utility of (18)O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of (18)O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM.

Development of a multiplex assay for the detection of antibodies to Borrelia burgdorferi in horses and its validation using Bayesian and conventional statistical methods

Lyme disease is a zoonotic, vector-borne disease and occurs in mammals including horses. The disease is induced by infection with spirochetes of the Borrelia burgdorferi sensu lato group. Infection of mammalian hosts requires transmission of spirochetes by infected ticks during tick bites. Lyme disease diagnosis is based on clinical signs, possible exposure to infected ticks, and antibody testing which is traditionally performed by ELISA and Western blotting (WB). This report describes the development and validation of a new fluorescent bead-based multiplex assay for the detection of antibodies to B. burgdorferi outer surface protein A (OspA), OspC and OspF antigens in horse serum. Testing of 562 equine sera was performed blindly and in parallel by using WB and the new multiplex assay. Because a true gold standard is missing for Lyme antibody testing, we performed and compared different statistical approaches to validate the new Lyme multiplex assay. One approach was to use WB results as a ‘relative gold standard’ in ROC-curve and likelihood-ratio analyses of the new test. Cut-off values and interpretation ranges of the multiplex assay were established by the analysis. The second statistical approach used a Bayesian model for the calculation of diagnostic sensitivities and specificities of the multiplex assay. The Bayesian analysis takes into consideration that no true gold standard exists for detecting antibodies to B. burgdorferi and estimated sensitivities and specificities of both tests that were compared. Therefore, the Bayesian analysis also resulted in an evaluation of diagnostic sensitivity and specificity of WB. Overall, the new assay was characterized by low background values and a wide dynamic quantification range for the detection of antibodies to OspA, OspC and OspF antigens of B. burgdorferi. The diagnostic sensitivity and specificity for the OspA bead-based assay were calculated as 49% and 85%, respectively, and by a standard ROC curve analysis only because the Bayesian model could not be run on this parameter. The Bayesian-derived diagnostic sensitivities of the OspC and OspF assays were 80% and 86%, respectively. For comparison, the Bayesian-derived estimates for WB resulted in sensitivities of 72% for OspC and 80% for OspF. The Bayesian diagnostic specificities of the multiplex assay were 79% and 69% for OspC and OspF, respectively. WB analysis had specificities of 92% for OspC and 77% for OspF. Although the analysis of a new assay in the absence of a true gold standard remains challenging, the approach used here can help to address this problem when new technologies and traditionally used test standards differ significantly in their analytical sensitivities, which consequently causes problems in the calculation of diagnostic sensitivity and sensitivity values for the new assay. In summary, the new multiplex assay for the detection of antibodies to B. burgdorferi OspA, OspC and OspF antigens in horse serum has improved analytical and diagnostic sensitivities compared to WB analysis. Multiplex analysis is a valuable quantitative tool that simultaneously detects antibodies indicative for natural infection with and/or vaccination against the Lyme pathogen.

Simultaneous separation and determination of 16 testosterone and nandrolone esters in equine plasma using ultra high performance liquid chromatography–tandem mass spectrometry for doping control

The potential for using testosterone and nandrolone esters in racehorses to boost the biological concentrations of these steroids and enhance athletic performance is very compelling and should be seriously considered in formulating regulatory policies for doping control. In order to regulate the use of these esters in racehorses, a sensitive and validated method is needed. In this paper, we report such a method for simultaneous separation, screening, quantification and confirmation of 16 testosterone and nandrolone esters in equine plasma by ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). Analytes were extracted from equine plasma by liquid–liquid extraction using a mixture of methyl tert-butyl ether and ethyl acetate (50:50, v/v) and separated on a sub-2 micron C 18 column. Detection of analytes was achieved on a triple-quadrupole mass spectrometer by positive electrospray ionization mode with selected reaction monitoring (SRM). Mobile phase comprised 2 mM ammonium formate and methanol. Deuterium-labeled testosterone enanthate and testosterone undecanoate were used as dual-internal standards for quantification. Limits of detection (LOD) and quantification (LOQ) were 25–100 pg/mL and 100–200 pg/mL, respectively. The linear dynamic range of quantification was 100–10,000 pg/mL. For confirmation of the presence of these analytes in equine plasma, matching of the retention time with mass spectrometric ion ratios from MS/MS product ions was used. The limit of confirmation (LOC) was 100–500 pg/mL. The method is sensitive, robust, selective and reliably reproducible.

Toward a standardized urine proteome analysis methodology

Urine is an easily accessible bodily fluid particularly suited for the routine clinical analysis of disease biomarkers. Actually, the urinary proteome is more diverse than anticipated a decade ago. Hence, significant analytical and practical issues of urine proteomics such as sample collection and preparation have emerged, in particular for large-scale studies. We have undertaken a systematic study to define standardized and integrated analytical protocols for a biomarker development pipeline, employing two LC-MS analytical platforms, namely accurate mass and time tags and selected reaction monitoring, for the discovery and verification phase, respectively. Urine samples collected from hospital patients were processed using four different protocols, which were evaluated and compared on both analytical platforms. Addition of internal standards at various stages of sample processing allowed the estimation of protein extraction yields and the absolute quantification of selected urinary proteins. Reproducibility of the entire process and dynamic range of quantification were also evaluated. Organic solvent precipitation followed by in-solution digestion provided the best performances and was thus selected as the standard method common to the discovery and verification phases. Finally, we applied this protocol for platforms' cross-validation and obtained excellent consistency between urinary protein concentration estimates by both analytical methods performed in parallel in two laboratories.

Quantification of Campylobacter spp. in pig feces by direct real-time PCR with an internal control of extraction and amplification

The rapid and direct quantification of Campylobacter spp. in complex substrates like feces or environmental samples is crucial to facilitate epidemiological studies on Campylobacter in pig production systems. We developed a real-time PCR assay for detecting and quantifying Campylobacter spp . directly in pig feces with the use of an internal control. Campylobacter spp. and Yersinia ruckeri primers–probes sets were designed and checked for specificity with diverse Campylobacter, related organisms, and other bacterial pathogens before being used in field samples. The quantification of Campylobacter spp. by the real-time PCR then was realized on 531 fecal samples obtained from experimentally and naturally infected pigs; the numeration of Campylobacter on Karmali plate was done in parallel. Yersinia ruckeri, used as bacterial internal control, was added to the samples before DNA extraction to control DNA-extraction and PCR-amplification. The sensitivity of the PCR assay was 10 genome copies. The established Campylobacter real-time PCR assay showed a 7-log-wide linear dynamic range of quantification (R 2 = 0.99) with a detection limit of 200 Colony Forming Units of Campylobacter per gram of feces. A high correlation was found between the results obtained by real-time PCR and those by culture at both qualitative and quantitative levels. Moreover, DNA extraction followed by real-time PCR reduced the time needed for analysis to a few hours (within a working day). In conclusion, the real-time PCR developed in this study provides new tools for further epidemiological surveys to investigate the carriage and excretion of Campylobacter by pigs.

Influence of baking time and matrix effects on the detection of milk allergens in cookie model food system by ELISA

Milk allergens are common allergens occurring in foods, therefore raising concern in allergic consumers. Enzyme-linked immunosorbent assay (ELISA) is, to date, the method of choice for the detection of food allergens by the food industry although, the performance of ELISA might be compromised when severe food processing techniques are applied to allergen-containing foods. In this paper we investigated the influence of baking time on the detection of milk allergens by using commercial ELISA kits. Baked cookies were chosen as a model food system and experiments were set up to study the impact of spiking a matrix food either before, or after the baking process. Results revealed clear analytical differences between both spiking methods, which stress the importance of choosing appropriate spiking methodologies for method validation purposes. Finally, since the narrow dynamic range of quantification of ELISA implies that dilution of samples is required, the impact of sample dilution on the quantitative results was investigated. All parameters investigated were shown to impact milk allergen detection by means of ELISA.

Rapid identification and quantification of Campylobacter coli and Campylobacter jejuni by real-time PCR in pure cultures and in complex samples

BackgroundCampylobacter spp., especially Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli), are recognized as the leading human foodborne pathogens in developed countries. Livestock animals carrying Campylobacter pose an important risk for human contamination. Pigs are known to be frequently colonized with Campylobacter, especially C. coli, and to excrete high numbers of this pathogen in their faeces. Molecular tools, notably real-time PCR, provide an effective, rapid, and sensitive alternative to culture-based methods for the detection of C. coli and C. jejuni in various substrates. In order to serve as a diagnostic tool supporting Campylobacter epidemiology, we developed a quantitative real-time PCR method for species-specific detection and quantification of C. coli and C. jejuni directly in faecal, feed, and environmental samples.ResultsWith a sensitivity of 10 genome copies and a linear range of seven to eight orders of magnitude, the C. coli and C. jejuni real-time PCR assays allowed a precise quantification of purified DNA from C. coli and C. jejuni. The assays were highly specific and showed a 6-log-linear dynamic range of quantification with a quantitative detection limit of approximately 2.5 × 102 CFU/g of faeces, 1.3 × 102 CFU/g of feed, and 1.0 × 103 CFU/m2 for the environmental samples. Compared to the results obtained by culture, both C. coli and C. jejuni real-time PCR assays exhibited a specificity of 96.2% with a kappa of 0.94 and 0.89 respectively. For faecal samples of experimentally infected pigs, the coefficients of correlation between the C. coli or C. jejuni real-time PCR assay and culture enumeration were R2 = 0.90 and R2 = 0.93 respectively.ConclusionThe C. coli and C. jejuni real-time quantitative PCR assays developed in this study provide a method capable of directly detecting and quantifying C. coli and C. jejuni in faeces, feed, and environmental samples. These assays represent a new diagnostic tool for studying the epidemiology of Campylobacter by, for instance, investigating the carriage and excretion of C. coli and C. jejuni by pigs from conventional herds.