Detection of Staphylococcal enterotoxin B via biomolecular interaction analysis mass spectrometry

Detection of Staphylococcus enterotoxin B (SEB) by biomolecular interaction analysis mass spectrometry (BIA/MS) is presented in this work. The BIA/MS experiments were based on a surface plasmon resonance (SPR) MS immunoassay that detects affinity-captured SEB both via SPR and by means of exact and direct mass measurement by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Experiments were performed with standard samples and food samples to assess the BIA/MS limit of detection for SEB and to set the experimental parameters for proper quantitation. Single and double SPR referencing was performed to accurately estimate the amount of the bound toxin. Reproducible detection of 1 ng of SEB per ml, corresponding to affinity capture and MS analysis of approximately 500 amol of SEB, was readily achieved from both the standard and mushroom samples. A certain amount of SEB degradation was indicated by the signals in the mass spectra. The combination of MS with SPR-based methods of detection creates a unique approach capable of quantifying and qualitatively analyzing protein toxins from pathogenic organisms.     

Analysis of native proteins from biological fluids by biomolecular interaction analysis mass spectrometry (BIA/MS): exploring the limit of detection, identification of non-specific binding and detection of multi-protein complexes

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a two-dimensional analytical technique that quantitatively and qualitatively detects analytes of interests. In the first dimension, surface plasmon resonance (SPR) is utilized for detection of biomolecules in their native environment. Because SPR detection is non-destructive, analyte(s) retained on the SPR-active sensor surface can be analyzed in a second dimension using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The qualitative nature of the MALDI-TOF MS analysis complements the quantitative character of SPR sensing and overcomes the shortcomings of the SPR detection stemming from the inability to differentiate and characterize multi-protein complexes and non-specific binding. In this work, the benefit of performing MS analysis following SPR sensing is established. Retrieval and detection of four markers present in biological fluids (cystatin C, beta-2-microglobulin, urinary protein 1 and retinol binding protein) was explored to demonstrate the effectiveness of BIA/MS in simultaneous detection of clinically related biomarkers and delineation of non-specific binding. Furthermore, the BIA/MS limit of detection at very low SPR responses was investigated. Finally, detection of in-vivo assembled protein complexes was achieved for the first time using BIA/MS.     

Practical considerations in BIA/MS: optimizing the biosensor-mass spectrometry interface

Biomolecular interaction analysis mass spectrometry (BIA/MS) is a multiplexed analytical technique that utilizes a unique combination of surface plasmon resonance (SPR) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection and analysis of small amounts of proteins residing in complex biological systems. In order to achieve high sensitivity during BIA/MS, certain experimental parameters and sequences of events need to be optimized and maintained. Immobilized ligand density, flow rate and biosensor control (in SPR-BIA) and matrix choice and application (in MALDI-TOF MS) have significant influence on the final outcome of the BIA/MS analysis and, consequently, need to be optimized and carefully controlled. In addition, chip washing and cutting are essential in converting the SPR-active sensor chips into target surfaces amenable to MALDI-TOF MS. Reviewed here are the prerequisites for successfully interfacing SPR-BIA with MALDI-TOF MS.     

Advances in surface plasmon resonance biomolecular interaction analysis mass spectrometry (BIA/MS)

Ongoing, worldwide efforts in genomic and protein sequencing, and the ability to readily access corresponding sequence databases, have emphatically driven the development of high‐performance bioanalytical instrumentation capable of characterizing proteins and protein–ligand interactions with great accuracy, speed and sensitivity. Two such analytical techniques have arisen over the past decade to play key roles in the characterization of proteins: surface plasmon resonance biomolecular interaction analysis (SPR‐BIA) and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF). SPR‐BIA is used in the real‐time investigation of biomolecular recognition events, and is thereby capable of providing details on the association and dissociation kinetics involved in the interaction, information ultimately leading to the determination of dissociation constants involved in the event. MALDI‐TOF is used in the structural characterization, identification and sensitive detection of biomolecules. Although the two techniques have found many independent uses in bioanalytical chemistry, the combination of the two, to form biomolecular interaction analysis mass spectrometry (BIA/MS), enables a technique of analytical capabilities greater than those of the component parts. Reviewed here are issues of concern critical to maintaining high‐levels of performance throughout the multiplexed analysis, as well as examples illustrating the potential analytical capabilities of BIA/MS. Copyright © 1999 John Wiley & Sons, Ltd.     

Coupling Immunomagnetic Separation on Magnetic Beads with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Detection of Staphylococcal Enterotoxin B

The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low-nanogram levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB antibody on the surface of para-toluene-sulfonyl-functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly “on beads” by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, “off beads” after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of Staphylococcus aureus strains and raw milk samples.     

Distribution,occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.     

Delineating protein-protein interactions via biomolecular interaction analysis-mass spectrometry

The utility of biomolecular interaction analysis-mass spectrometry (BIA/MS) in screening for protein-protein interactions was explored in this work. Experiments were performed in which proteins served as ligands for screening of possible interactions with other proteins from human plasma and urine. The proteins utilized were beta-2-microglobulin, cystatin C (cysC), retinol binding protein (RBP), transthyretin (TTR), alpha-1-microglobulin, C-reactive protein, transferrin and papain. The immobilization of functionally active proteins was confirmed via interactions with antibodies to the corresponding proteins. Various dilutions of human urine and plasma were injected over the protein-derivatized surfaces. It was observed that the urine injections generally yielded smaller SPR responses than those observed after the plasma injections. The BIA/MS experiments did not reveal novel protein-protein interactions, although several established interactions (such as those between RBP and TTR, and cysC and papain) were validated. Few protein ligand deficiencies (such as truncations) leading to false negative and false positive BIA/MS results were also discovered.     

Characterization of antibody-antigen interactions: comparison between surface plasmon resonance measurements and high-mass matrix-assisted laser desorption/ionization mass spectrometry

The interaction between the bovine prion protein (bPrP) and a monoclonal antibody, 1E5, was studied with high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and surface plasmon resonance (SPR). In the case of MS a cross-linking stabilization was used prior to the analysis, whereas for SPR the antibody was immobilized and bPrP was injected. We compared the determination of parameters such as the epitope, the kinetics and binding strength, and the capacity of the antigen to bind two different antibodies. The two methods are highly complementary. SPR measurements require a lower amount of sample but are more time-consuming due to all of the necessary side steps (e.g., immobilization, regeneration). High-mass MALDI MS needs a higher overall amount of sample and cannot give direct access to the kinetic constants, but the analysis is faster and easier compared with SPR.     

Qualitative Profiling and Quantification of Neonicotinoid Metabolites in Human Urine by Liquid Chromatography Coupled with Mass Spectrometry

Neonicotinoid pesticides have been widely applied for the production of fruits and vegetables, and occasionally detected in conventionally grown produce. Thus oral exposure to neonicotinoid pesticides may exist in the general population; however, neonicotinoid metabolites in human body fluids have not been investigated comprehensively. The purpose of this study is the qualitative profiling and quantitative analysis of neonicotinoid metabolites in the human spot urine by liquid chromatography coupled with mass spectrometry (LC/MS). Human urine samples were collected from three patients suspected of subacute exposure to neonicotinoid pesticides. A qualitative profiling of urinary metabolites was performed using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) with a database of nominal molecular weights of 57 known metabolites of three neonicotinoid pesticides (acetamiprid, Imidacloprid, and clothianidin), as well as the parent compounds. Then a quantitative analysis of selected urinary metabolites was performed using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a standard pesticide and metabolite, which were detected by the qualitative profiling. The result of qualitative profiling showed that seven metabolites, i.e. an acetamiprid metabolite, N-desmethyl-acetamiprid; three Imidacloprid metabolites, 5-hydroxy-Imidacloprid, 4,5-dihydroxy-imidacloprid, 4,5-dehydro-Imidacloprid; a common metabolite of acetamiprid and Imidacloprid, N-(6-chloronicotinoyl)-glycine; and two clothianidin metabolites, N-desmethyl-clothianidin, N-(2-(methylsulfanyl)thiazole-5-carboxyl)-glycine, as well as acetamiprid, were detected in the urine of three cases. The result of the quantitative analysis showed N-desmethyl-acetamiprid was determined in the urine of one case, which had been collected on the first visit, at a concentration of 3.2 ng/mL. This is the first report on the qualitative and quantitative detection of N-desmethyl-acetamiprid in the human urine. The results suggest that the one case with detection of N-desmethyl-acetamiprid was exposed to acetamiprid through the consumption of contaminated foods. Urinary N-desmethyl-acetamiprid, as well as 5-hydroxy-Imidacloprid and N-desmethyl-clothianidin, may be a good biomarker for neonicotinoid exposure in humans and warrants further investigation.     

Simultaneous determination of sulfamethoxazole and trimethoprim in biological fluids for high-throughput analysis: comparison of HPLC with ultraviolet and tandem mass spectrometric detection

The comparison of two methods based on online solid phase extraction-liquid chromatography with UV (SPE-LC-UV) or mass spectrometry detection (SPE-LC-MS/MS) for the simultaneous quantification of sulfamethoxazole (SMZ) and trimethoprim (TMP) is presented. The methods were validated and proved to be accurate. The analysis of standard samples for SMZ at concentrations of 0.5, 1.5, 25 and 50microg/mL demonstrated a relative standard deviation of less than 6% for both methods (n=18), while TMP samples at concentrations of 0.05, 0.15, 1.5 and 5.0microg/mL were analyzed with R.S.D. of less than 4% (n=18). The method with mass spectrometric detection was approximately six times more sensitive than the method with ultraviolet detection. The total run time for the SPE-LC-MS/MS was 2.5min per sample as opposed to 18.0min for the SPE-LC-UV method. The method with MS detection in comparison with UV detection proved to be more rugged and was successfully applied to pharmacokinetics studies.     

Coupling immunomagnetic separation on magnetic beads with matrix-assisted laser desorption ionization-time of flight mass spectrometry for detection of staphylococcal enterotoxin B

The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low-nanogram levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB antibody on the surface of para-toluene-sulfonyl-functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly "on beads" by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, "off beads" after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of Staphylococcus aureus strains and raw milk samples.     

Quantitative analysis of [Dmt(1)]DALDA in ovine plasma by capillary liquid chromatography-nanospray ion-trap mass spectrometry

The synthetic opioid peptide analog Dmt-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA; [Dmt= 2',6'-dimethyltyrosine) is a highly potent and selective mu opioid-receptor agonist. A very sensitive and robust capillary liquid chromatography/nanospray ion-trap (IT) mass spectrometry method has been developed to quantify [Dmt(1)]DALDA in ovine plasma, using deuterated [Dmt(1)]DALDA as the internal standard. The standard MS/MS spectra of d(0)- and d(5)-[Dmt(1)]DALDA were obtained, and the collision energy was experimentally optimized to 25%. The product ion [ M + 2H-NH(3)](2+) (m/z 312.2) was used to identify and to quantify the synthetic opioid peptide analog in ovine plasma samples. The MS/MS detection sensitivity for [Dmt(1)]DALDA was 625 amol. A calibration curve was constructed, and quantitative analysis was performed on a series of ovine plasma samples.     

Liquid chromatography high‐resolution mass spectrometry for fatty acid profiling

Quantification of fatty acids has been crucial to elucidate lipid biosynthesis pathways in plants. To date, fatty acid identification and quantification has relied mainly on gas chromatography (GC) coupled to flame ionization detection (FID) or mass spectrometry (MS), which requires the derivatization of samples and the use of chemical standards for annotation. Here we present an alternative method based on a simple procedure for the hydrolysis of lipids, so that fatty acids can be quantified by liquid chromatography mass spectrometry (LC‐MS) analysis. Proper peak annotation of the fatty acids in the LC‐MS‐based methods has been achieved by LC‐MS measurements of authentic standard compounds and elemental formula annotation supported by ¹³C isotope‐labeled Arabidopsis. As a proof of concept, we have compared the analysis by LC‐MS and GC‐FID of two previously characterized Arabidopsis thaliana knock‐out mutants for FAD6 and FAD7 desaturase genes. These results are discussed in light of lipidomic profiles obtained from the same samples. In addition, we performed untargeted LC‐MS analysis to determine the fatty acid content of two diatom species. Our results indicate that both LC‐MS and GC‐FID analyses are comparable, but that because of higher sensitivity and selectivity the LC‐MS‐based method allows for a broader coverage and determination of novel fatty acids.     

Determination of serum uric acid using high-performance liquid chromatography (HPLC)/isotope dilution mass spectrometry (ID-MS) as a candidate reference method

Uric acid is an important diagnostic marker of catabolism of the purine nucleosides, and accurate measurements of serum uric acid are necessary for proper diagnosis of gout or renal disease appearance. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) has been described. An isotopically labeled internal standard, [1,3-(15)N(2)] uric acid, was added to serum, followed by equilibration and protein removal clean up to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) analyses. (M-H)(-) ions at m/z 167 and 169 for uric acid and its labeled internal standard were monitored for LC/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for uric acid (Standard Reference Materials SRM909b) with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added uric acid. The method performed well against the established reference method of ion-exchange followed by derivatization isotope dilution (ID) gas chromatography mass spectrometry (ID-GC/MS). The results of this method for uric acid agreed well with the certified values and were within 0.10%. The amounts of uric acid recovered and added were in good agreement for the three concentrations. This method was applied to determine uric acid in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.08-0.18% and between-set CVs of 0.02-0.07% for LC/MS analyses. Liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analysis was also performed. The LC/MS and LC/MS/MS results were in very good agreement (within 0.14%). This LC/MS method, which demonstrates good accuracy and precision, and is in the speed of analysis without the need for a derivatization stage, qualifies as a candidate reference method. This method can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared.     

Automated SPR-LC-MS/MS system for protein interaction analysis

We have developed a novel automated system to analyze protein complexes by integrating a surface plasmon resonance (SPR) biosensor with highly sensitive nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS). A His-tagged protein, which is also tagged with FLAG and biotinylated sequences, was expressed in mammalian cells. After purification by using the His tag from the cell lysate, the sample protein mixture was applied to an SPR biosensor and the protein complex was captured on the sensor chip. The automated SPR-LC-MS/MS was then performed: (1) two-step on-chip purification of the protein complex by using the FLAG and the biotinylated tags, (2) on-chip protease digestion of the complex, and (3) online nanoflow LC-MS/MS analysis of the resulting peptide fragments for protein identification. All of these processes could be monitored in real-time by the SPR biosensor. We validated the performance of the system using either FK506-binding protein 52 kDa (FKBP52) or ribosomal protein S19 (rpS19) as bait. Thus, the fully automated SPR-LC-MS/MS system appeared to be a powerful tool for functional proteomics studies, particularly for snapshot analysis of functional cellular complexes and machines.     

Simultaneous quantification of tiloronoxim and tilorone in human urine by liquid chromatography-tandem mass spectrometry

A simple, sensitive and specific HPLC method with tandem mass spectrometry (HPLC/MS/MS) detection has been developed and validated for the simultaneous quantification of tiloronoxim and its major active metabolite, tilorone, in human urine. The analytes, together with metoprolol, which was employed as an internal standard (IS), were extracted with a mixture solvent of chloroform/ethyl ether (1/2, v/v). The chromatographic separation was performed on a narrow-bore reversed phase HPLC column with a gradient mobile phase of methanol/water containing 15 mM ammonium bicarbonate (pH 10.5). The API 3,000 mass spectrometer was equipped with a TurboIonSpray interface and was operated on positive-ion, multiple reaction-monitoring (MRM) mode. The mass transitions monitored were m/z 426.3-->100.0, m/z 411.3-->100.0 and m/z 268.3-->116.1 for tiloronoxim, tilorone and the IS, respectively. The assay exhibited a linear dynamic range of 1-100 ng/ml for both tiloronoxim and tilorone based on the analysis of 0.2 ml aliquots of urine. The lower limit of quantification was 1 ng/ml for both compounds. Acceptable precision and accuracies were obtained for concentrations over the standard curve ranges. Run time of 8 min for each injection made it possible to analyze a high throughput of urine samples. The assay has been successfully used to analyze human urine samples from healthy volunteers.     

High-performance liquid chromatography-tandem mass spectrometry for the analysis of bile acid profiles in serum of women with intrahepatic cholestasis of pregnancy

A simple, sensitive, and specific high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the analysis of the bile acid profile has been developed. Fifteen bile acids, including free and conjugated bile acids, were separated and detected by HPLC-MS/MS. The MS detection was performed by electrospray ionization (ESI) in negative ion mode. Quantification was achieved in multiple reaction monitoring (MRM) mode with external standard curve methods. Total analysis time was 15 min for one sample including re-equilibration time of the column. The assay was linear in the range 0.02-100.0 micromol/L with correlation coefficients of standard curves for all bile acids better than 0.999. The detection limits ranged from 0.001 to 0.006 micromol/L for different bile acids. The precisions for each bile acid were CVs<3.8% for within-day and CVs<6.1% for between-day. The average recoveries for all bile acids studied were in the range of 86-110.0%. The developed method was applied to the analysis of clinic samples consisting of 53 women with healthy pregnancies and 43 women with intrahepatic cholestasis of pregnancy (ICP). The results revealed that the bile acid profile was markedly different between women with ICP and women with healthy pregnancies.     

A miniature fiber optic surface plasmon resonance sensor for fast detection of Staphylococcal enterotoxin B

A fiber optic surface plasmon resonance (SPR) biosensor for detection of Staphylococcal enterotoxin B (SEB) is reported. The sensor is based on spectral interrogation of surface plasmons in a miniature sensing element based on a side-polished single-mode optical fiber with a thin metal overlayer. For specific detection of SEB, the SPR sensor is functionalized with a covalently crosslinked double-layer of antibodies against SEB. The SPR biosensor is demonstrated to be able to detect ng/ml concentrations of SEB in less than 10 min.     

Use of an ion-pairing reagent for high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry determination of anionic anticoagulant rodenticides in body fluids

The on-line combination of high-performance liquid chromatography with mass spectrometry (HPLC–MS) has become a powerful tool for trace analysis thanks to the developments in interface techniques. However, non-volatile salts such as ion-pairing reagents are considered to be incompatible with HPLC–MS systems; they cause drops in analyte signals because of contamination of mass analyzers and also because of blocking of the capillary transferring ions from atmospheric pressure to the vacuum manifold. In this work, a new type of ion-pairing reagent, di-n-butylammonium acetate (DBA), was evaluated for use in HPLC–MS. DBA did not cause these problems to HPLC–MS systems; a possible explanation might be that DBA decomposed to volatile compounds under APCI conditions. In addition, DBA was very useful for obtaining sharp peaks, which resulted in high sensitivity. With this ion-pairing reagent, we developed a procedure for the measurement of five (including internal standard) anticoagulant rodenticides in whole blood and urine samples by SIM detection of [M−H]⁻ ions. Calibration range, recoveries and precision of the method were examined; detection limits as low as 1–5 ng/ml blood sample or 0.5–2.5 ng/ml urine sample were achieved.