Optimal Signal Intensity Research Articles

Optimal Threshold and Interpatient Variability in Left Atrial Ablation Scar Assessment by Dark-Blood LGE CMR

BackgroundDark-blood late gadolinium enhancement (LGE) cardiac magnetic resonance (CMR) has better correlation with bipolar voltage (BiV) to define ablation scar in the left atrium (LA) compared to conventional bright-blood LGE CMR. ObjectivesThis study sought to determine the optimal signal intensity threshold of dark-blood LGE CMR to identify LA ablation scar. MethodsIn 54 patients scheduled for atrial fibrillation ablation, image intensity ratios (IIRs) were derived from preprocedural dark-blood LGE CMR. In 26 patients without previous ablation, the upper limit of normal was derived from the 95th and 98th percentiles of pooled IIR values. In 28 patients with previous atrial fibrillation ablation, BiV was compared with the corresponding IIR. Receiver-operating characteristics analyses were employed to determine the optimal IIR threshold (ie, the point with the smallest distance to the upper left corner of the receiver-operating characteristics) for LA ablation scar (BiV ≤0.15 mV). ResultsUpper limit of normal corresponded to IIR values 1.16 and 1.21, yielding low sensitivities of 0.32 and 0.09 to detect LA ablation scar. Receiver-operating characteristics analysis of IIR and BiV comparison achieved a median area under the curve of 0.77. Median optimal IIR threshold for LA ablation scar was 1.09, with an average sensitivity of 0.73, specificity of 0.75, and accuracy of 0.71. Median IIR thresholds of 1.00 and 1.10 corresponded to 80% sensitivity and 80% specificity, respectively. There was considerable interpatient variability: optimal IIR thresholds per patient ranged from 1.01 to 1.22. ConclusionsThe optimal IIR threshold to identify LA ablation scar by dark-blood LGE CMR is 1.09. Because of interpatient variability, the investigators recommend using a lower (1.00) and upper (1.10) threshold to prevent over- or underestimation of ablation scar.

Variation of b Value for Axial DWI Prostate MR Imaging in Cancer: Which b Value is Optimal?

Background: Diffusion Weighted Imaging (DWI) is a technique that visualizes the movement of water molecules from areas of higher to lower concentration. In DWI sequences, the b-value parameter affects the signal intensity in the images. The objective of this study is to describe the differences in anatomical image information between b-value variations of 1500s/mm2, 2000s/mm2, and 2500s/mm2 in MRI prostate case cancer and to determine the most optimal b-value. Method: This study used a pre-exeachimental approach with a 1.5T Philips MRI machine at a private hospital in South Jakarta from March to April 2023. The sample consisted of 12 Prostate MRI images with DWI sequences and different b-values. Visual grading assessment was eachformed by three radiology specialists, and the data were analyzed using the Friedman test in SPSS. Results: The results showed a significant difference in anatomical image information based on the b-value variations used, with a p-value of 0.05 (0.02). The highest mean rank was obtained from the Friedman test for the b-value of 2000s/mm2. Conclusion: The significant difference in anatomical image information occurred due to differences in signal intensity and noise levels. Prostate cancer tissues appeared more hyperintense with lower noise in images with higher b-values, and vice versa. Additionally, the b-value of 2000s/mm2 resulted in the most optimal signal intensity and noise level. In conclusion, the use of b-value variations in axial DWI images of prostate cancer Prostate MRI to differences in anatomical image information, with the most optimal image information observed at a b-value of 2000s/mm2.

Theoretical and simulation investigation of practical QKD for both BB84 and SARG04 protocols

In this paper, we provide a simple approach for determining the rate of secure key production when single-photon and two-photon pulses are used to generate the secure key. For both BB84 and SARG04, we offer estimation and optimization of decoy-state parameters for a single decoy state protocol and two decoy state protocols. This method can be used to determine the lower bound on the fraction of single-photon counts ([Formula: see text]), the fraction of two-photon counts ([Formula: see text]), the upper bound on the QBER of single-photon pulses ([Formula: see text]), the upper bound on the QBER of two-photon pulses ([Formula: see text]), and the lower bound on the key generation rate for both BB84 and SARG04. Due to the limited data size in real-world experiments, we estimate the statistical fluctuations of several parameters, including the gain and QBER of the signal state, as well as the gain and QBER of the decoy state, which are used to estimate the lower bound of key generation rate. We propose formulae that specify the optimal decoy and signal state percentages and intensities. Then we can determine the maximum and optimal key generation rate. For both SARG04 and BB84, we simulated a free space-based QKD system using our decoy state strategy. The simulation results show that free space-based QKD systems that use the suggested approach for BB84 can achieve a higher secret key rate and a greater safe distance than SARG04-based systems.

Optimal pixel signal intensity threshold for the assessment of endocardial left-ventricular scar by 3D dark blood MRI compared to bipolar voltage electro-anatomical mapping

Abstract Background Late gadolinium enhancement (LGE) MRI is considered the standard for noninvasive assessment of 3D scar architecture. Intraprocedural integration of 3D scar models improve the outcome of catheter ablation for ventricular tachycardia (VT). A pixel signal intensity (PSI) cut-off of 40 to 60% has been widely used for conventional bright-blood LGE MRI. Novel 3D MRI acquisitions with dark-blood (DB) nulling with higher spatial resolution and superior detection of (subendocardial) scar patterns are increasingly available, but the optimal pixel signal intensity (PSI) value remains unknown. Purpose To compare the accuracy of the 3D DB MRI-derived endocardial scar reconstructions at three different PSI cut-offs with endocardial electro-anatomical contact mapping in patients with scar-related VT. Methods Preprocedural 3D DB LGE MRIs were retrospectively compared to high-density endocardial left-ventricular (LV) bipolar voltage mapping (cut-off 0.5-1.5mV) obtained during endocardial mapping at sinus rhythm using the ADAS3D software. The LV scar models were aligned using the mitral valve, left ventricular apex and aorta cusps. Points with a Euclidean node-to-node distance of ≥10 mm were excluded. Variable upper PSI thresholds were set at 50%, 60% and 70%; the lower threshold at two-thirds of the upper limit. The 10% LV layer from the 3D MRI was used. The pairs of points were checked for agreement for scar or healthy (%). The Pearson correlation was used to analyze the agreement between core scar, border zone and healthy tissue area (in %) at three thresholds. Results Ten patients (90% male, mean age 65±11 year, 8 ischemic/2 nonischemic, LV ejection fraction 41±6%) were analyzed. A mean of 2024 (range 385-6810) pairs were analyzed per patient at three PSI thresholds. A correct correlation for scar or healthy was present in 67%, 69% and 69% for the upper PSI thresholds of 50%, 60% and 70% respectively. The optimal match was obtained for an upper PSI cutoff value of 70%, for healthy (r2=0.57; P=0.01), border zone (r2=0.73; P=0.01), 50% for core (r2=0.09; P=0.39). Using a 60% PSI cutoff the scar core % increased significantly compared to the 70% cutoff (12.8 ±13.8 versus 4.9±7.3, P=0.001). Conclusion The optimal depiction of the boundary between healthy and border zone scar by 3D DB MRI is best depicted at an upper PSI threshold of 70%.

An Image-Based Workflow for Objective Vessel Wall Enhancement Quantification in Intracranial Aneurysms.

Background: VWE in contrast-enhanced magnetic resonance imaging (MRI) is a potential biomarker for the evaluation of IA. The common practice to identify IAs with VWE is mainly based on a visual inspection of MR images, which is subject to errors and inconsistencies. Here, we develop and validate a tool for the visualization, quantification and objective identification of regions with VWE. Methods: N = 41 3D T1-MRI and 3D TOF-MRA IA images from 38 patients were obtained and co-registered. A contrast-enhanced MRI was normalized by the enhancement intensity of the pituitary stalk and signal intensities were mapped onto the surface of IA models generated from segmented MRA. N = 30 IAs were used to identify the optimal signal intensity value to distinguish the enhancing and non-enhancing regions (marked by an experienced neuroradiologist). The remaining IAs (n = 11) were used to validate the threshold. We tested if the enhancement area ratio (EAR—ratio of the enhancing area to the IA surface-area) could identify high risk aneurysms as identified by the ISUIA clinical score. Results: A normalized intensity of 0.276 was the optimal threshold to delineate enhancing regions, with a validation accuracy of 81.7%. In comparing the overlap between the identified enhancement regions against those marked by the neuroradiologist, our method had a dice coefficient of 71.1%. An EAR of 23% was able to discriminate high-risk cases with an AUC of 0.7. Conclusions: We developed and validated a pipeline for the visualization and objective identification of VWE regions that could potentially help evaluation of IAs become more reliable and consistent.

Neutrophil myeloperoxidase harbors distinct site-specific peculiarities in its glycosylation

Anti-neutrophil cytoplasmic autoantibodies (ANCAs) are directed against lysosomal components of neutrophils. ANCAs directed to proteinase 3 and myeloperoxidase (MPO) in particular are associated with distinct forms of small vessel vasculitides. MPO is an abundant neutrophil-derived heme protein that is part of the antimicrobial defense system. The protein is typically present in the azurophilic granules of neutrophils, but a large portion may also enter the extracellular space. It remains unclear why MPO is frequently the target of antibody-mediated autoimmune responses. MPO is a homodimeric glycoprotein, posttranslationally modified with complex sugars at specific sites. Glycosylation can strongly influence protein function, affecting its folding, receptor interaction, and backbone accessibility. MPO potentially can be heavily modified as it harbors 5 putative N-glycosylation sites (10 in the mature dimer). Although considered important for MPO structure and function, the full scope and relative abundance of the glycans attached to MPO is unknown. Here, combining bottom-up glycoproteomics and native MS approaches, we structurally characterized MPO from neutrophils of healthy human donors. We quantified the relative occupancy levels of the glycans at each of the five sites and observed complex heterogeneity and site-specific glycosylation. In particular, we detected glycosylation phenotypes uncommon for glycoproteins in the extracellular space, such as a high abundance of phosphorylated high-mannose species and severely truncated small glycans having the size of paucimannose or smaller. We hypothesize that the atypical glycosylation pattern found on MPO might contribute to its specific processing and presentation as a self-antigen by antigen-presenting cells.

Capillary zone electrophoresis-native mass spectrometry for the quality control of intact therapeutic monoclonal antibodies

Therapeutic monoclonal antibodies (mAbs) are complex glycoproteins and ensuring their safety, efficacy and quality is still challenging. Indeed, during their manufacturing process, they are exposed to several stresses that can lead to their denaturation, misfolding or dimerization. We report here a new method based on capillary electrophoresis coupled to native mass spectrometry (MS) with a sheath liquid interface to analyze an intact therapeutic mAb, Infliximab, under non-denaturing conditions that preserve its conformational heterogeneity as well as self-association without inducing further unfolding / denaturation. For capillary zone electrophoresis (CZE) separation, a triple layer coating using polybrene-dextran sulfate-polybrene was employed. A sheath liquid composed of isopropanol - water - acetic acid with a flow rate of 10 μL min−1 and mild MS conditions allowed optimal signal intensities. A specific mass spectrum was obtained for each Infliximab conformation in a “stressed” formulated preparation. This is the first time that within a single analysis different conformational states, i.e. native and unfolded monomers as well as dimers are simultaneously detected. The results and the lack of analytical bias arising from the CZE-MS conditions were confirmed by using atomic force microscopy (AFM) as an orthogonal technique. A middle-up approach combined to CZE-MS analysis of the stressed samples suggested that the dimer formation involved mostly Fab-Fab interactions.

Development and validation of a solid-phase extraction method coupled with LC-MS/MS for the simultaneous determination of 16 antibiotic residues in duck meat.

The present study was carried out to determine 16 antibiotics belonging to seven different groups (tetracyclines, sulfonamides, penicillins, fluoroquinolones, macrolides, lincosamides and trimethoprims) in duck meat. A solid-phase extraction method based on Oasis HLB cartridges coupled with liquid chromatography-electrospray ionization tandem mass spectrometry was developed. Solutions of 0.1m ethylenediaminetetraacetic acid disodium salt and 2% trifluoroacetic acid were used for the preliminary extraction of the target antibiotics from duck meat and n-hexane was used for purification prior to solid-phase extraction. Mobile phases composed of 0.1% trifluoroacetic acid in distilled water (solvent A) and 0.1% trifluoroacetic acid in methanol (solvent B), combined with a reversed-phase C18 analytical column, provided the optimal separation and signal intensity. The linearity of the method was assessed using six concentrations (5, 10, 20, 30, 40, and 50 μg/kg), and the recoveries, which were calculated at three spiking concentrations (5, 10 and 20 μg/kg), were in the range 69.8-103.3% with relative standard deviations (RSDs) ≤ 6.9% for the 16 tested antibiotics. Matrix effects ranging from -47.2 to -13.5% were observed for all the analytes, and the limits of quantitation (LOQ), which ranged from 4.93 to 26.21 μg/kg, were much lower than the maximum residue limits (MRLs) set by various regulatory authorities. Ten samples from a market were tested, and none of the target analytes were detected. Thus, a simple and versatile protocol has been developed to detect and quantify 16 antibiotics in duck meat samples.

Water/Oil Interfacial Self-Assembled Gold Nanoarrays Modified on Transparent Tape for In Situ Surface-Enhanced Raman Scattering

Herein, a novel in situ surface-enhanced Raman scattering (SERS) method was proposed based on the prepared SERS substrate with good flexibility, excellent optical transparency, and high SERS activity. The SERS substrate was fabricated through the Au colloids injected with an ethanol and hexane mixture to form Au monolayer film (Au MLF) on the water/oil interface and followed by transferred to the sticky side of transparent tape after hexane volatilization. Transmission electron microscopy and scanning electron microscopy revealed that Au NPs with particle sizes of 25 nm were densely assembled and evenly distributed on the flexible and transparent tape. This Au MLF/tape was directly covered on the irregular shape samples and the detection signal was collected by the laser transmitting into the tape and interacting with the analytes. The optimal SERS signal intensity was obtained by tuning the concentration of Au colloid and addition volume of the mixture of ethanol and hexane to regulate the numbers of Au NPs on water/oil interface. The SERS intensity of six sample points selected randomly show a good repeatability with RSD = 4.92% (n = 6 × 8) and satisfying uniformity with RSD of 8.6% within an area of 4.0 × 4.0 μm. Also, the obtained Au MLF/tape SERS substrate showed good detection accuracy and recovery when used to detect residual malachite green (MG) residue on the surfaces of real fish samples with detection limit of 0.04 μmol/L (S/N = 3). Therefore, the developed Au MLF/tape SERS substrate has potential applications in the in situ SERS detection.

The requirements for low-temperature plasma ionization support miniaturization of the ion source.

Ambient ionization mass spectrometry (AI-MS), the ionization of samples under ambient conditions, enables fast and simple analysis of samples without or with little sample preparation. Due to their simple construction and low resource consumption, plasma-based ionization methods in particular are considered ideal for use in mobile analytical devices. However, systematic investigations that have attempted to identify the optimal configuration of a plasma source to achieve the sensitive detection of target molecules are still rare. We therefore used a low-temperature plasma ionization (LTPI) source based on dielectric barrier discharge with helium employed as the process gas to identify the factors that most strongly influence the signal intensity in the mass spectrometry of species formed by plasma ionization. In this study, we investigated several construction-related parameters of the plasma source and found that a low wall thickness of the dielectric, a small outlet spacing, and a short distance between the plasma source and the MS inlet are needed to achieve optimal signal intensity with a process-gas flow rate of as little as 10mL/min. In conclusion, this type of ion source is especially well suited for downscaling, which is usually required in mobile devices. Our results provide valuable insights into the LTPI mechanism; they reveal the potential to further improve its implementation and standardization for mobile mass spectrometry as well as our understanding of the requirements and selectivity of this technique. Graphical abstract Optimized parameters of a dielectric barrier discharge plasma for ionization in mass spectrometry. The electrode size, shape, and arrangement, the thickness of the dielectric, and distances between the plasma source, sample, and MS inlet are marked in red. The process gas (helium) flow is shown in black.

Characterization of conformers and dimers of antithrombin by capillary electrophoresis-quadrupole-time-of-flight mass spectrometry

Antithrombin (AT) is a plasma glycoprotein which possesses anticoagulant and anti-inflammatory properties. AT exhibits various forms, among which are native, latent and heterodimeric ones. We studied the potential of capillary electrophoresis-mass spectrometry (CE-MS) using a sheath liquid interface, electrospray ionization (ESI), and a quadrupole-time-of-flight (Q-TOF) mass spectrometer to separate and quantify the different AT forms. For CE separation, a neutral polyvinyl alcohol (PVA) coated capillary was employed. The protein conformation was preserved by using a background electrolyte (BGE) at physiological pH. A sheath liquid of isopropanol-water 50:50 (v/v) with 14 mM ammonium acetate delivered at a flow rate of 120 μL h−1 resulted in optimal signal intensities. Each AT form exhibited a specific mass spectrum, allowing unambiguous distinction. Several co-injection experiments proved that latent AT had a higher electrophoretic mobility (μep) than native AT, and that these conformers could associate to form a heterodimer during the CE analysis. The developed CE-MS method enabled the detection and quantitation of latent and heterodimeric forms in a commercial AT preparation stored at room temperature for three weeks.

Reporters for Single-Cell Analysis of Colicin Ib Expression in Salmonella enterica Serovar Typhimurium.

Colicins are toxins that mediate interference competition in microbial ecosystems. They serve as a “common good” for the entire producer population but are synthesized by only few members which pay the costs of colicin production. We have previously shown that production of colicin Ib (cib), a group B colicin, confers a competitive advantage to Salmonella enterica serovar Typhimurium (S. Tm) over commensal E. coli strains. Here, we studied regulation of S. Tm cib expression at the single cell level. Comparative analysis of a single- and a multicopy gfp-reporter for the colicin Ib promoter (Pcib) revealed that the latter yielded optimal signal intensity for a diverse range of applications. We further validated this reporter and showed that gfp expression correlated well with colicin Ib (ColIb) protein levels in individual cells. Pcib is negatively controlled by two repressors, LexA and Fur. Only a small fraction of S. Tm expressed cib under non-inducing conditions. We studied Pcib activity in response to mitomycin C mediated DNA damage and iron limitation. Both conditions, if applied individually, lead to an increase in the fraction of GFP+ S. Tm, albeit an overall low fluorescence intensity. When both conditions were applied simultaneously, the majority of S. Tm turned GFP+ and displayed high fluorescence intensity. Thus, both repressors individually confine cib expression to a subset of the population. Taken together, we provide the first thorough characterization of a conventional gfp-reporter to study regulation of a group B colicin at the single cell level. This reporter will be useful to further investigate the costs and benefits of ColIb production in human pathogenic S. Tm and analyze cib expression under environmental conditions encountered in the mammalian gut.

Quantitative liquid chromatography–electrospray ionization-mass spectrometry analysis of amine-containing metabolites derivatized with cyanuric chloride and methylamine isotopologues

Electrospray ionization (ESI) is the most useful interface for mass spectrometry associated with liquid chromatography (LC). However, analyses of polar metabolites become a challenge because the high polarity impairs the separation of metabolites in reversed-phase liquid chromatography and ionization of metabolites by ESI. In this article, we have used cyanuric chloride to couple the amine-containing molecules with methylamine and found that both resolution on LC chromatogram and ionization by ESI are greatly improved. Derivatives would be obtained in 2-h coupling reactions, and then resolved by LC–ESI-MS in 15min for each sample. Most amino acids can be quantified with linear range from 1nM to 1μM and with an R2 above 0.979. Although reversed-phase chromatography is suitable for resolving the derivatives, phenyl columns with methanol elution provide optimal separation and signal intensity. Moreover, most structural isomers are well separated following cyanuric chloride and methylamine derivatization. Instead of synthesizing a stable isotope-labeled cyanuric chloride, we can take advantage of using commercially available methyl-d3-amine for a novel stable isotope-coded derivatization method. Each metabolite can be directly quantified by the peak intensity ratio of each derivative isotopologue pair in a single LC–MS analysis. The coupling reactions are relatively easy and accessible to most investigators to generate multiple stable isotope-labeled derivatives of amine-containing compounds for a differential metabolomic analysis.

TH-C-19A-11: Toward An Optimized Multi-Point Scintillation Detector

Purpose: The purpose of this work is to characterize a 2-points mPSDs' optical chain using a spectral analysis to help selecting the optimal components for the detector. Methods: Twenty different 2-points mPSD combinations were built using 4 plastic scintillators (BCF10, BCF12, BCF60, BC430; St-Gobain) and quantum dots (QDs). The scintillator is said to be proximal when near the photodetector, and distal otherwise. A 15m optical fiber (ESKA GH-4001) was coupled to the scintillating component and connected to a spectrometer (Shamrock, Andor and QEPro, OceanOptics). These scintillation components were irradiated at 125kVp; a spectrum for each scintillator was obtained by irradiation of individual scintillator and shielding the second component, thus talking into account light propagation in all components and interfaces. The combined total spectrum was also acquired and involved simultaneous irradiation of the two scintillators for each possible combination. The shape and intensity were characterized. Results: QDs in proximal position absorb almost all the light signal from distal plastic scintillators and emit in its own emission wavelength, with 100% of the signal in the QD range (625–700nm) for the combination BCF12/QD. However, discrimination is possible when QD is in distal position in combination with blue scintillators, total signal being 73% in the blue range (400-550nm) and 27% in QD range. Similar results are obtained with the orange scintillator (BC430). For optimal signal intensity, BCF12 should always be in proximal position, e.g. having 50% more intensity when coupled with BCF60 in distal position (BCF12/BCF60) compared to the BCF60/BCF12 combination. Conclusion: Different combinations of plastic scintillators and QD were built and their emission spectra were studied. We established a preferential order for the scintillating components in the context of an optimized 2-points mPSD. In short, the components with higher wavelength emission spectrum should be distal and lower wavelength in the proximal position.

Study on effective probe depth of optical coherence tomography system by Monte Carlo simulation

The intensities of Class I and Class II signals at several different probing depths and different coherence lengths are analyzed using a Monte Carlo model with a pencil beam, and the result shows that the longitudinal resolution has influence on the effective probe depth. A Monte Carlo model for optical coherence tomography system with a focused Gaussian beam is proposed. The intensities of Class I and Class II signals at different probing depth for different radius and depth of focus of the beam are simulated using this model. We found that increasing of the depth of focus and decreasing of the beam radius can finitely increase effective probing depth. When the effective probing depth is fixed, optimal signal intensity can be achieved by altering the beam radius or the depth of focus.

Performance analysis of decoy-state quantum key distribution with a heralded pair coherent state photon source

从有效性、稳定性和可行性三个方面, 对基于标记配对相干态光源的诱骗态量子密钥分配的性能进行了全面分析. 采用四组实验数据对基于标记配对相干态光源的三强度诱骗态方案的密钥生成效率、量子比特误码率和最优信号态强度与安全传输距离之间的关系进行了仿真和分析; 考虑到光源涨落, 对方案的稳定性进行了讨论和仿真; 并对基于标记配对相干态光源设计简单易实现方案的可行性进行了分析. 结论表明: 基于标记配对相干态光源的诱骗态方案性能在安全传输距离和密钥生成效率两方面都优于现有基于弱相干态光源和预报单光子源的诱骗态方案; 在光源强度涨落相同条件下, 标记配对相干态光源的稳定性逊于预报单光子源, 而优于相干态光源. 但是标记配对相干态光源在有效性上的优势可弥补其在稳定性上的不足; 且标记配对相干态光源的双模特性为设计简单易实现的被动诱骗态方案提供了条件.

Velocity Profile Measurements in Hypersonic Flows Using Sequentially Imaged Fluorescence-Based Molecular Tagging

Nitric-oxide planar laser-induced fluorescence was used to perform velocity measurements in hypersonic flows by generatingmultiple tagged lines thatfluoresce as they convect downstream.Determination of axial velocitywasmade by application of a cross-correlation analysis of the horizontal shift of individual tagged lines. A single interline, progressive scan-intensified charge-coupled device camera was used to obtain two sequential images of the nitricoxide molecules that had been tagged by the laser. The charge-coupled device allowed for submicrosecond acquisition of both images, resulting in submicrosecond temporal resolution as well as submillimeter spatial resolution (0.5 mm horizontal, 0.7 mm vertical). Quantification of systematic errors, the contribution of gating/ exposure duration errors, and the influence of collision rate on temporal uncertainty were made. This velocity measurement technique has been demonstrated for two hypersonic flow experiments: 1) a reaction control system jet on an Orion crew exploration vehicle wind-tunnel model and 2) a 10 deg half-angle wedge with a 2-mm-tall 4-mmwide cylindrical boundary-layer trip. Mean-velocity uncertainties below 30 m=s (2.7% of the measured average velocity) and single-shot uncertainties below 100 m=s (9.7%of themeasured average velocity) have been obtained in regions with optimal signal intensities using this technique.

Capillary electrophoresis–mass spectrometry of intact basic proteins using Polybrene–dextran sulfate–Polybrene-coated capillaries: System optimization and performance

A capillary electrophoresis–mass spectrometry (CE–MS) method using sheath liquid electrospray ionization interfacing was studied and optimized for the analysis of intact basic proteins. To prevent protein adsorption, capillaries with a noncovalent positively charged coating were utilized. Capillaries were coated by subsequent rinsing with solutions of Polybrene, dextran sulfate and Polybrene. The coating proved to be fully compatible with MS detection, causing no background signals and ionization suppression. The composition of the sheath liquid and BGE was optimized using the model proteins α-chymotrypsinogen A, ribonuclease A, lysozyme and cytochrome c. A sheath liquid of isopropanol–water–acetic acid (75:25:0.1, v/v/v) at 2 μL min −1 resulted in optimal signal intensities for most proteins, but caused dissociation of the heme group of cytochrome c. Optimum protein responses were obtained with a BGE of 50 mM acetic acid (pH 3.0), which allowed a baseline separation of the test protein mixture. Several minor impurities present in the mixture could be detected and provisionally identified using accurate mass and a protein modification database. The selectivity of the CE–MS system was investigated by the analysis of acetylated lysozyme. Eight highly related species, identified as non-acetylated lysozyme and lysozyme acetylated in various degrees, could be distinguished. The CE–MS system showed good reproducibility yielding interday (three weeks period) RSDs for migration time and peak area within 2% and 10%, respectively. With the CE–MS system, determination coefficients ( R 2) for protein concentration and peak area were higher than 0.996, whereas detection limits were between 11 and 19 nM.

Determination of methylmercury and inorganic mercury by coupling short-column ion chromatographic separation, on-line photocatalyst-assisted vapor generation, and inductively coupled plasma mass spectrometry

We have combined short-column ion chromatographic separation and on-line photocatalyst-assisted vapor generation (VG) techniques with inductively coupled plasma mass spectrometry to develop a simple and sensitive hyphenated method for the determination of aqueous Hg(2+) and MeHg(+) species. The separation of Hg(2+) and MeHg(+) was accomplished on a cation-exchange guard column using a glutathione (GSH)-containing eluent. To achieve optimal chromatographic separation and signal intensities, we investigated the influence of several of the operating parameters of the chromatographic and photocatalyst-assisted VG systems. Under the optimized conditions of VG process, the shortcomings of conventional SnCl(2)-based VG techniques for the vaporization of MeHg(+) was overcome; comparing to the concentric nebulizer-ICP-MS system, the analytical sensitivity of ICP-MS toward the detection of Hg(2+) and MeHg(+) were also improved to 25- and 7-fold, respectively. With the use of our established HPLC-UV/nano-TiO(2)-ICP-MS system, the precision for each analyte, based on three replicate injections of 2 ng/mL samples of each species, was better than 15% RSD. This hyphenated method also provided excellent detection limits--0.1 and 0.03 ng/mL for Hg(2+) and MeHg(+), respectively. A series of validation experiments--analysis of the NIST 2672a Standard Urine Reference Material and other urine samples--confirmed further that our proposed method could be applied satisfactorily to the determination of inorganic Hg(2+) and MeHg(+) species in real samples.

Signal loss in 1D magic-angle spinning exchange NMR (CODEX): radio-frequency limitations and intermediate motions

The popular 1D MAS exchange experiment CODEX suffers limitations due to signal loss during the finite recoupling periods, during which the magnetization evolves in the transverse plane. Here, we address the origins and possible improvements of this problem, aimed at (i) an optimization of the signal-to-noise ratio in the experiments, as well as harnessing intermediate-motion induced signal loss for obtaining approximate information on (ii) correlation times and (iii) potential distributions, where the latter are often found in polymeric systems. First, we show that the intensity of the signal is sensitive to the radiofrequency (rf) parameters of the carbon recoupling and proton decoupling, and care must be taken to gain optimal signal intensity. Optimum conditions are found for recoupling pulses being as short as possible for large chemical shift anisotropy (CSA) values, and approaching a ratio of 3 between the nutation frequencies for protonated carbons, calling for an individual adjustment in each case. Second, we demonstrate that the effect of intermediate motions can be studied semi-quantitatively by combining CODEX data with its constant-time modification CONTRA, which allows for a tuning of the signal loss due to intermediate motions. Third, for the case of samples featuring a distribution of correlation times, we propose a procedure to obtain an estimate of the proportion of molecular segments in the sample for which the CODEX data are representative, i.e., which share of segments moves truly in the slow-motion regime. The procedure involves the combination of CODEX data with a cross-polarisation (CP) reference experiment for an estimate of the full sample magnetization; it is demonstrated on the example of semi-crystalline poly(ethylene oxide).