Analytical and Bioanalytical Chemistry最新文献

Amyloidosis collectively describes a heterogeneous group of protein aggregation-based diseases involving the misfolding and extracellular accumulation of fibril-forming amyloid proteins. Diagnosing amyloidosis is difficult due to its many subtypes (e.g., AA, AL, ATTR), with varying symptoms. Current diagnosis often involves Congo red staining, but it has limitations in quantification and specificity. A novel method called iprm-PASEF exploits MALDI imaging and offers a faster, spatially resolved, antibody-independent technique for identifying peptides while preserving tissue structure. In this study, iprm-PASEF was used to further evaluate its applicability on amyloidosis. FFPE slides of an amyloidosis TMA including biopsies of 18 amyloidosis-positive tissues were prepared for tryptic peptide MALDI imaging. An initial MALDI TIMS MS1 measurement was performed, followed by the manual generation of a precursor list containing mass-to-charge ratios and ion mobility windows. In a second iprm-PASEF measurement, the selected precursors are analyzed in a multiplexed MALDI MS/MS mode. Peptide identification was achieved through peptide-to-spectrum matching using MASCOT. Within the course of this study, we characterized an amyloidosis TMA consisting of AA, AL, and ATTR amyloidosis diseased tissue with MALDI imaging of tryptic peptides. We successfully identified eight amyloidosis-related peptides derived from serum amyloid A, vitronectin, apolipoprotein E, serum amyloid P component, and transthyretin receptor in one single iprm-PASEF measurement. Peptide signals mapped to amyloidogenic plaques determined in a Congo red staining. Some of these peptides were specifically found in ATTR and AA amyloidosis. This represents a significant step towards integrating MALDI imaging into the diagnostic process for amyloidosis.

Extracellular vesicles (EVs) are membrane-bound nanosized particles excreted by all cells and are of significant interest for biomedical applications such as diagnostic testing and as vectors for therapeutic delivery. EVs are abundant in biofluids, including urine, saliva, blood, and cell culture media, but must be isolated from their complex matrix for use. Once isolated, a primary challenge is determining the EV size distribution and the total number of particles recovered. Multiple detection methods are currently used to characterize EV recoveries in terms of sizing and number density determinations, the most common being nanoparticle tracking analysis (NTA), flow cytometry, and electron microscopy (transmission (TEM) or scanning (SEM)). Addressed here is a practical assessment of three common light scattering-based methods for the determination of EV population sizing and number densities. NTA, multi-angle light scattering (MALS), and nano-flow cytometry (nFCM) are directly compared. Specifically, the baseline practical advantages and disadvantages of each technique are evaluated via analysis of silica nanoparticle standards. Subsequently, EVs isolated from human embryonic kidney (HEK) cell culture supernatant using a hydrophobic interaction chromatography-based separation on the previously developed polyester (PET) capillary-channeled polymer (C-CP) fiber column platform were characterized. This isolation of high-purity EVs from HEK cell culture matrix components was validated using UV chromatograms, Bradford protein assays, SEM, TEM, and fluorescence nFCM analysis. The relative attributes of these important light scattering methods are presented in terms of their fitness for specific applications and overall effectiveness in EV size and concentration analysis.

Bile acids (BAs) are endogenous signaling molecules with diverse biological functions. In cholestasis, conjugated BAs are markedly elevated, making their ratio to unconjugated BAs a critical diagnostic biomarker. Accurate quantification of BAs is pivotal for assessing cholestatic disease progression. Conjugated BAs contain a fragile amide bond that is susceptible to hydrolysis in biological samples, resulting in an artificially low ratio of conjugated-to-unconjugated BAs. Here, we systematically investigated the benchtop stability of BAs in murine biological samples using liquid chromatography-mass spectrometry. Strikingly, pronounced degradation of conjugated BAs occurred in liver and ileum samples from wild-type and Mdr2 knockout mice within 1 h on the benchtop at 25 ℃. In liver samples, tauro-α/β-muricholic acid (T-α/β-MCA), the predominant murine conjugated BAs, decreased by over 70%. Other conjugated BAs, such as taurochenodeoxycholic acid (TCDCA) and tauroursodeoxycholic acid (TUDCA), also showed significant degradation. Concurrently, unconjugated BAs increased by 1-12-fold. In ileum samples, conjugated BAs exhibited a 5%-40% reduction concomitant with up to a 30-fold increase in unconjugated BAs. In contrast, BAs remained stable in serum samples. Mechanistic studies using deuterium-labeled conjugated BAs confirmed amide bond hydrolysis as the primary degradation pathway. Several optimized protocols, such as immediate storage on ice, enzymatic inactivation, and liquid nitrogen snap-freezing, effectively mitigated the hydrolysis. These findings suggest that the hydrolysis of conjugated BAs in untreated liver and ileum samples leads to serious underestimation of conjugated BAs and inflation of unconjugated BA levels, highlighting a preanalytical pitfall in BA quantification. Stabilizing protocols are essential immediately upon sample collection.

Natural variability in stable isotope ratios provides critical constraints on elemental cycling in nature without the need for the introduction of artificial tracers. While such data are widely used in environmental studies, they are not as widely employed in biomedical research, despite vast potential. One critical hurdle to the adoption of such techniques in biomedical studies is sample throughput. Elemental purification via ion-exchange chromatography and isotopic analysis via multiple collector inductively coupled plasma mass spectrometry (MC-ICP-MS) are time-consuming, requiring long hours from experienced researchers to generate datasets. Here we present new methods to improve the throughput of both elemental purification and sample introduction to mass spectrometers. We use an automated, low-pressure ion exchange chromatography system to isolate purified fractions of potassium, magnesium, and calcium from one sample in a single sequence with high yields (80-100%) and low blanks (<0.5% carryover). Modification of flow rates and column volumes also enables recovery of purified strontium, lithium, and sodium in the same routine. Solutions are introduced to the MC-ICP-MS via syringe injection and with automated removal of vial caps to minimize evaporation. We find that syringe injection from capped vials gives a >10 × more stable signal (0.7% RSD) over a 9-h sequence than self-aspirated, uncapped solutions (8.0% RSD). Syringe injection also enables modification of signal intensity by changing the injection rate, with a linear response of signal to flow rate. We demonstrate the potential of these methods by analyzing calcium, magnesium, and potassium isotope ratios at high precision (<0.1 ‰) from single 0.5 mL aliquots of urine samples from individuals with chronic kidney disease. These data show a change in calcium reabsorption, highlighting avenues for further research as well as the value of these multi-isotopic analysis methods.

The mother machine (MM) is a microfluidic device designed to trap and measure single bacteria for long-term experiments, enabling the study of bacterial growth and cell cycle dynamics. In this work, the concept of a MM is adapted to fit the requirements for investigating Mycobacterium smegmatis, a bacterium used extensively as a model system in tuberculosis research. A MM chip was fabricated using soft lithography, and a protocol for sample preparation and filling of the side channels for mycobacteria was established. Trapped bacteria were measured using Raman spectroscopy to introduce a new analysis approach inside the MM. The subsequent data evaluation demonstrates the potential for obtaining spatially resolved chemical information on a single cell within a side channel of the MM through Raman imaging. Furthermore, the combination of Raman imaging and stable isotope labelling, as applied in this study, demonstrates the viability of bacteria inside a side channel and opens up possibilities to study cell cycles in long-term experiments.

Gadolinium (Gd) complexes, used as contrast agents in magnetic resonance imaging (MRI), along with other rare earth elements (REE), are emerging contaminants in natural waters. The quantification of these compounds is a challenge due to their low concentrations. In this study, an extraction and preconcentration method was optimized and validated for the simultaneous determination of REE, including anthropogenic Gd, in tap water by inductively coupled plasma mass spectrometry (ICP-MS). Initially, two solid-phase preconcentration methods (NOBIAS-chelate PA-1® resin columns and SEP-PAK Classic C₁₈ columns) were tested univariately. According to the % recovery, NOBIAS-chelate PA-1® columns was chosen. Chemometric tools, including fractional factorial design and Doehlert matrix, were applied to evaluate the parameters influencing the extraction and preconcentration steps. Under optimal conditions, the method was validated, achieving low limits of detection (0.02-0.14 ng L^-1) and quantification (0.06-0.48 ng L^-1), as well as good recoveries (92-99%) and precision (0.7-5.9%). The practicality and sustainability of the method were demonstrated using blue applicability grade index (BAGI) and sustainability and practicality metrics system (SPMS) metrics. Finally, the optimized and validated method was applied to six tap water samples from Brazil and Germany. ∑REE concentrations ranged from 37.9 to 596 ng L^-1 (Brazil) and 47.6 to 80.5 ng L^-1 (Germany), and the method demonstrated its suitability for the precise determination of REE, producing high recoveries and low uncertainties. The approach is straightforward, efficient in reagent and sample use and produces minimal waste.

A self-calibrated ratiometric electrochemical aptasensor was developed for the detection of ochratoxin A. This sensor is based on DNA-mediated gold/prussian blue nanoflowers (Au/PB NFs), methylene blue/platinum/zinc-cobalt metal-organic framework (MB/Pt/ZnCo MOF), and an exonuclease III (Exo III)-driven bipedal DNA walker cyclic amplification strategy. The self-calibration mechanism of this sensor relies on the ratio between the response signal of MB/Pt/ZnCo MOF (I_MB) and the internal reference signal of Au/PB NFs (I_PB). This ratio can effectively resist external interference factors. DNA-mediated Au/PB NFs with a flower-like structure provide stable internal reference signals and promote electron transfer. MB/Pt/ZnCo MOF, leveraging ZnCo MOF with a porous structure and bimetallic synergistic effects, and Pt with electrocatalytic activity, serve as response signals. An Exo III-driven biped DNA walker was used to realize the cyclic amplification strategy. The sensor showed a wide linear range of 0.001~500 ng/mL, with a low limit of detection of 0.17 pg/mL. The detection capability of spiked corn flour, wine, and coffee powder samples was satisfactory, and the detection results for the quality control samples of coarse rice flour and wheat flour were not significantly different from those of high-performance liquid chromatography (HPLC), showing the potential for practical application.

Trace metals and metal homeostasis play an essential role in cell metabolism, and an imbalance in this careful balance leads to pathological changes. In neurodegenerative diseases such as Parkinson's disease, trace metals and metal species are increasingly recognized as key factors in disease progression in mechanisms such as ferroptosis. In this study, we combined modern analytical techniques like Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) with metal-free high-pressure liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) to analyze metabolomic and metallomic changes in the model organism Caenorhabditis elegans under conditions of induced metal homeostasis imbalance. Our results identified zinc as a key player in the regulation of free metal species of iron and manganese. Furthermore, iron exposure resulted in distinct metabolomic patterns indicative of impaired energy metabolism, suggesting an exhaustion of cellular metabolic energy capacity.

Human myoglobin (hMYO) is a sensitive early biomarker for acute myocardial infarction, rising within 1~3 h after myocardial injury and offering high predictive value for excluding infarction. To standardize its measurement, a precise quantitative method combining magnetic bead extraction with liquid chromatography-isotope dilution tandem mass spectrometry (LC‑IDMS/MS) was established. ¹⁵NLabeled myoglobin was spiked into serum as an internal standard, and magnetic beads coated with monoclonal antibodies (mAbs) against hMYO were used for the extraction of hMYO from serum. The mAb employed in this study demonstrated comparable binding affinity for both native myoglobin and its isotopically labeled counterpart, as confirmed by surface plasmon resonance measurements. After extraction, the magnetic beads were washed and digested, and two signature peptides, VEADIPGHGQEVLIR (VR) and HGATVLTALGGILK (HGK), were selected for quantification of hMYO. The incubation time, bead and enzyme amounts, and digestion time were optimized to establish optimal sample treatment conditions. Digested peptides were analyzed by LC‑IDMS/MS and recovery based on the VR peptide was 95.4-101.6% (RSD = 2.3%), and based on the HGK peptide was 100.1-105.5% (RSD = 2.0%). Limits of detection and quantification of hMYO were 1.04 ng/g and 3.43 ng/g by VR, and 1.45 ng/g and 4.74 ng/g by HGK. The proposed method enables accurate serum hMYO quantification, supports reference material development and clinical standardization, and also provides an example for MS‑based quantification of clinical protein biomarkers.

Protein determination is of importance to characterise insect composition and quality. While different factors (kp) to convert nitrogen to protein have been proposed to report the concentration of crude protein (CP) in insect applications, the N × 6.25 is still utilised by many commercial laboratories. Different data-dependent conversion factors have been proposed to report the concentration of CP in different insect species, including black soldier fly larvae (BSFL). The objective of this paper was to evaluate the effect of using different nitrogen to protein conversion factors (kp) to predict CP in BSFL from different commercial sources using near-infrared spectroscopy. The coefficient of determination in cross-validation (R² _CV) and the standard error in cross-validation (SECV) for the prediction of CP% in the BSFL were 0.75 (SECV, 4.51%), 0.75 (SECV, 4.03%), and 0.75 (SECV, 3.43%), using the kp_6.25, kp_5.65, and kp_4.76, respectively. The study showed that the different kp used can affect the cross-validation statistics (SECV) for the prediction of CP in BSFL using NIR spectroscopy. Additionally, the lower accuracies obtained for the prediction of CP are not only associated with the kp used to calculate the CP but also with the amount of chitin in the BSFL. Understanding the variables, such as reference data, that influence the calibration results using NIR spectroscopy is of importance to better provide consistent QC methods for the industry. The limitations of this study are the few numbers of samples used to develop the calibration models, although different waste streams and larvae stages were evaluated.