Species In Biological Samples Research Articles

Establishment of a targeted analysis method for gangliosides in mouse tissues by HILIC-ESI-MS/MS.

Gangliosides play an imperative role in cell signaling, neuronal recovery, apoptosis, and other physiological processes. For example, GM3 can regulate hypothalamic leptin resistance and control energy homeostasis, GD3 can mediate cell proliferation and differentiation and induce apoptosis, and GQ1b can stimulate neurogenesis. Therefore, the present study sought to establish and optimize the targeted analysis method for ganglioside subclasses and their molecular species using hydrophilic interaction liquid chromatography-triple quadrupole-MS/MS (HILIC-QQQ-MS/MS). Additionally, the fragmentation pattern of different ganglioside subclasses and their retention time patterns were analyzed, providing more accurate qualitative results. The limit of quantitation (LOQ) was as low as 10-4ng. Moreover, the molecular species of gangliosides in the liver, cortex, and hypothalamus of C57BL/6 mice were analyzed using the established method. A total of 23 ganglioside subclasses with 164 molecular species, including 40 O-acetylated ganglioside molecular species and 28 NeuGc ganglioside molecular species, were identified using the semi-quantitative analysis method of anexternal standard curve corrected by an internal standard. In addition to NeuGc gangliosides, the contents of ganglioside subclasses were more abundant in the mouse brain than those in the mouse liver; especially, the contents of unsaturated gangliosides in the hypothalamus were much higher than those in the liver. Among them, O-acetylated gangliosides were detected only in the cortex and hypothalamus at a concentration of up to 100μg/mg protein (40 molecular species). Overall, the proposed method expanded the detectable number of ganglioside subclasses and molecular species in biological samples and provided more opportunities for further study of the biological functions of gangliosides.

LiLA: lipid lung-based ATLAS built through a comprehensive workflow designed for an accurate lipid annotation

Accurate lipid annotation is crucial for understanding the role of lipids in health and disease and identifying therapeutic targets. However, annotating the wide variety of lipid species in biological samples remains challenging in untargeted lipidomic studies. In this work, we present a lipid annotation workflow based on LC-MS and MS/MS strategies, the combination of four bioinformatic tools, and a decision tree to support the accurate annotation and semi-quantification of the lipid species present in lung tissue from control mice. The proposed workflow allowed us to generate a lipid lung-based ATLAS (LiLA), which was then employed to unveil the lipidomic signatures of the Mycobacterium tuberculosis infection at two different time points for a deeper understanding of the disease progression. This workflow, combined with manual inspection strategies of MS/MS data, can enhance the annotation process for lipidomic studies and guide the generation of sample-specific lipidome maps. LiLA serves as a freely available data resource that can be employed in future studies to address lipidomic alterations in mice lung tissue.

Holo/apo conversion two-dimensional urea PAGE for speciation of Fe3+-bound transferrin in serum.

This paper presents holo/apo conversion two-dimensional urea polyacrylamide gel electrophoresis (HAC-2D urea PAGE) as a novel method for speciating Fe3+-bound transferrin (Tf) species in biological samples, with a combination of metal ion contaminant sweeping (MICS) technique and Fe3+ detection PAGE. In the HAC-2D urea MICS-PAGE approach, HAC was performed to dissociate all the Fe3+ ions bound to Tf from the Fe-Tf species, during a two-step urea PAGE. Using this method, Fe2-Tf, FeN-Tf, and FeC-Tf (holo-Tf, Fe3+-bound Tf attached to N-lobe, and Fe3+-bound Tf attached C-lobe, respectively) were completely isolated based on the difference in the higher-order structure of Tf, visible as horizontally aligned spots off the diagonal. The Fe3+ ions bound to Tf in each gel fraction were determined using PAGE with a fluorescent probe. Without the MICS technique, which electrophoretically removes all contaminant Fe3+ ions from the gel medium to ensure accurate determination of the Fe3+ concentration, it becomes challenging to precisely measure the distribution of metalloprotein species owing to the contaminants. Finally, the distribution of each Fe-bound Tf in a standard human serum sample was successfully determined by complete separation from large amounts of coexisting proteins, and the free Fe3+ concentration in the serum was estimated.

Matrix Development for the Detection of Phosphorylated Amyloid-β Peptides by MALDI-TOF-MS.

Amyloid-β (Aβ) peptides, including post-translationally modified variants thereof, are believed to play a key role in the onset and progression of Alzheimer's disease. Suggested modified Aβ species with potential disease relevance include Aβ peptides phosphorylated at serine in position eight (pSer8-Aβ) or 26 (pSer26-Aβ). However, the published studies on those Aβ peptides essentially relied on antibody-based approaches. Thus, complementary analyses by mass spectrometry, as shown for other modified Aβ variants, will be necessary not only to unambiguously verify the existence of phosphorylated Aβ species in brain samples but also to reveal their exact identity as to phosphorylation sites and potential terminal truncations. With the aim of providing a novel tool for addressing this still-unresolved issue, we developed a customized matrix formulation, referred to as TOPAC, that allows for improved detection of synthetic phosphorylated Aβ species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. When TOPAC was compared with standard matrices, we observed higher signal intensities but minimal methionine oxidation and phosphate loss for intact pSer8-Aβ(1-40) and pSer26-Aβ(1-40). Similarly, TOPAC also improved the mass spectrometric detection and sequencing of the proteolytic cleavage products pSer8-Aβ(1-16) and pSer26-Aβ(17-28). We expect that TOPAC will facilitate future efforts to detect and characterize endogenous phosphorylated Aβ species in biological samples and that it may also find its use in phospho-proteomic approaches apart from applications in the Aβ field.

Comparison of Workflows for Milk Lipid Analysis: Phospholipids.

Milk is a rich source of lipids, with the major components being triglycerides (TAG) and phospholipids (mainly phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI)). Liquid chromatography-mass spectrometry (LC-MS) is the predominant technique for lipid identification and quantification across all biological samples. While fatty acid (FA) composition of the major lipid classes of milk can be readily determined using tandem MS, elucidating the regio-distribution and double bond position of the FA remains difficult. Various workflows have been reported on the quantification of lipid species in biological samples in the past 20 years, but no standard or consensus methods are currently available for the quantification of milk phospholipids. This study will examine the influence of several common factors in lipid analysis workflow (including lipid extraction protocols, LC stationary phases, mobile phase buffers, gradient elution programmes, mass analyser resolution and isotope correction) on the quantification outcome of bovine milk phospholipids. The pros and cons of the current LC-MS methods as well as the critical problems to be solved will also be discussed.

Synthesis and characterization of molecularly imprinted polymers for detection of the local anesthetic lidocaine in urine

AbstractMolecularly imprinted polymers are materials that can capture target analytes using molecular recognition. These polymers can efficiently detect trace target chemical species in biological samples, such as urine and blood, containing various matrix components. We aimed to analyze the interaction of molecularly imprinted polymers with the target molecule lidocaine, which is an amino amide pharmaceutical. Lidocaine‐templated molecularly imprinted polymers were synthesized by bulk polymerization. Gas chromatography‐mass spectrometry showed there was a strong interaction between lidocaine and the recognition site in the molecularly imprinted polymers. Non‐selective adsorption of lidocaine on the molecularly imprinted polymers was confirmed and characterized by Scatchard analysis. The washing solution was optimized for the separation of lidocaine from the structurally similar anesthetics procaine and tetracaine by taking advantage of differences in binding strength. The molecularly imprinted polymers could adsorb lidocaine in urine samples and were not affected by interfering compounds. Consequently, they could be applied to the analysis of biological materials. These results suggest that the molecularly imprinted polymers can be used for separation and detection with high selectivity and could be applied in forensic and clinical analysis.

Prevalence of three Mycoplasma sp. by multiplex PCR in cattle with and without respiratory disease in central Mexico.

This study aimed to identify Mycoplasma bovis, Myc. dispar, and Myc. bovirhinis, which are involved in bovine respiratory disease through a multiplex PCR as an alternative to culture's features that hamper Mycoplasma isolation. Nasal swabs were taken from 335 cattle with and without respiratory disease background (RDB) from dairy herds in the central region of Mexico. Each sample was divided in two; the first part was processed for the direct DNA extraction of the nasal swab and the second for Mycoplasma isolation, culture, and then the multiplex PCR was performed. In the nasal swabs, Myc. bovis was identified in 21.1%; Myc. dispar, in 11.8%; and Myc. bovirhinis, in 10.8% in cattle with RDB. Isolates were identified as Myc. bovis, 20.1%; Myc. dispar, 11.8%; and Myc. bovirhinis, 6.1%. There is a strong correlation between the presence of Mycoplasma identified by PCR and the clinical history of the disease (ρ < 0.0000). In animals without RDB, Myc. bovirhinis was the only species detected in 6.1% of the samples processed directly for multiplex PCR, and in 2% of the isolates. There is an excellent correlation (kappa 0.803) between the isolation and the 16S PCR and a high correlation (kappa 0.75) between the isolation and the multiplex PCR. Therefore, we conclude that the PCR multiplex test is highly sensitive and may be used for the diagnosis and surveillance of the three species in biological samples and mycoplasma isolates.

Labelfree mapping and profiling of altered lipid homeostasis in the rat hippocampus after traumatic stress: Role of oxidative homeostasis

Oxidative and lipid homeostasis are altered by stress and trauma and post-traumatic stress disorder (PTSD) is associated with alterations to lipid species in plasma. Stress-induced alterations to lipid and oxidative homeostasis may exacerbate PTSD pathology, but few preclinical investigations of stress-induced lipidomic changes in the brain exist. Currently available techniques for the quantification of lipid species in biological samples require tissue extraction and are limited in their ability to retrieve spatial information. Raman imaging can overcome this limitation through the quantification of lipid species in situ in minimally processed tissue slices. Here, we utilized a predator exposure and psychosocial stress (PE/PSS) model of traumatic stress to standardize Raman imaging of lipid species in the hippocampus using LC-MS based lipidomics and these data were confirmed with qRT-PCR measures of mRNA expression of relevant enzymes and transporters. Electron Paramagnetic Resonance Spectroscopy (EPR) was used to measure free radical production and an MDA assay to measure oxidized polyunsaturated fatty acids. We observed that PE/PSS is associated with increased cholesterol, altered lipid concentrations, increased free radical production and reduced oxidized polyunsaturated fats (PUFAs) in the hippocampus (HPC), indicating shifts in lipid and oxidative homeostasis in the HPC after traumatic stress.

A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins.

Protein ubiquitylation is an important posttranslational modification affecting a wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain (UBD) derived from an Orientia tsutsugamushi deubiquitylase (DUB). We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As proof of principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the budding yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for the E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin research with specific advantages over certain other methods, such as in efficiently detecting monoubiquitylation or ubiquitin linkages to noncanonical sites.

DIA Proteomics and Machine Learning for the Fast Identification of Bacterial Species in Biological Samples.

Identification of bacterial species in biological samples is essential in many applications. However, the standard methods usually use a time-consuming bacterial culture (24-48h) and sometimes lack in specificity. To overcome these limitations, we developed a new protocol, combining LC-MS/MS analysis in Data Independent Acquisition mode and machine learning algorithms, enabling the accurate identification of the bacterial species contaminating a sample in a few hours without bacterial culture. In this chapter, we describe the three steps of the protocol (spectral libraries generation, training step, identification step) to generate customized peptide signatures and use them for bacterial identification in biological samples through targeted proteomics analyses and prediction models.

Sensitive online speciation analysis of arsenic in biological samples by ambient mass spectrometry

Various arsenic species in biological samples were directly molecularly analyzed by ambient EESI-HG-MPT-MS with enhanced sensitivity.

Assay of selenol species in biological samples by the fluorescent probe Sel-green.

Selenium (Se) is an essential trace element for diverse cellular functions. The biological significance of Se is predominantly dependent on its incorporation into the selenocysteine (Sec) for synthesis of selenoproteins (SePs), such as thioredoxin reductase family enzymes and glutathione peroxidase family enzymes. In general, the hyperactivity of the selenol group in Sec confers the Sec residue critical for functions of SePs. The Sec is much less abundant than its sulfur analog cysteine (Cys), and it remains a high challenge to detect Sec, especially in complex biological samples. We recently reported a selective fluorescent probe Sel-green for selenols and summarized the principles for design of selenol (and thiophenol) probes. Sel-green discriminates selenols from other biological species, especially thiols, under physiological conditions, and has been applied to detect both endogenous and exogenous selenol species in live cells. In this chapter, we describe a protocol and guideline for the selective detection of Sec by applying the Sel-green. This protocol is also suitable for detection of other selenol species. This practical and convenient assay would assist scientists to better understand the pivotal roles of Sec as well as SePs.

Effect of protease XXIII on selenium species interconversion during their extraction from biological samples

Protease XIV is the most common used agent for selenium species extraction from biological samples. In this work, the interconversion of selenocystine during extraction with Tris−HCl buffer containing protease XIV was demonstrated. The 29 % of selenocystine was converted to species with retention time corresponding to Se-methyl-selenocysteine. In addition, selenomethionine, selenoethionine, and Se-methyl-selenocysteine were oxidized to their oxo-analogues when 0.1 % Triton X-100 or 0.1 % sodium dodecyl sulphate was used for improvement of extraction efficiency. The interconversion of these species was suppressed by using a protease XXIII, which application for the extraction of selenium species is described here for the first time. The method for speciation analysis of nine Se species (selenate, selenite, selenomethionine, selenocystine, Se-methyl-selenocysteine, selenoethionine, trimethylselenonium, methylselenocysteine oxide, and selenomethionine Se-oxide) using extraction with protease XXIII was developed and validated (limits of quantitation 0.21–0.70 μg/L Se, linearity up to 1000 μg/L Se, spike recovery 92.3–110.1 %). It has also been shown that the extraction efficiency of protease XXIII (average 53 %) is only slightly lower in comparison to protease XIV (average 65 %). The main Se species in biological samples, selenomethionine, selenocystine, and Se-methyl-selenocysteine, were stable in extract for three days at room temperature.

Justicia adhatoda L. mediated green synthesis of silver nanoparticles and assessment of their antioxidant, hydrogen peroxide sensing and optical properties

ABSTRACT In this report, a green synthesis practice has been adopted to fabricate silver nanoparticles (AgNPs) using Justicia adhatoda L. leaf extract (JALE). The phytosynthesized AgNPs present a characteristic SPR peak at 446 nm. Comparative FTIR analyses of JALE and AgNPs revealed the input of phytochemicals in the reduction, capping and stabilisation of J. adhatoda-mediated silver nanoparticles (JA@AgNPs). The crystalline nature and face-centred cubic lattice plane of JA@AgNPs were explained by XRD. SEM and TEM revealed a spherical shape having about ~19 nm mean NPs size. JA@AgNPs displayed significant inhibition potency against DPPH and ABTS free radicals and thus could be convenient in biomedical applications. Furthermore, the green-synthesised JA@AgNPs presented SPR-based colorimetric detection of H2O2 with exceptional sensing, which exhibited its usefulness in accessing reactive oxygen species in biological samples and waterbodies. The bandgap energy of JA@AgNPs was calculated as 3.3 eV, which revealed its semiconductor behaviour.

TAILOR-MS, a Python Package that Deciphers Complex Triacylglycerol Fatty Acyl Structures: Applications for Bovine Milk and Infant Formulas.

Liquid chromatography tandem mass spectrometry (LC/MS) and other mass spectrometric technologies have been widely applied for triacylglycerol profiling. One challenge for targeted identification of fatty acyl moieties that constitute triacylglycerol species in biological samples is the numerous combinations of 3 fatty acyl groups that can form a triacylglycerol molecule. Manual determination of triacylglycerol structures based on peak intensities and retention time can be highly inefficient and error-prone. To resolve this, we have developed TAILOR-MS, a Python (programming language) package that aims at assisting: (1) the generation of targeted LC/MS methods for triacylglycerol detection and (2) automating triacylglycerol structural determination and prediction. To assess the performance of TAILOR-MS, we conducted LC/MS triacylglycerol profiling of bovine milk and two infant formulas. Our results confirmed dissimilarities between bovine milk and infant formula triacylglycerol composition. Furthermore, we identified 247 triacylglycerol species and predicted the possible existence of another 317 in the bovine milk sample, representing one of the most comprehensive reports on the triacylglycerol composition of bovine milk thus far. Likewise, we presented here a complete infant formula triacylglycerol profile and reported >200 triacylglycerol species. TAILOR-MS dramatically shortened the time required for triacylglycerol structural identification from hours to seconds and performed decent structural predictions in the absence of some triacylglycerol constituent peaks. Taken together, TAILOR-MS is a valuable tool that can greatly save time and improve accuracy for targeted LC/MS triacylglycerol profiling.

Quantitative Analysis of Polyphosphoinositide, Bis(monoacylglycero)phosphate, and Phosphatidylglycerol Species by Shotgun Lipidomics After Methylation.

Phospholipids play important roles in biological process even at a very low level. For example, bis(monoacylglycerol)phosphate (BMP) is involved in the pathogenesis of lysosomal storage diseases, and polyphosphoinositides (PPI) play critical roles in cellular signaling and functions. Phosphatidylglycerol (PG), a structural isomer of BMP, mediates lipid-protein and lipid-lipid interactions, and inhibits platelet activating factor and phosphatidylcholine transferring. However, due to their low abundance, the analysis of these phospholipids from biological samples is technically challenging. Therefore, the cellular function and metabolism of these phospholipids are still elusive. This chapter overviews a novel method of shotgun lipidomics after methylation with trimethylsilyl-diazomethane (TMS-D) for accurate and comprehensive analysis of these phospholipid species in biological samples. Firstly, a modified Bligh and Dyer procedure is performed to extract tissue lipids for PPI analysis, whereas modified methyl-tert-butylether (MTBE) extraction and modified Folch extraction methods are described to extract tissue lipids for PPI analysis. Secondly, TMS-D methylation is performed to derivatize PG/BMP and PPI, respectively. Then, we described the shotgun lipidomics strategies that can be used as cost-effective and relatively high-throughput methods to determine BMP, PG, and PPI species and isomers with different phosphate position(s) and fatty acyl chains. The described method of shotgun lipidomics after methylation achieves feasible and reliable quantitative analysis of low-abundance lipid classes. The application of this novel method should enable us to reveal the metabolism and functions of these phospholipids in healthy and disease states.

Aptamer-based nanofilter interface for small-biomarker detection with potentiometric biosensor

In this paper, we report the concept of an aptamer (Ap)-based nanofilter interface for the suppression of nonspecific signals generated by interfering species in biological samples. As a model of the target small biomolecule, l-3,4-dihydroxyphenylalanine (l-DOPA) is used to demonstrate its electrical discrimination from dopamine (DA) despite their similar chemical structures using a DA–Ap nanofilter-coated Au gate field-effect transistor (FET). A Au gate electrode is employed to assess the electrical response of the FET to l-DOPA on the basis of oxidative reactions. In particular, the proposed nanofilter interface is based on the DA–Ap layer immobilized at the aryl-diazonium-based anchor monolayer, which is electrochemically grafted on the Au gate electrode via diazonium chemistry. As a result, the electrical signal from l-DOPA is clearly discriminated from that from DA at a concentration of 1 μM or higher using the DA–Ap nanofilter-coated Au gate FET. That is, DA is trapped by the DA–Ap nanofilter through the formation of complexes, the molecular charges of which are shielded by counter ions that are not in contact with the Au gate surface, whereas l-DOPA reaches and electrochemically reacts with the Au gate surface, generating an electrical signal regardless of the nanofilter. The surface properties of the DA–Ap nanofilter modified on the Au gate electrode are investigated by cyclic voltammetry, chronocoulometry, atomic force microscopy, and X-ray photoelectron spectroscopy. A platform based on the FET biosensor with the Ap-based nanofilter interface contributes to the electrical monitoring of small biomarkers while suppressing the nonspecific signals of interfering species in biological samples.

Whole Mount In Situ Hybridization of Mid-Gestation Mouse Embryos.

In situ hybridization is a powerful technique that allows the visualization of specific RNA species in biological samples in exquisite detail. It has been particularly well explored in the field of developmental genetics. The spatial and temporal patterns of RNA expression provide us with critical information on likely gene function during embryonic development, and often inform the decision on whether to attempt further gene manipulation approaches. Furthermore, once a mouse strain with altered gene function has been created, in situ hybridization is a critical tool for revealing how the development of embryos with the mutation differs from that of wild-type embryos, and thus infer the function of the altered gene. Here, a well-tested protocol used to visualize RNA expression in whole-mount mid-gestation mouse embryos ranging from 8.5 to 14.5 days post-coitum (dpc) is described. © 2020 Wiley Periodicals LLC. Basic Protocol 1: RNA probe synthesis Alternate Protocol: Preparation of DNA template by PCR Basic Protocol 2: Embryo dissection Basic Protocol 3: Whole mount in situ hybridization Support Protocol: Generation of embryo powder.

Nucleic Acid Quantitation with Log-Linear Response Hybridization Probe Sets.

Concentrations of different nucleic acid species in biological samples span many orders of magnitude. A real-time polymerase chain reaction maps the concentration of a target nucleic acid sequence log-linearly into cycle threshold to enable quantitation with a wide dynamic range but suffers from enzymatic biases. Here, we present a general design for constructing hybridization probe sets with highly log-linear response curves to enable accurate enzyme-free quantitation across large ranges (more than 6 logs) of target DNA concentrations. The sensitivity of each component probe is accurately adjusted via formulation stoichiometry to reduce the standard error of target quantitation down to 7%. As a proof of concept, we show multiplexed quantitation of three microRNA species in total RNA of the human brain and liver.

Real-time polymerase chain reaction based algorithm for differential diagnosis of Kinetoplastidean species of zoonotic relevance

Kinetoplastids are a group of flagellated protozoa that infect a vast repertoire of mammals and insect vectors. From a zoonotic point of view, domestic animals are critical reservoirs for transmission of Kinetoplastidean parasites. Due to their proximity to humans, they assume substantial epidemiological importance in the context of these zoonoses and consequently in public health. Their reliable identification is relevant to understand their eco-epidemiological involvement in transmission cycles. This work aimed to develop an algorithm based on sequential Real-Time PCR (qPCR) assays targeted to different loci (24S alpha rDNA, ITS1 and Hsp70) allowing distinction among Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma evansi and Leishmania species in biological samples collected from mammalian reservoirs and triatomine vectors. The algorithm includes a first qPCR test targeted to endogenous genes conserved within mammals and within triatomine vectors as internal controls of DNA sample integrity and/or qPCR inhibition. This algorithm was evaluated in biological samples from domestic cattle (N = 14), dogs (N = 19) and triatomines (N = 19). Analytical sensitivity of 24S alpha rDNA for detection of T. rangeli was 10 fg of DNA, with a linear range between 10 fg and 10 ng. For T. cruzi it varied depending on the Discrete typing unit. The ITS1 qPCR showed an analytical sensitivity of 100 pg/reaction and 100 fg/reaction of Leishmania spp. and T. evansi DNAs. In mammal field samples, four T. cruzi 24S alpha rDNA sequences and fourteen ITS1 amplicons specific for T. evansi were detected. qPCR-HRM analysis directed to the Hsp70 gene diagnosed two dogs with Leishmania infantum infection. Among 19 triatomine field samples, T. cruzi was detected in five; T. rangeli in eight and one specimen showed a mixed infection. This diagnostic algorithm can provide more accurate records of kinetoplastidean infection burden in vectors and reservoirs, relevant to update current eco-epidemiological maps in co-endemic regions.