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Viral capsids are dynamic protein assemblies surrounding viral genomes. Despite the high-resolution structures determined by X-ray crystallography and cryo-electron microscopy, their in-solution structure and dynamics can be probed by hydrogen exchange. We report here using hydrogen exchange combined with protein enzymatic fragmentation and mass spectrometry to determine the capsid structure and dynamics of a human rhinovirus, HRV14. Capsid proteins (VP1-4) were labeled with deuterium by incubating intact virus in D2O buffer at neutral pH. The labeled proteins were digested by immobilized pepsin to give peptides analyzed by capillary reverse-phase HPLC coupled with nano-electrospray mass spectrometry. Deuterium levels incorporated at amide linkages in peptic fragments were measured for different exchange times from 12 sec to 30 h to assess the amide hydrogen exchange rates along each of the four protein backbones. Exchange results generally agree with the crystal structure of VP1-4,with extended, flexible terminal and surface-loop regions in fast exchange and folded helical and sheet structures in slow exchange. In addition, three α-helices, one from each of VP1-3, exhibited very slow exchange, indicating high stability of the protomeric interface. The β-strands at VP3 N terminus also had very slow exchange, suggesting stable pentamer contacts. It was noted, however, that the interface around the fivefold axis had fast and intermediate exchange, indicating relatively more flexibility. Even faster exchange rates were found in the N terminus of VP1 and most segments of VP4, suggesting high flexibilities, which may correspond to their potential roles in virus uncoating. [ABSTRACT FROM AUTHOR]

The largest subunit of RNA polymerase (Pol) II harbors an evolutionarily conserved C‐terminal domain (CTD), composed of heptapeptide repeats, central to the transcriptional process. Here, we analyze the transcriptional phenotypes of a CTD‐Δ5 mutant that carries a large CTD truncation in human cells. Our data show that this mutant can transcribe genes in living cells but displays a pervasive phenotype with impaired termination, similar to but more severe than previously characterized mutations of CTD tyrosine residues. The CTD‐Δ5 mutant does not interact with the Mediator and Integrator complexes involved in the activation of transcription and processing of RNAs. Examination of long‐distance interactions and CTCF‐binding patterns in CTD‐Δ5 mutant cells reveals no changes in TAD domains or borders. Our data demonstrate that the CTD is largely dispensable for the act of transcription in living cells. We propose a model in which CTD‐depleted Pol II has a lower entry rate onto DNA but becomes pervasive once engaged in transcription, resulting in a defect in termination. Synopsis: RNA polymerase II is essential for gene expression. This study shows that its Carboxy‐Terminal Domain plays a previously unappreciated role in the control of transcription termination and is dispensable for transcription in human cells. RNA polymerase II Carboxy‐Terminal Domain (CTD) is dispensable for transcription.Transcription without CTD displays a termination defect at both 5′ and 3′ ends of the genes.RNA polymerase II without CTD interacts less with Mediator and Integrator complexes. [ABSTRACT FROM AUTHOR]

Surfactants are surface‐active agents widely used in numerous applications in our daily lives as personal care products, domestic, and industrial detergents. To determine complex mixtures of surfactants and their degradation products, unselective and rather insensitive methods, based on colorimetric and complexometric analyses are no longer employable. Analytical methodologies able to determine low concentration levels of surfactants and closely related compounds in complex matrices are required. The recent introduction of robust, sensitive, and selective mass spectrometry (MS) techniques has led to the rapid expansion of the surfactant research field including complex mixtures of isomers, oligomers, and homologues of surfactants as well as their chemically and biodegradation products at trace levels. In this review, emphasis is given to the state‐of‐the‐art MS‐based analysis of surfactants and their degradation products with an overview of the current research landscape from traditional methods involving hyphenate techniques (gas chromatography‐MS and liquid chromatography‐MS) to the most innovative approaches, based on high‐resolution MS. Finally, we outline a detailed explanation on the utilization of MS for mechanistic purposes, such as the study of micelle formation in different solvents. [ABSTRACT FROM AUTHOR]

We have partially purified the U2 snRNP of Saccharomyces cerevisiae. Identification of some proteins consistently found in the purified fractions by nano-electrospray mass spectrometry indicated the presence of a novel splicing factor named Rse1p. The RSE1 gene is essential and codes for a 148.2 kDa protein. We demonstrated that Rse1p associates specifically with U2 snRNA at low salt concentrations. In addition, we showed that Rse1p is a component of the pre-spliceosome. Depletion of Rse1p and analysis of a conditional mutant indicated that Rse1p was required for efficient splicing in vivo. In vitro Rse1p is required for the formation of pre-spliceosomes. Database searches revealed that Rse1p is conserved in humans and that it belongs to a large protein family that includes polyadenylation factors and DNA repair proteins. The characteristics of Rse1p suggest that its human homologue could be a subunit of the SF3 splicing factor. [ABSTRACT FROM AUTHOR]

Due to their involvement in numerous biochemical pathways, neuropeptides have been the focus of many recent research studies. Unfortunately, classic analytical methods, such as western blots and enzyme‐linked immunosorbent assays, are extremely limited in terms of global investigations, leading researchers to search for more advanced techniques capable of probing the entire neuropeptidome of an organism. With recent technological advances, mass spectrometry (MS) has provided methodology to gain global knowledge of a neuropeptidome on a spatial, temporal, and quantitative level. This review will cover key considerations for the analysis of neuropeptides by MS, including sample preparation strategies, instrumental advances for identification, structural characterization, and imaging; insightful functional studies; and newly developed absolute and relative quantitation strategies. While many discoveries have been made with MS, the methodology is still in its infancy. Many of the current challenges and areas that need development will also be highlighted in this review. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common neurodegenerative disorders. Pathologically, AD and PD are characterized by the accumulation of misfolded proteins. Hence, they are also called as proteinopathy diseases. Gender is considered as one of the risk factors in both diseases. Estrogens are widely accepted to be neuroprotective in several neurodegenerative disorders. Estrogens can be produced in the central nervous system, where they are called as neurosteroids. Estrogens mediate their neuroprotective action mainly through their actions on estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). However, ERα is mainly involved in the growth and development of the primary and secondary sexual organs in females. Hence, the activation of ERα is associated with undesired side effects such as gynecomastia and increase in the risk of breast cancer, thromboembolism, and feminization. Therefore, selective activation of ERβ is often considered to be safer. In this review, we explore the role of ERβ in regulating the expression and functions of AD- and PD-associated genes. Additionally, we discuss the association of these genes with the amyloid-beta peptide (Aβ) and α-synuclein mediated toxicity. Ultimately, we established a correlation between the importance of ERβ activation and the process underlying ERβ's neuroprotective mechanisms in AD and PD., (© 2024 John Wiley & Sons Ltd.)

Many analytical challenges in biomedicine arise from the generally high heterogeneity and complexity of glycan‐ and glycoconjugate‐containing samples, which are often only available in minute amounts. Therefore, highly sensitive workflows and detection methods are required. In this review mass spectrometric workflows and detection methods are evaluated for glycans and glycoproteins. Furthermore, glycomic methodologies and innovations that are tailored for enzymatic treatments, chemical derivatization, purification, separation, and detection at high sensitivity are highlighted. The discussion is focused on the analysis of mammalian N‐linked and GalNAc‐type O‐linked glycans. [ABSTRACT FROM AUTHOR]

A pituitary adenoma (PA) is a common intracranial neoplasm, and is a complex, chronic, and whole‐body disease with multicausing factors, multiprocesses, and multiconsequences. It is very difficult to clarify molecular mechanism and treat PAs from the single‐factor strategy model. The rapid development of multiomics and systems biology changed the paradigms from a traditional single‐factor strategy to a multiparameter systematic strategy for effective management of PAs. A series of molecular alterations at the genome, transcriptome, proteome, peptidome, metabolome, and radiome levels are involved in pituitary tumorigenesis, and mutually associate into a complex molecular network system. Also, the center of multiomics is moving from structural genomics to phenomics, including proteomics and metabolomics in the medical sciences. Mass spectrometry (MS) has been extensively used in phenomics studies of human PAs to clarify molecular mechanisms, and to discover biomarkers and therapeutic targets/drugs. MS‐based proteomics and proteoform studies play central roles in the multiomics strategy of PAs. This article reviews the status of multiomics, multiomics‐based molecular pathway networks, molecular pathway network‐based pattern biomarkers and therapeutic targets/drugs, and future perspectives for personalized, predeictive, and preventive (3P) medicine in PAs. [ABSTRACT FROM AUTHOR]

Neospora caninum is an obligate intracellular protozoan parasite of the phylum Alveolata (subphylum Apicomplexa) which has not been studied extensively in a biochemical context. N. caninum is a primary cause of reproductive disorders causing mummification and abortion not only in cattle but also in other small ruminant species resulting in a substantial economic impact on the livestock industry. In canids, which are the final hosts of N. caninum, clinical disease includes neuromuscular symptoms, ataxia, and ascending paralysis. Fatal outcomes of neosporosis have also been reported depending on the host species, age and immune status, however, its zoonotic potential is still uncertain. Therefore, N. caninum should be thoroughly investigated. Matrix‐assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) and MS imaging (MSI) were used, combined with high‐performance liquid chromatography (HPLC) to investigate these intracellular parasites. The aim of this study was to identify molecular biomarkers for N. caninum tachyzoite‐infected host cells and to further clarify their functions. By atmospheric‐pressure scanning microprobe MALDI MS(I), sections of N. caninum‐infected and non‐infected host cell pellets were examined in order to determine potential markers. In vivo, N. caninum infects different types of nucleated cells, such as endothelial cells which represent a highly immunoreactive cell type. Therefore, primary bovine umbilical vein endothelial cells were here used as a suitable infection system. For comparison, the permanent MARC‐145 cell line was used as an additional, simplified in vitro cell culture model. HPLC‐tandem MS (HPLC‐MS/MS) experiments combined with database search were employed for structural verification of markers. The statistically relevant biomarkers found by MS and identified by HPLC‐MS/MS measurements were partly also found in infected monolayers. Marker signals were imaged in cell layers of N. caninum‐infected and non‐infected host cells at 5 µm lateral resolution. [ABSTRACT FROM AUTHOR]

The liquid extraction surface analysis technique is a new high‐throughput instrument for ambient mass spectrometry. The benefits of the liquid extraction surface analysis‐mass spectrometry approach are the high throughput screening of samples and the absence of sample preparation. liquid extraction surface analysis‐mass spectrometry also consumes less solvent for extraction, making it more environmentally friendly and there is no substrate restriction. It utilizes advanced instrumentation like the use of robotic pipettes, nanoelectrospray systems, electronspray ionization chips which makes it highly efficient. In recent years, liquid extraction surface analysis‐mass spectrometry has seen widespread use in a variety of analytical fields including drug metabolite analysis, mapping drug distribution in tissues, protein and lipid characterization, etc. In this review, we have summarized the basic working principles of the liquid extraction surface analysis‐mass spectrometry approach in detail along with a detailed description of the recently reported applications in the analysis of proteins, lipids, drugs and foods. The investigated analytes along with detection methodologies and significant outcomes of various research reports have been presented with the help of tables. This tool has also been utilized in clinical investigations of biological fluids, fingerprint analysis and authentication of agarwood. [ABSTRACT FROM AUTHOR]

The bacterial heterodimeric ATP‐binding cassette (ABC) multidrug exporter PatAB has a critical role in conferring antibiotic resistance in multidrug‐resistant infections by Streptococcus pneumoniae. As with other heterodimeric ABC exporters, PatAB contains two transmembrane domains that form a drug translocation pathway for efflux and two nucleotide‐binding domains that bind ATP, one of which is hydrolysed during transport. The structural and functional elements in heterodimeric ABC multidrug exporters that determine interactions with drugs and couple drug binding to nucleotide hydrolysis are not fully understood. Here, we used mass spectrometry techniques to determine the subunit stoichiometry in PatAB in our lactococcal expression system and investigate locations of drug binding using the fluorescent drug‐mimetic azido‐ethidium. Surprisingly, our analyses of azido‐ethidium‐labelled PatAB peptides point to ethidium binding in the PatA nucleotide‐binding domain, with the azido moiety crosslinked to residue Q521 in the H‐like loop of the degenerate nucleotide‐binding site. Investigation into this compound and residue's role in nucleotide hydrolysis pointed to a reduction in the activity for a Q521A mutant and ethidium‐dependent inhibition in both mutant and wild type. Most transported drugs did not stimulate or inhibit nucleotide hydrolysis of PatAB in detergent solution or lipidic nanodiscs. However, further examples for ethidium‐like inhibition were found with propidium, novobiocin and coumermycin A1, which all inhibit nucleotide hydrolysis by a non‐competitive mechanism. These data cast light on potential mechanisms by which drugs can regulate nucleotide hydrolysis by PatAB, which might involve a novel drug binding site near the nucleotide‐binding domains. [ABSTRACT FROM AUTHOR]

Boldenone is an anabolic‐androgenic steroid that is prohibited in equine sports. However, in certain situations, it is endogenous or is believed to be formed by microbes in urine, and therefore, an approach for the differentiation is required. Following the identification of Δ1‐progesterone and 20(S)‐hydroxy‐Δ1‐progesterone as potential biomarkers of microbial activity, the presence of six steroids was investigated in the postrace urine of castrated male horses (geldings, n = 158). In line with endogenous findings from several other species when ultrasensitive methods are employed, boldenone was detected at low concentrations in all urine samples (27.0–1330 pg/ml). Furthermore, testosterone and androstenedione were detected in 157 samples (≤12,400 and 944 pg/ml, respectively), boldienone in two samples (≤22.0 pg/ml) and 20(S)‐hydroxy‐Δ1‐progesterone in 20 samples (≤66.0 pg/ml). Δ1‐Progesterone was not detected in any population samples analysed on arrival at the laboratory. The ex vivo transformation of boldienone, boldenone, androstenedione, Δ1‐progesterone and 20(S)‐hydroxy‐Δ1‐progesterone was induced following the storage of urine samples at room temperature for 7 days but not after refrigeration. Because the administration of inappropriately stored feed sources also resulted in an increase in 20(S)‐hydroxy‐Δ1‐progesterone concentrations, a biomarker approach to distinguish steroid administrations was proposed. In situations where the presence of boldenone would exceed a proposed action limit, the presence of Δ1‐progesterone and 20(S)‐hydroxy‐Δ1‐progesterone would be investigated. If either Δ1‐progesterone or 20(S)‐hydroxy‐Δ1‐progesterone would exceed 50 and 100 pg/ml, respectively, for instance, then this would indicate ex vivo transformation or consumption of altered feed rather than steroid administration. [ABSTRACT FROM AUTHOR]

Rationale: Saliva has been widely accepted as a more convenient alternative to serum or plasma in the field of clinical diagnosis. However, the detection of trace components in saliva has been a bottleneck problem. The aim of this work was to develop a highly sensitive and reliable method for simultaneously determining the trace steroid hormones including some with poor ionization efficiency in human saliva by liquid chromatography/tandem mass spectrometry (LC/MS). Methods: Saliva was deproteinated by acetonitrile containing mixed isotope internal standards and extracted with methyl tert-butyl ether. The extraction solution was dried under a stream of nitrogen and the residue was derivatized using 50 mM Oethylhydroxylamine hydrochloride in 80% methanol/water solution (v/v). The processed sample was determined by LC/MS in multiple reaction monitoring (MRM) mode. Results: The method was successfully established for the simultaneous quantification of seven steroid hormones in human saliva and showed excellent specificity and sensitivity. The limits of quantification (LOQs) of all steroid hormones were below 5 pg/mL, in particular, the LOQ of progesterone was as low as 0.15 pg/mL. The linear correlation coefficients (r) were greater than 0.9990 in the range of 2-200 pg/mL for T, DHEA, A4, P4, P5, and 17OHP4 and in the range of 5-500 pg/mL for 17OHP5. The intra-day and inter-day variability ranged from 1.86% to 7.83% and 1.95% to 10.4%, respectively. The recovery of the method ranged from 86.9% to 111.1% for all steroid hormones using three spiked concentrations. Conclusions: A novel LC/MS/MS method was developed for the simultaneous quantification of seven kinds of trace steroid hormones in human saliva. The results of the methodological study showed that the method exhibited excellent sensitivity and reliability for the evaluation of free steroid hormones in the human body. It is believed that this method could provide useful information of steroid hormone metabolism for auxiliary diagnosis of some endocrine disorders. [ABSTRACT FROM AUTHOR]

Excitatory synapses of principal hippocampal neurons are frequently located on dendritic spines. The dynamic strengthening or weakening of individual inputs results in structural and molecular diversity of dendritic spines. Active spines with large calcium ion (Ca2+) transients are frequently invaded by a single protrusion from the endoplasmic reticulum (ER), which is dynamically transported into spines via the actin‐based motor myosin V. An increase in synaptic strength correlates with stable anchoring of the ER, followed by the formation of an organelle referred to as the spine apparatus. Here, we show that myosin V binds the Ca2+ sensor caldendrin, a brain‐specific homolog of the well‐known myosin V interactor calmodulin. While calmodulin is an essential activator of myosin V motor function, we found that caldendrin acts as an inhibitor of processive myosin V movement. In mouse and rat hippocampal neurons, caldendrin regulates spine apparatus localization to a subset of dendritic spines through a myosin V‐dependent pathway. We propose that caldendrin transforms myosin into a stationary F‐actin tether that enables the localization of ER tubules and formation of the spine apparatus in dendritic spines. SYNOPSIS: Activity‐dependent formation of the spine apparatus organelle, a marker of large and stable spines, involves synaptic anchoring of an ER protrusion. Here, this process is shown to depend on caldendrin, a brain‐specific calmodulin homolog modulating myosin V function. Caldendrin binds myosin V in a calcium‐dependent manner via a region containing myosin's first calmodulin binding site, IQ1.Binding of calmodulin or caldendrin to IQ1‐containing region is mutually exclusive.While calmodulin promotes myosin V motility on actin filaments, caldendrin impairs processive movement, but does not affect myosin V ability to bind to F‐actin.In rodent hippocampal neurons, caldendrin increases dwell‐time of spine‐inserted ER tubules transported by myosin V, and facilitates the formation of the spine apparatus organelle. [ABSTRACT FROM AUTHOR]

Close homolog of L1 (CHL1) is a cell adhesion molecule of the immunoglobulin superfamily. It promotes neuritogenesis and survival of neurons in vitro. In vivo, CHL1 promotes nervous system development, regeneration after trauma, and synaptic function and plasticity. We identified programmed cell death 6 (PDCD6) as a novel binding partner of the CHL1 intracellular domain (CHL1‐ICD). Co‐immunoprecipitation, pull‐down assay with CHL1‐ICD, and proximity ligation in cerebellum and pons of 3‐day‐old and 6‐month‐old mice, as well as in cultured cerebellar granule neurons and cortical astrocytes indicate an association between PDCD6 and CHL1. The Ca2+‐chelator BAPTA‐AM inhibited the association between CHL1 and PDCD6. The treatment of cerebellar granule neurons with a cell‐penetrating peptide comprising the cell surface proximal 30 N‐terminal amino acids of CHL1‐ICD inhibited the association between CHL1 and PDCD6 and PDCD6‐ and CHL1‐triggered neuronal survival. These results suggest that PDCD6 contributes to CHL1 functions in the nervous system. [ABSTRACT FROM AUTHOR]

Intact phase II steroid metabolites have poor product ion mass spectra under collision‐induced dissociation (CID) conditions. Therefore, we present herein the liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/(MS)) behavior of intact phase II metabolites of oxosteroids after derivatization. Based on the fact that Girard's reagent T (GRT), as derivatization reagent, was both convenient and efficient in terms of the enhancement in the ionization efficiency and the production of diagnostic product ions related to the steroid moiety, the latter was preferably selected between methoxamine and hydroxylamine upon the model compounds of androsterone glucuronide and androsterone sulfate. Sixteen different glucuronides and 29 sulfate conjugated metabolites of anabolic androgenic steroids (AASs), available either as pure reference materials or synthesized/extracted from administration studies, were derivatized with GRT, and their product ion spectra are presented. Product ion spectra include in all cases high number of product ions that in some cases are characteristic for certain structures of the steroid backbone. More specifically, preliminary results have shown major differences in fragmentation pattern for 17α/17β‐isomers of the sulfate conjugates, but limited differentiation for 17α/17β‐isomers of glucuronide conjugates and for 3α/3β‐ and 5α/5β‐stereoisomers of both sulfate and glucuronide conjugates. Further to the suggestion of the current work, application on mesterolone administration studies confirmed—according to the World Anti‐Doping Agency (WADA) TD2015IDCR—the presence of seven intact phase II metabolites, one glucuronide and six sulfates with use of LC–ESI–MS/(MS). [ABSTRACT FROM AUTHOR]

The present contribution discusses recent advances in ultra‐high‐pressure liquid chromatography (UHPLC) and multi‐dimensional liquid chromatography (MDLC) technology. First, new developments in UHPLC column technology and system design are highlighted. The latter includes a description of a novel injector concept enabling method speed‐up, emerging detectors, and instrument diagnostics approaches. Next, online MDLC workflows are reviewed and advances in modulation technology are highlighted. Finally, key applications published in 2020 are reviewed. [ABSTRACT FROM AUTHOR]

Rationale: A calibration solution for mass spectrometry needs to cover the range of interest with intense and sufficiently narrowly spaced peaks. Limited options fulfilling this may lead to compromises between performance and ease of use. SpheriCal®‐ESI was designed to combine high calibration performance for electrospray ionization (ESI) mass spectrometric analysis of peptides in positive mode with quick and easy use. Methods: The developed calibration solution was tested using three mass spectrometers: two ion traps and one tandem quadrupole. The m/z errors of SpheriCal®‐ESI itself and of a tryptic digest of cytochrome C were measured after calibration. The results were compared with those achieved with ESI Tuning Mix. The memory effects of the dendrimers, and contamination from Na+ in the calibration solution, were evaluated. Results: SpheriCal®‐ESI showed good shelf life as powder and was quickly reconstituted for use. Achieving intense and stable signals was straightforward. The accuracies and precisions were as expected for the instruments. SpheriCal®‐ESI was more precise and at least as accurate as ESI Tuning Mix. The memory effects and Na+ contamination were found to be negligible in typical peptide solvents. In addition, the dendrimers showed predictable dissociations with product ions common to collision‐induced dissociation in both ion trap and tandem quadrupole mass spectrometers. Conclusions: SpheriCal®‐ESI provided easily accessible calibration by showing intense signals at low infusion rates and at source settings equal or similar to those used in peptide analysis. Nine calibration points in the range of interest gave precise and accurate results. Memory effects and contamination were negligible even without rinsing. [ABSTRACT FROM AUTHOR]

β‐propeller proteins are common in nature, where they are observed to adopt 4‐ to 10‐fold internal rotational pseudo‐symmetry. This size diversity can be explained by the evolutionary process of gene duplication and fusion. In this study, we investigated a distorted β‐propeller protein, an apparent intermediate between two symmetries. From this template, we created a perfectly symmetric 9‐bladed β‐propeller named Cake, using computational design and ancestral sequence reconstruction. The designed repeat sequence was found to be capable of generating both 8‐fold and 9‐fold propellers which are highly stable. Cake variants with 2–10 identical copies of the repeat sequence were characterised by X‐ray crystallography and in solution. They were found to be highly stable, and to self‐assemble into 8‐ or 9‐fold symmetrical propellers. These findings show that the β‐propeller fold allows sufficient structural plasticity to permit a given blade to assemble different forms, a transition from even to odd changes in blade number, and provide a potential explanation for the wide diversity of repeat numbers observed in natural propeller proteins. Database: Structural data are available in Protein Data Bank database under the accession numbers 6TJB, 6TJC, 6TJD, 6TJE, 6TJF, 6TJG, 6TJH and 6TJI. [ABSTRACT FROM AUTHOR]

Introduction: Agarwood is a highly valuable fragrant resinous wood which is widely used as traditional Chinese medicines, perfumes, incense and decorations. Due to its high economic value and excessive demand, this leads to a rising price and proliferation of fake commodities. Thus, strict authenticity identification and quality evaluation of agarwood are of great significance. Objective: To establish a simple, rapid and non‐destructive technique for identifying the authenticity of agarwood. Methods: Liquid extraction surface analysis mass spectrometry (LESA‐MS) was firstly proposed to identify the authenticity of 62 agarwood samples without sample preparation. In addition, multivariate statistical models and thin‐layer chromatography (TLC) method were used to analyse and verify the results of LESA‐MS. Results: Representative compounds of agarwood were detected by LESA‐MS. A characteristic 2‐(2‐phenylethyl)chromone compound (m/z 319.1) was treated as a key chemical marker to identify agarwood and its counterfeits rapidly. Several other chromones ions were identified and used as additional evidence for authentic samples. A total of 62 samples were visually discriminated as two groups by principal component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS‐DA), and the specific characteristic marker was highlighted. Moreover, the qualitative results of the conventional TLC method were in agreement with the LESA‐MS approach. Conclusion: The proposed LESA‐MS method was successfully applied in the direct qualitative analysis of agarwood from different sources. This study indicated great feasibility and practicality of LESA‐MS in the rapid identification of agarwood, and provided a non‐destructive and meaningful preliminary screening tool for the agarwood industry. Agarwood is a highly valuable fragrant resinous wood and has been listed as endangered species in recent years. A new LESA‐MS method was established to rapidly identify authenticity of agarwood without sample preparation, some 2‐(2‐phenylethyl)chromone derivatives were detected and the characteristic 5,6,7,8‐tetrahydro‐2‐(2‐phenylethyl)chromone (m/z 319.1) was the most important compound to differentiate authentic agarwood from counterfeits. The developed LESA‐MS approach was demonstrated to be a simple, automated and non‐destructive technique for direct qualitative determination of agarwood. [ABSTRACT FROM AUTHOR]

N‐glycans from glycoproteins are complex, branched structures whose structural determination presents many analytical problems. Mass spectrometry, usually conducted in positive ion mode, often requires extensive sample manipulation, usually by derivatization such as permethylation, to provide the necessary structure‐revealing fragment ions. The newer but, so far, lesser used negative ion techniques, on the contrary, provide a wealth of structural information not present in positive ion spectra that greatly simplify the analysis of these compounds and can usually be conducted without the need for derivatization. This review describes the use of negative ion mass spectrometry for the structural analysis of N‐linked glycans and emphasises the many advantages that can be gained by this mode of operation. Biosynthesis and structures of the compounds are described followed by methods for release of the glycans from the protein. Methods for ionization are discussed with emphasis on matrix‐assisted laser desorption/ionization (MALDI) and methods for producing negative ions from neutral compounds. Acidic glycans naturally give deprotonated species under most ionization conditions. Fragmentation of negative ions is discussed next with particular reference to those ions that are diagnostic for specific features such as the branching topology of the glycans and substitution positions of moieties such as fucose and sulfate, features that are often difficult to identify easily by conventional techniques such as positive ion fragmentation and exoglycosidase digestions. The advantages of negative over positive ions for this structural work are emphasised with an example of a series of glycans where all other methods failed to produce a structure. Fragmentation of derivatized glycans is discussed next, both with respect to derivatives at the reducing terminus of the molecules, and to methods for neutralization of the acidic groups on sialic acids to both stabilize them for MALDI analysis and to produce the diagnostic fragments seen with the neutral glycans. The use of ion mobility, combined with conventional mass spectrometry is described with emphasis on its use to extract clean glycan spectra both before and after fragmentation, to separate isomers and its use to extract additional information from separated fragment ions. A section on applications follows with examples of the identification of novel structures from lower organisms and tables listing the use of negative ions for structural identification of specific glycoproteins, glycans from viruses and uses in the biopharmaceutical industry and in medicine. The review concludes with a summary of the advantages and disadvantages of the technique. © 2020 John Wiley & Sons Ltd. Mass Spec Rev [ABSTRACT FROM AUTHOR]

Acyl‐quinic acids (chlorogenic acids) are produced by many plants, including fruits, vegetables, and herbal remedies, with coffee and maté particularly rich dietary sources. Epidemiological and intervention studies suggest that they can reduce the risk of developing type 2 diabetes and cardiovascular disease. This review addresses their metabolic handling after oral consumption to provide a mechanistic basis to explain their possible effects on health. Intact acyl‐quinic acids are absorbed only to a small extent in the small intestine, but the cinnamic acids are efficiently absorbed after hydrolysis by either digestive or microbial enzymes in the colon. Metabolism results in phenolic conjugates in the blood and urine, but varying dependent on the acyl‐quinic acid, and subject to significant interperson variability. The balance between hydrogenation and complete β‐oxidation of the cinnamic acids, both by liver and gut microbiota, determines the profile of metabolites. Pharmacokinetic data suggest that some metabolites are bound to human serum albumin and/or sequestered in tissues, and some exhibit biological activity in vitro, consistent with proposed protective action in vivo. Significant gaps in the literature include lack of plasma and urinary data for free‐living individuals, and pharmacokinetic data for groups who consume coffee or maté at regular short intervals. Data are required for cis isomers. There is a critical need for precise urinary biomarkers of consumption of acyl‐quinic acids, accounting for variability in individual metabolism and in beverage composition, thus facilitating better translation of urinary metabolite measurements into accurate coffee consumption data to improve the outcomes of future epidemiological and intervention studies. [ABSTRACT FROM AUTHOR]

Although the multiplicative and growth‐arrested states play key roles in Leishmania development, the regulators of these transitions are largely unknown. In an attempt to gain a better understanding of these processes, we characterised one member of a family of protein kinases with dual specificity, LinDYRK1, which acts as a stasis regulator in other organisms. LinDYRK1 overexpressing parasites displayed a decrease in proliferation and in cell cycle re‐entry of arrested cells. Parasites lacking LinDYRK1 displayed distinct fitness phenotypes in logarithmic and stationary growth phases. In logarithmic growth phase, LinDYRK1−/− parasites proliferated better than control lines, supporting a role of this kinase in stasis, while in stationary growth phase, LinDYRK1−/− parasites had important defects as they rounded up, accumulated vacuoles and lipid bodies and displayed subtle but consistent differences in lipid composition. Moreover, they expressed less metacyclic‐enriched transcripts, displayed increased sensitivity to complement lysis and a significant reduction in survival within peritoneal macrophages. The distinct LinDYRK1−/− growth phase phenotypes were mirrored by the distinct LinDYRK1 localisations in logarithmic (mainly in flagellar pocket area and endosomes) and late stationary phase (mitochondrion). Overall, this work provides first evidence for the role of a DYRK family member in sustaining promastigote stationary phase phenotype and infectivity. [ABSTRACT FROM AUTHOR]

Boldenone (1‐dehydrotestosterone) is an exogenous anabolic‐androgenic steroid (AAS) but is also known to be endogenous in the entire male horse and potentially formed by microbes in voided urine, the gastrointestinal tract, or feed resulting in its detection in urine samples. In this study, equine fecal and urine samples were incubated in the presence of selected stable isotope labeled AAS precursors to investigate whether microbial activity could result in 1‐dehydrogenation, in particular the formation of boldenone. Fecal matter was initially selected for investigation because of its high microbial activity, which could help to identify potential 1‐dehydrogenated biomarkers that might also be present in low quantities in urine. Fecal incubations displayed Δ1‐dehydrogenase activity, as evidenced by the use of isotope labeled precursors to show the formation of boldenone and boldione from testosterone and androstenedione, as well as the formation of Δ1‐progesterone and boldione from progesterone. Unlabeled forms were also produced in unspiked fecal samples with Δ1‐progesterone being identified for the first time. Subsequent incubation of urine samples with the labeled AAS precursors demonstrated that Δ1‐dehydrogenase activity can also occur in this matrix. In all urine samples where labeled boldenone or boldione were detected, labeled Δ1‐progesterone was also detected. Δ1‐progesterone was not detected any non‐incubated urine samples or following an administration of boldenone undecylenate to one mare/filly. Δ1‐progesterone appears to be a candidate for further investigation as a suitable biomarker to help evaluate whether boldenone present in a urine sample may have arisen due to microbial activity rather than by its exogenous administration. [ABSTRACT FROM AUTHOR]

A better understanding of nanoelectrospray ionization (nano‐ESI) would be beneficial in further improving the performances of nano‐ESI. In this work, the pulsed high‐voltage (HV) nano‐ESI has been electrically modeled and then systematically characterized by both voltage‐current and mass spectrometry measurements. First, the equivalent resistance of a nano‐ESI source changes with respect to both emitter tip diameter and the HV applied. Increased voltage could improve both spray current and ionization efficiency of the pulsed HV nano‐ESI. Compared with conventional DC HV method, a pulsed HV has less heating effect on the capillary tip and thus allowing the application of a much higher voltage onto a nano‐ESI source. As a result, a pulsed HV nano‐ESI could further boost the ionization efficiency of nano‐ESI by employing even higher voltages than conventional DC nano‐ESI sources. [ABSTRACT FROM AUTHOR]

Time‐dependent mass spectra of mixture solutions of cytochrome c and cholesterol were measured by probe electrospray ionization (PESI) mass spectrometry and the PESI characteristics for several metal needles were investigated with different tip conditions. For a mixture solution of cytochrome c and cholesterol in 0.1% formic acid methanol/water (1/1) at a motion frequency of 1 Hz, an acupuncture needle with a sharp tip and 0.5‐mm‐diameter titanium and stainless‐steel wire needles gave strong cytochrome c and extremely weak cholesterol ion signals. When the frequency was lowered, and the high‐voltage duration increased, the titanium and stainless‐steel needles provided both cytochrome c and cholesterol ion signals with high intensities well separated over time. [ABSTRACT FROM AUTHOR]

The use of testosterone and its pro‐drugs, such as dehydroepiandrosterone (DHEA), is currently regulated in horseracing by the application of international testosterone thresholds. However, additional steroidomic approaches, such as steroid ratios, to distinguish overall adrenal stimulation from drug administrations and an equine biological passport for longitudinal steroid profiling of individual animals could be advantageous in equine doping testing. Thus, DHEA concentrations and related ratios (testosterone [T] to DHEA and DHEA to epitestosterone [E]) were assessed in the reference population by quantitative analysis of 200 post‐race gelding urine samples using liquid chromatography–tandem mass spectrometry. DHEA concentrations ranged between 0.9 and 136.6 ng/mL (mean 12.8 ng/mL), T:DHEA ratios between 0.06 and 1.85 (mean 0.43), and DHEA:E ratios between 0.21 and 13.56 (mean 2.20). Based on the reference population statistical upper limits of 5.4 for T:DHEA ratio and 48.1 for DHEA:E ratio are proposed with a risk of 1 in 10 000 for a normal outlier exceeding the value. Analysis of post‐administration urine samples collected following administrations of DHEA, Equi‐Bolic® (a mix of DHEA and pregnenolone) and testosterone propionate to geldings showed that the upper limit for T:DHEA ratio was exceeded following testosterone propionate administration and DHEA:E ratio following DHEA administrations and thus these ratios could be used as additional biomarkers when determining the cause of an atypical testosterone concentration. Additionally, DHEA concentrations and ratios can be used as a starting point to establish reference ranges for an equine biological passport. Steroidomic approaches, such as steroid ratios and an equine biological passport for longitudinal steroid profiling, could be advantageous in equine doping testing. In the current study, references ranges for DHEA concentrations, testosterone to DHEA ratios and DHEA to epitestosterone ratios were established and these were compared with samples taken following administrations of testosterone and related steroids. The results showed that these steroid ratios could be used as additional biomarkers when determining the cause of atypically high testosterone concentrations in geldings. [ABSTRACT FROM AUTHOR]

Rationale: The toxic metabolites of pyrrolizidine alkaloids (PAs) are initially formed by cytochrome P450‐mediated oxidation reactions and primarily eliminated as glutathione (GSH) conjugates. Although the reaction between the reactive metabolites and GSH can occur spontaneously, the role of the cytosolic enzymes in the process has not been studied. Methods: The toxic metabolites of selected PAs (retrorsine, monocrotaline, senecionine, lasiocarpine, heliotrine or senkirkine) were generated by incubating them in 100 mM phosphate buffer (pH 7.4) containing liver microsomes of human, pig, rat or sheep, NADPH and reduced GSH in the absence or presence of human, pig, rat or sheep liver cytosolic fraction. The supernatants were analyzed using liquid chromatography connected to Finnigan LTQ ion‐trap, Agilent QTOF or Thermo Scientific Q Exactive Focus quadrupole‐orbitrap mass spectrometers. Results: Retrorsine, senecionine and lasiocarpine yielded three GSH conjugates producing [M − H]− ions at m/z 439 (7‐GSH‐DHP (CHO)), m/z 441 (7‐GSH‐DHP (OH)) and m/z 730 (7,9‐diGSH‐DHP) in the presence of human liver cytosolic fraction. 7‐GSH‐DHP (CHO) was a novel metabolite. Monocrotaline, heliotrine and senkirkine did not produce this novel 7‐GSH‐DHP (CHO) conjugate. 7‐GSH‐DHP (CHO) disappeared when incubated with hydroxylamine, and a new oxime derivative was formed. This metabolite was formed only by the human liver cytosolic enzymes but not in the presence of rat or sheep liver cytosolic fractions under otherwise identical reaction conditions. Conclusions: 7‐GSH‐DHP (CHO) has not been reported before, and thus it was considered as a novel metabolite of PAs. This may clarify the mechanisms involved in PA detoxification and widely observed but less understood species differences in response to PA exposure. [ABSTRACT FROM AUTHOR]

Rationale: Liquid extraction surface analysis (LESA) can be used to generate spatially directed protein identifications in an imaging mass spectrometry (IMS) workflow. This approach involves the use of robotic micro‐extractions coupled to online liquid chromatography (LC). We have characterized the extraction efficiency of this method as well as its ability to identify proteins from a matrix assisted laser/desorption ionization (MALDI) IMS experiment. Methods: Proteins and peptides were extracted from transverse sections of a rat brain and sagittal sections of a mouse pup using liquid surface extractions. Extracts were either analyzed by online LC coupled to a high mass resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometer or collected offline and analyzed by traditional LC/MS methods. Identifications were made using both top‐down and bottom‐up methodologies. MALDI images were acquired on a 15T FTICR mass spectrometer at 125 μm spatial resolution. Results: Robotic liquid surface extractions are reproducible across various tissue types, providing significantly improved spatial resolution, with respect to extractions, while still allowing for a robust number of protein identifications. A single 2‐μL extract can provide identification of over 14,000 peptides with little sample preparation, increasing throughput for spatially targeted workflows. Surface extractions from tissue were coupled directly to LC to gather spatially relevant proteomics data. Conclusions: Robotic liquid surface extractions can be used to interrogate discrete regions of tissue to provide protein identifications with high throughput, accuracy, and robustness. The direct coupling of tissue surface extractions and LC offers a new and effective approach to provide spatial proteomics data in an imaging experiment. [ABSTRACT FROM AUTHOR]

The small heat shock protein (sHsp) chaperones are important for stress survival, yet the molecular details of how they interact with client proteins are not understood. All sHsps share a folded middle domain to which is appended flexible N- and C-terminal regions varying in length and sequence between different sHsps which, in different ways for different sHsps, mediate recognition of client proteins. In plants there is a chloroplast-localized sHsp, Hsp21, and a structural model suggests that Hsp21 has a dodecameric arrangement with six N-terminal arms located on the outside of the dodecamer and six inwardly-facing. Here, we investigated the interactions between Hsp21 and thermosensitive model substrate client proteins in solution, by small-angle X-ray scattering (SAXS) and crosslinking mass spectrometry. The chaperone-client complexes were monitored and the Rg-values were found to increase continuously during 20 min at 45°, which could reflect binding of partially unfolded clients to the flexible N-terminal arms of the Hsp21 dodecamer. No such increase in Rg-values was observed with a mutational variant of Hsp21, which is mainly dimeric and has reduced chaperone activity. Crosslinking data suggest that the chaperone-client interactions involve the N-terminal region in Hsp21 and only certain parts in the client proteins. These parts are peripheral structural elements presumably the first to unfold under destabilizing conditions. We propose that the flexible and hydrophobic N-terminal arms of Hsp21 can trap and refold early-unfolding intermediates with or without dodecamer dissociation. [ABSTRACT FROM AUTHOR]

The dismantling and elimination of excess neurons and their connections (pruning) is essential for brain development and may be aberrantly reactivated in some neurodegenerative diseases. Growing evidence implicates caspase-mediated apoptotic and nonapoptotic cascades in the dysfunction and death of neurons in neurodegenerative disorders such as Alzheimer's, Parkinson, and Huntington's diseases. It is the cleaved caspase substrates that are the effectors of synapse elimination. However, their identities, specific cleavage sites, and functional consequences of cleavage are largely unknown. An important gap in our knowledge is a comprehensive catalog of synapse-specific or synapse-enriched caspase targets. Traditional biochemical approaches have revealed only a small number of neuronal caspase targets. Instead, we utilized a gel-based proteomics approach to enable the first global analysis of caspase-mediated cleavage events in mammalian brain synapses, employing both an in vitro system with recombinant activated caspases and an in vivo model of ethanol-induced neuronal apoptosis. Of the more than 70 putative cleavage substrates that were identified, 22 were previously known caspase substrates. Among the novel targets identified and validated by Western blot were the proton pump ATPase subunit ATP6V1B2 and the N-ethylmaleimide-sensitive fusion protein (NSF). Our work represents the first comprehensive, proteome-wide screen for proteolytic targets of caspases in neuronal synapses. Our discoveries will have significance for both furthering basic understanding of roles of caspases in synaptic plasticity and synaptic loss in neurodegeneration, and on a more immediately practical level, may provide candidate biomarkers for measuring synapse loss in human disease states. [ABSTRACT FROM AUTHOR]

Rationale Affinity mass spectrometry (AMS) is an emerging tool in the field of the study of protein•carbohydrate complexes. However, experimental obstacles and data analysis are preventing faster integration of AMS methods into the glycoscience field. Here we show how analysis of direct electrospray ionization mass spectrometry (ESI-MS) AMS data can be simplified for screening purposes, even for complex AMS spectra. Methods A direct ESI-MS assay was tested in this study and binding data for the galectin-3C•lactose complex were analyzed using a comprehensive and simplified data analysis approach. In the comprehensive data analysis approach, noise, all protein charge states, alkali ion adducts and signal overlap were taken into account. In a simplified approach, only the intensities of the fully protonated free protein and the protein•carbohydrate complex for the main protein charge state were taken into account. Results In our study, for high intensity signals, noise was negligible, sodiated protein and sodiated complex signals cancelled each other out when calculating the Kd value, and signal overlap influenced the Kd value only to a minor extent. Influence of these parameters on low intensity signals was much higher. However, low intensity protein charge states should be avoided in quantitative AMS analyses due to poor ion statistics. Conclusions The results indicate that noise, alkali ion adducts, signal overlap, as well as low intensity protein charge states, can be neglected for preliminary experiments, as well as in screening assays. One comprehensive data analysis performed as a control should be sufficient to validate this hypothesis for other binding systems as well. [ABSTRACT FROM AUTHOR]

Detection of testosterone and/or its pro-drugs in the gelding is currently regulated by the application of an international threshold for urinary testosterone of 20 ng/mL. The use of steroid ratios may provide a useful supplementary approach to aid in differentiating between the administration of these steroids and unusual physiological conditions that may result in atypically high testosterone concentrations. In the current study, an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify testosterone (T) and epitestosterone (E). The method was used to analyze 200 post-race urine samples from geldings in order to generate the ratios for the reference population. Following statistical analysis of the data, an upper limit of 5 for T:E ratio in geldings is proposed. Samples collected from 15 geldings with atypical urinary testosterone concentrations (>15 ng/mL) but otherwise normal steroid profile, had T:E ratios within those observed for the reference population. The applicability of an upper T:E ratio to detect an administration was demonstrated by the analysis of a selection of incurred samples from testosterone propionate, dehydroepiandrosterone (DHEA), and a mixture of DHEA and pregnenolone (Equi-Bolic®) administrations. These produced testosterone concentrations above the threshold of 20 ng/mL, but also T:E ratios above the proposed limit of 5. In conclusion, consideration of the T:E ratio appears to be a valuable complementary aid to evaluate whether an atypical testosterone concentration could be caused by a natural biological outlier as opposed to the administration of these steroids. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The small heat shock protein (sHsp) chaperones are crucial for cell survival and can prevent aggregation of client proteins that partially unfold under destabilizing conditions. Most investigations on the chaperone activity of sHsps are based on a limited set of thermosensitive model substrate client proteins since the endogenous targets are often not known. There is a high diversity among sHsps with a single conserved β-sandwich fold domain defining the family, the α-crystallin domain, whereas the N-terminal and C-terminal regions are highly variable in length and sequence among various sHsps and conserved only within orthologues. The endogenous targets are probably also varying among various sHsps, cellular compartments, cell type and organism. Here we have investigated Hsp21, a non-metazoan sHsp expressed in the chloroplasts in green plants which experience huge environmental fluctuations not least in temperature. We describe how Hsp21 can also interact with the chloroplast thylakoid membranes, both when isolated thylakoid membranes are incubated with Hsp21 protein and when plants are heat-stressed. The amount of Hsp21 associated with the thylakoid membranes was precisely determined by quantitative mass spectrometry after metabolic 15N-isotope labeling of either recombinantly expressed and purified Hsp21 protein or intact Arabidopsis thaliana plants. We found that Hsp21 is among few proteins that become associated with the thylakoid membranes in heat-stressed plants, and that approximately two thirds of the pool of chloroplast Hsp21 is affected. We conclude that for a complete picture of the role of sHsps in plant stress resistance also their association with the membranes should be considered. [ABSTRACT FROM AUTHOR]

Direct analysis in real time (DART) represents a new generation of ion source which is used for rapid ionization of small molecules under ambient conditions. The combination of DART and various mass spectrometers allows analyzing multiple food samples with simple or no sample treatment, or in conjunction with prevailing protocolized sample preparation methods. Abundant applications by DART-MS have been reviewed in this paper. The DART-MS strategy applied to food supply chain (FSC), including production, processing, and storage and transportation, provides a comprehensive solution to various food components, contaminants, authenticity, and traceability. Additionally, typical applications available in food analysis by other ambient ionization mass spectrometers were summarized, and fundamentals mainly including mechanisms, devices, and parameters were discussed as well. © 2015 Wiley Periodicals, Inc. Mass Spec Rev. 36:161-187, 2017 [ABSTRACT FROM AUTHOR]

The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 ( Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 ( BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth-instar ecdysis and larval-pupal metamorphosis. By immunoprecipitation with the anti-BmNep1 antibody and liquid chromatography-tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20-hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval-pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx. [ABSTRACT FROM AUTHOR]