Extracellular vesicles (EVs), the key players in inter‐cellular communication, are produced by all cell types and are present in all body fluids. Analysis of the proteome content is an important ...approach in structural and functional studies of these vesicles. EVs circulating in human plasma are heterogeneous in size, cellular origin, and functions. This heterogeneity and the potential presence of contamination with plasma components such as lipoprotein particles and soluble plasma proteins represent a challenge in profiling the proteome of EV subsets by mass spectrometry. An immunocapture strategy prior to mass spectrometry may be used to isolate a homogeneous subpopulation of small EVs (sEV) with a specific endocytic origin from plasma or other biofluids. Immunocapture selectively separates EV subpopulations in biofluids based on the presence of a unique protein carried on the vesicle surface. The advantages and disadvantages of EV immune capture as a preparative step for mass spectrometry are discussed.
Time‐of‐flight secondary‐ion mass spectrometry (TOF‐SIMS), a powerful analytical technique sensitive to all components of perovskite solar cell (PSC) materials, can differentiate between the various ...organic species within a PSC absorber or a complete device stack. The ability to probe chemical gradients through the depth of a device (both organic and inorganic), with down to 100 nm lateral resolution, can lead to unique insights into the relationships between chemistry in the absorber bulk, at grain boundaries, and at interfaces as well as how they relate to changes in performance and/or stability. In this review, the technique is described; then, from the literature, several examples of how TOF‐SIMS have been used to provide unique insight into PSC absorbers and devices are covered. Finally, the common artifacts that can be introduced if the data are improperly collected, as well as methods to mitigate these artifacts are discussed.
Time of flight secondary ion mass spectrometry (TOF‐SIMS) is a versatile characterization technique which can provide key insights into the spatial location of all components of perovskite solar cell materials, and how those distributions change with performance/degradation. The technique is summarized here, past uses from the literature are covered, and example data and mitigation of known measurement artifacts are described.
The fact that ions of macromolecular complexes produced by electrospray ionization can be maintained intact in a mass spectrometer has stimulated exciting new lines of research. In this review we ...chart the progress of this research from the observation of simple homo-oligomers to complex heterogeneous macromolecular assemblies of mega-Dalton proportions. The applications described herein not only confirm the status of mass spectrometry (MS) as a structural biology approach to complement X-ray analysis or electron microscopy, but also highlight unique attributes of the methodology. This is exemplified in studies of the biogenesis of macromolecular complexes and in the exchange of subunits between macromolecular complexes. Moreover, recent successes in revealing the overall subunit architecture of complexes are set to promote MS from a complementary approach to a structural biology tool in its own right.
•Cardiovascular disease is a leading cause of morbidity and mortality.•There is notable interindividual variation in response to cardiovascular drugs.•An HPLC-MS/MS assay to quantify atorvastatin, ...bisoprolol and clopidogrel has been developed.•The assay has been applied to 1279 plasma samples from 1024 patients.
Cardiovascular disease is a leading cause of morbidity, mortality, and healthcare expenditure worldwide. Importantly, there is interindividual variation in response to cardiovascular medications, leading to variable efficacy and adverse events. Therefore a rapid, selective, sensitive and reproducible multi-analyte HPLC-MS/MS assay for the quantification in human plasma of atorvastatin, its major metabolites 2-hydroxyatorvastatin, atorvastatin lactone and 2-hydroxyatorvastatin lactone, plus bisoprolol and clopidogrel-carboxylic acid has been developed, fully validated, and applied to a large patient study. Fifty microliter plasma samples were extracted with a simple protein precipitation procedure involving acetonitrile with acetic acid (0.1%, v/v). Chromatographic separation was via a 2.7 μm Halo C18 (50 × 2.1 mm ID, 90 Å) column and gradient elution at a flow rate of 500 μL/min consisting of a mobile phase of water (A) and acetonitrile (B), each containing 0.1% formic acid (v/v), over a 6.0 min run time. The six analytes and their corresponding six deuterated internal standards underwent positive ion electrospray ionisation and were detected with multiple reaction monitoring. The developed method was fully validated with acceptable selectivity, carryover, dilution integrity, and within-run and between-run accuracy and precision. Mean extraction recovery for the analytes was 92.7–108.5%, and internal standard-normalised matrix effects had acceptable precision (coefficients of variation 2.2–12.3%). Moreover, all analytes were stable under the tested conditions. Atorvastatin lactone to acid interconversion was assessed and recommendations for its minimisation are made. The validated assay was successfully applied to analyse 1279 samples from 1024 patients recruited to a cardiovascular secondary prevention prospective study.
Molecular networking is a tandem mass spectrometry (MS/MS) data organizational approach that has been recently introduced in the drug discovery, metabolomics, and medical fields. The chemistry of ...molecules dictates how they will be fragmented by MS/MS in the gas phase and, therefore, two related molecules are likely to display similar fragment ion spectra. Molecular networking organizes the MS/MS data as a relational spectral network thereby mapping the chemistry that was detected in an MS/MS-based metabolomics experiment. Although the wider utility of molecular networking is just beginning to be recognized, in this review we highlight the principles behind molecular networking and its use for the discovery of therapeutic leads, monitoring drug metabolism, clinical diagnostics, and emerging applications in precision medicine.
Liquid chromatography-mass spectrometry (LC-MS)-based peptidomics methods allow for the detection and identification of many peptides in a complex biological mixture in an untargeted manner. ...Quantitative peptidomics approaches allow for comparisons of peptide abundance between different samples, allowing one to draw conclusions about peptide differences as a function of experimental treatment or physiology. While stable isotope labeling is a powerful approach for quantitative proteomics and peptidomics, advances in mass spectrometry instrumentation and analysis tools have allowed label-free methods to gain popularity in recent years. In a general label-free quantitative peptidomics experiment, peak intensity information for each peptide is compared across multiple LC-MS runs. Here, we outline a general approach for label-free quantitative peptidomics experiments, including steps for sample preparation, LC-MS data acquisition, data processing, and statistical analysis. Special attention is paid to address run-to-run variability, which can lead to several major problems in label-free experiments. Overall, our method provides researchers with a framework for the development of their own quantitative peptidomics workflows applicable to quantitation of peptides from a wide variety of different biological sources.
Mass spectrometry is a high throughput technique widely used for metabolic fingerprinting of plant material. Among the diverse plant metabolites, pigments such as anthocyanins play a determinant role ...in plant defence mechanisms, protecting them from biotic and abiotic stresses. Anthocyanins are phenolic water-soluble glycosides or acyl-glycosides of anthocyanidins which could be accurately detected and quantified through mass spectrometry. This chapter describes how to extract anthocyanins from higher plant materials and quantify them through a liquid chromatography-mass spectrometry (LC-MS) based method.
One of the major problems in quantitative analysis of pesticide residues in food samples by gas chromatography–tandem mass spectrometry (GC–MS/MS) is the enhancement or the suppression, of the target ...analyte signals in matrix extracts. Potentially positive samples, which had previously been identified by a rapid screening method, were quantified using standard addition to compensate matrix effects. As example we performed a systematic study on the application of the standard addition calibration (SAC) method for the determination of 12 pesticides (acephate, bromopropylate, chlorpyrifos, cypermethrin, diazinon, etrimfos, heptenophos, iprodione, methamidophos, procymidone, tetradifon, and triadimefon) in two matrices (cucumber and orange) in the range of initial concentrations of 10–200
μg
kg
−1. The influence of some factors, such as the minimum number of standard additions used (single, two, three or four points calibration), as well as the known amount of analyte added to the sample, is evaluated in order to obtain reliable results. Accurate quantification is achieved when a single point SAC at 200
μg
kg
−1 was used, obtaining for all the cases recoveries between 70 and 120%. The proposed analytical approach only needs two injections per sample (blank and spiked extracted sample) to determine the final concentration of pesticide in positive samples.
We have developed a new mass spectrometry (MS) technology, the Single-probe MS, capable of real-time, in situ metabolomic analysis of individual living cells. The Single-probe is a miniaturized ...multifunctional sampling and ionization device that is directly coupled to the mass spectrometer. With a sampling tip smaller than individual eukaryotic cells (<10 μm), the Single-probe can be inserted into single cells to sample the intracellular compounds for real-time MS analysis. We have used the Single-probe to detect several cellular metabolites and the anticancer small molecules paclitaxel, doxorubicin, and OSW-1 in individual cervical cancer cells (HeLa). Single cell mass spectrometry (SCMS) is an emerging scientific technology that could reshape the analytical science of many research disciplines, and the Single-probe MS technology is a novel method for SCMS that, through its accessible fabrication protocols, can be broadly applied to different research areas.
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