Submicrogram Quantities Research Articles

Holistic analysis of glyphosate formulations with nuclear magnetic resonance, similarity calculations and differential NMR

Glyphosate is the most widely used herbicide in the world. Due to varying formulations, common adulteration and illegal trade there is an extreme need to monitor the composition of formulations to ensure the safety. Similarity calculations and differential NMR allowed the assessment of all organic compounds of the mixture, down to the detection limit in the submicrogram range. Glyphosate, isopropylamine and polyoxyethylene tallow amines (POEA) were among the compounds detected together with components classified as impurities such as methanol and acetone. The quantities of the analyzed compounds range from several dozens of milligrams of glyphosate down to submicrogram quantities of methanol and acetone in the NMR tube. Glyphosate was quantified using a fitted calibration curve that, in comparison to classic qNMR experiments, needed shorter measuring times. Among the co-formulants, the one of greatest environmental interest is polyoxyethylene tallow amine especially because it might be more harmful than glyphosate itself. This work illustrates the value of NMR, with calculation of similarities and differential NMR for the rapid comparison and assessment of samples, taking into account major and minor components in the formulations, many of these being industrial secrets.

Recent advances in the synthesis of insect pheromones: an overview from 2013 to 2022.

Covering: 2013 to June 2022Pheromones are usually produced by insects in sub-microgram amounts, which prevents the elucidation of their structures by nuclear magnetic resonance (NMR). Instead, a synthetic reference material is needed to confirm the structure of the natural compounds. In addition, the provision of synthetic pheromones enables large-scale field trials for the development of environmentally friendly pest management tools. Because of these potential applications in pest control, insect pheromones are attractive targets for the development of synthetic procedures and the synthesis of these intraspecific chemical messengers has been at the core of numerous research efforts in the field of pheromone chemistry. The present review is a quick reference guide for the syntheses of insect pheromones published from 2013 to mid-2022, listing the synthesized compounds and highlighting current methodologies in organic synthesis, such as carbon-carbon coupling reactions, organo-transition metal chemistry including ring-closing olefin metathesis, asymmetric epoxidations and dihydroxylations, and enzymatic reactions.

Direct, multielement determinations from cotton swipes via plate express microextraction coupled to an inductively coupled plasma mass spectrometer (μEx-ICP-MS)

Use of a microextraction device allows for ready sampling of solid substrates, such as cotton swipes, allowing transfer of sub-microgram quantities for multielement ICP-MS analysis.

Shotgun Lipidomics of Rodent Tissues.

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Proteomics-Compatible Fourier Transform Isotopic Ratio Mass Spectrometry of Polypeptides.

Measuring the relative abundances of heavy stable isotopes of the elements C, H, N, and O in proteins is of interest in environmental science, archeology, zoology, medicine, and other fields. The isotopic abundance measurements of the fine structure of immonium ions with ultrahigh resolution mass spectrometry obtained in gas-phase fragmentation of polypeptides have previously uncovered anomalous deuterium enrichment in (hydroxy)proline of bone collagen in marine mammals. Here, we provide a detailed description and validation of this approach and demonstrate per mil-range precision of isotopic ratio measurements in aliphatic residues from proteins and cell lysates. The analysis consists of proteomics-type experiment demanding sub-microgram amounts of a protein sample and providing concomitantly protein sequence data allowing one to verify sample purity and establish its identity. A novel software tool protein amino acid-resolved isotopic ratio mass spectrometry (PAIR-MS) is presented for extracting isotopic ratio data from the raw data files acquired on an Orbitrap mass spectrometer.

A review of suspension trapping digestion method in bottom-up proteomics.

The standard bottom-up proteomic workflow is comprised of sample preparation, data acquisition, and data analysis. While the latter two parts have made considerable advances in the last decade, sample preparation has remained an important challenge within the workflow due to the multi-step nature of complex biological samples, and still requires much development. Several sample preparation methods have been developed and used in the last two decades, including in-gel, in-solution, on-bead, filter-aided sample preparation, and suspension trapping, to improve reproducibility, efficiency, scalability, and reduce the handling time of this process. One of the most recent methods developed and applied in proteomics studies in recent years is suspension trapping, which combines rapid detergent removal, reactor-type protein digestion, and peptide clean-up in a tip or spin column. Suspension trapping is a simple, rapid, and reproducible digestion method that can effectively handle proteins in low microgram or sub-microgram amounts. This review discusses the benefits of the suspension trapping digestion method in relation to its development and application in bottom-up proteomics studies. We also discuss recent applications of suspension trapping digestion to different sample types and the features of the suspension trapping digestion method compared with other sample preparation methods.

De novo transcriptome assembly and identification of G-Protein-Coupled-Receptors (GPCRs) in two species of monogenean parasites of fish.

Genomic resources for Platyhelminthes of the class Monogenea are scarce, despite the diversity of these parasites, some species of which are highly pathogenic to their fish hosts. This work aimed to generate de novo-assembled transcriptomes of two monogenean species, Scutogyrus longicornis (Dactylogyridae) and Rhabdosynochus viridisi (Diplectanidae), providing a protocol for cDNA library preparation with low input samples used in single cell transcriptomics. This allowed us to work with sub-microgram amounts of total RNA with success. These transcriptomes consist of 25,696 and 47,187 putative proteins, respectively, which were further annotated according to the Swiss-Prot, Pfam, GO, KEGG, and COG databases. The completeness values of these transcriptomes evaluated with BUSCO against Metazoa databases were 54.1% and 73%, respectively, which is in the range of other monogenean species. Among the annotations, a large number of terms related to G-protein-coupled receptors (GPCRs) were found. We identified 109 GPCR-like sequences in R. viridisi, and 102 in S. longicornis, including family members specific for Platyhelminthes. Rhodopsin was the largest family according to GRAFS classification. Two putative melatonin receptors found in S. longicornis represent the first record of this group of proteins in parasitic Platyhelminthes. Forty GPCRs of R. viridisi and 32 of S. longicornis that were absent in Vertebrata might be potential drug targets. The present study provides the first publicly available transcriptomes for monogeneans of the subclass Monopisthocotylea, which can serve as useful genomic datasets for functional genomic research of this important group of parasites.

MRNA: A Novel Avenue to Antibody Therapy?

First attempts to use exogenous mRNA for protein expression invivo were made more than 25 years ago. However, widespread appreciation of invitro transcribed mRNA as a powerful technology for supplying therapeutic proteins to the body has evolved only during the past few years. Various approaches to turning mRNA into a potent therapeutic have been developed. All of them share utilization of specifically designed, rather than endogenous, sequences and thorough purification protocols. Apart from this, there are two fundamental philosophies, one promoting the use of chemically modified nucleotides, the other advocating restriction to unmodified building blocks. Meanwhile, both strategies have received broad support by successful mRNA-based protein treatments in animal models. For such invivo use, specifically optimized mRNA had to be combined with potent formulations to enable efficient invivo delivery. The present review analyzes the applicability of mRNA technology to antibody therapy in all main fields: antitoxins, infectious diseases, and oncology.

Proteomics in Neural Crest Cell Research.

Detailed investigation of the neural crest cell proteome has lingered behind that of the transcriptome due to the challenge of obtaining sufficient starting material for subsequent proteomic investigation. Compounded by the complexity of protein abundance, large number of posttranslational modifications, and the stochastic nature of proteomics approaches, little data so far exists describing the true neural crest cell proteome. However, recent advances in instrument sensitivity and recovery of material during sample preparation have alleviated many of these problems and make proteomics analysis an underutilized tool to study neural crest cell biology. Here we present a quantitative proteomics protocol for deep analysis of both whole proteome and posttranslational modifications and a separate protocol for ultrasensitive proteomic profiling from submicrogram amounts of protein.

A color-code for glycosaminoglycans identification by means of polyacrylamide gel electrophoresis stained with the cationic carbocyanine dye Stains-all.

Cationic dyes such as toluidin blue are commonly employed to visualize glycosaminoglycans (GAGs) on electrophoresis gels; however, the carbocyanine-based dye Stains-all have been increasingly used to stain the non-sulfated hyaluronic acid and other GAGs in submicrogram quantities. In this short communication, we demonstrate that Stains-all is able to stain the most common GAGs on polyacrylamide gels with distinct and contrasting colors in a reproducible manner. We also show that this staining method is useful to identify GAGs present both in mixtures and in submicrogram quantities. Therefore, Stains-all has shown to be useful in identifying GAGs on polyacrylamide gels with basis on their specific colors, at least on screening level.

Quantitative Mass-Spectrometric Sequencing of Chitosan Oligomers Revealing Cleavage Sites of Chitosan Hydrolases.

Partially acetylated chito-oligosaccharides (paCOS) have diverse bioactivities that turn them into promising compounds especially for medical and agricultural applications. These properties likely arise from different acetylation patterns, but determining the sequences of paCOS and producing paCOS with patterns of interest have proven difficult. We present a novel method for sequencing submicrogram amounts of paCOS using quantitative mass spectrometry, allowing one to rapidly analyze the substrate specificities of chitosan hydrolases that can be used to produce paCOS. The method involves four major steps: (i) acetylation of free amino groups in paCOS using a deuterated reagent; (ii) labeling the reducing end with an 18O-tag; (iii) quantifying paCOS using [13C2, 2H3]-labeled isotopologs as internal standards; (iv) sequencing paCOS by tandem MS. Eventually, this method will aid in developing enzymes with cleavage patterns optimized for producing paCOS with defined patterns of acetylation and specific bioactivities.

Petrochronology of Zircon and Baddeleyite in Igneous Rocks: Reconstructing Magmatic Processes at High Temporal Resolution

Zircon (ZrSiO4) and baddeleyite (ZrO2) are common accessory minerals in igneous rocks of felsic to mafic composition. Both minerals host trace elements substituting for Zr, among them Hf, Th, U, Y, REEs and many more. The excellent chemical and physical resistivity of zircon makes this mineral a perfect archive of chemical and temporal information to trace geological processes in the past, utilizing the outstanding power and temporal resolution of the U–Pb decay schemes. Baddeleyite is a chemically and physically much more fragile mineral. It preserves similar information only where it is shielded from dissolution and physical fragmentation as an inclusion in other minerals or in a fine-grained or non-reactive rock matrix. It offers the potential for dating the solidification of mafic rocks with high-precision through its crystallization in small pockets of Zr-enriched melt, after extensive olivine and pyroxene fractionation. Zircon and baddelelyite U–Pb dates are, for an overwhelming majority of cases and where we can assume a closed system, considered to reflect the time of crystallization. The development of the U–Pb dating tool CA-ID-TIMS (chemical abrasion-isotope dilution-thermal ionization mass spectrometry) since 2005 has led to unprecedented precision of better than 0.1% in 206Pb/238U dates (Bowring et al. 2005). Increased sensitivity of mass spectrometers and low laboratory blanks due to reduction of acid volumes allow routine U–Pb age determinations of micrograms of material at sufficiently high radiogenic/common lead ratios (see Schoene and Baxter 2017, this volume). In situ U–Pb age analysis using laser ablation or primary ion beam sputtering allows analysis of sub-microgram quantities of zircon material from polished internal sections or zircon surfaces with spot diameters ranging from ~30 μm for laser-ablation, inductively coupled plasma mass spectrometry (LA-ICP-MS) to 10 μm for secondary ion mass spectrometry (SIMS), lateral resolutions of 2–5 μm for NanoSIMS …

Cu-decorated Raschig-TiO2 rings inducing MB repetitive discoloration without release of Cu-ions under solar light

The methylene blue (MB) discoloration on innovative photocatalysts made up of Raschig-rings coated by pre-formed TiO2 nanoparticles decorated with CuOx (RR@TiO2-Cu) is presented. The MB-discoloration was drastically accelerated by the subsequent addition of 0.004% Cu. This Cu-content being ∼3000 times below the TiO2 present in the RR@TiO2 samples led to MB-discoloration in shorter times. The Cu-ions released from the RR@TiO2-Cu 0.004% were determined ≤1ppb by inductively coupled plasma mass-spectrometry (ICP-MS). Cu-percentages >0.018% decreased the MB-discoloration kinetics due to the Cu acting as recombination sites for the photogenerated charges. The total organic carbon (TOC) reduction concomitant with the MB-discoloration is reported as well as the effect of the initial MB-concentration and pH on the discoloration kinetics. The Cu-addition shifted significantly the TiO2 absorption into the visible region even when added in sub-microgram quantities (0.004%) to RR@TiO2 as detected by diffuse reflectance spectroscopy (DRS). The Cu level added to RR@TiO2 was too low to be detected by X-ray diffraction (XRD). The roughness of the RR@TiO2-Cu (0.018%–0.081% Cu) presented Rg-values between 11.8nm and 23.9nm. The RR@TiO2Cu presented agglomerate sizes of ∼200nm for TiO2 and for the Cu aggregates sizes of ∼20nm. The MB discoloration kinetics and reuse of the RR@TiO2-Cu suggest the potential as a supported catalyst in environmental cleaning process. A reaction mechanism is suggested taking into consideration the role of the Cu1+/2+ intra-gap states involving redox catalysis as detected by X-ray photoelectron-spectroscopy (XPS).

Versatile cell for in-situ spectroelectrochemical and ex-situ nanomorphological characterization of both water soluble and insoluble phthalocyanine compounds

A novel cell for complex spectroelectrochemical and nanomorphological characterization of phthalocyanine has been developed. The applicability and universality of the cell allows performing both in-situ and ex-situ characterization of the same sample, in solid and solution state, respectively, which was demonstrated on tetramethyltetra-3,4-pyridine-porphyrazino cobalt as water soluble and tetraneopentoxyphthalocyanine cobalt as water insoluble phthalocyanine based compound. The cell is composed of a planar freely replaceable bottom formed by a basal plane of highly ordered pyrolytic graphite. It allows complete characterization of modified electrode on same electrode, from spectral observation of modification process via atomic force microscopy to spectroelectrochemical characterization of redox and catalytic behavior. The relatively inert HOPG electrode serves as a substrate for phthalocyanine compounds and also as a backscattering mirror for in-situ reflection spectroscopy. This makes the cell suitable for spectroelectrochemical studies of phthalocyanine both as HOPG-supported thin film and in the solution. Moreover, the cell can take the advantage of optional utilization of supported thin layer interface of two immiscible electrolyte solutions allowing spectroelectrochemical analysis of sub-microgram quantities of water insoluble compounds. .

High temperature mass detection using a carbon nanotube bilayer modified quartz crystal microbalance as a GC detector.

A small, portable gas chromatograph (GC) was assembled for the trace detection of controlled substances using a novel quartz crystal microbalance sensor (QCM). The QCM crystal surface was modified with a variety of sorption materials to increase adsorption thereby amplifying mass detection. Single polymer thin film coatings increased the QCM response by 1-2 orders of magnitude, while operating at over 100 °C. Adding a layer of carbonaceous nanomaterial (graphene or carbon nanotubes) above such a film dramatically increased sensitivity by up to 3 orders of magnitude compared to uncoated crystals. Separation and detection of submicrogram quantities of controlled substances was carried out within minutes by employing a GC column and detector temperature ramp up to 220 °C. An additional 10-fold enhancement in sensitivity was achieved by mechanical abrasion of the sample swabs used in the sample introduction process. This study demonstrated a novel use of a polymer composite modified QCM as a chemical sensor at high temperatures.

Visualization of Unstained Protein Bands on PVDF.

In 1988, two separate investigators reported a novel method of detecting unstained protein bands on PVDF membranes using white light transillumination. This simple method exploits the intrinsic hydrophobicity of PVDF membrane, which enables the visual observation of transferred protein bands due to differential wetting patterns between protein bands and the membrane itself. This method applies only to hydrophobic PVDF membranes, because hydrophilic membranes such as nitrocellulose wet out completely, rendering the protein bands invisible by transillumination. Transilluminational protein visualization can detect submicrogram quantities of proteins while circumventing the use of protein stains, which can potentially interfere with downstream analysis such as N-terminal sequencing. In this chapter, we demonstrate efficient transilluminational protein visualization on a recently introduced low fluorescence PVDF membrane, normally used for downstream fluorescent immunodetection.

Copy number variation analysis based on AluScan sequences.

BackgroundAluScan combines inter-Alu PCR using multiple Alu-based primers with opposite orientations and next-generation sequencing to capture a huge number of Alu-proximal genomic sequences for investigation. Its requirement of only sub-microgram quantities of DNA facilitates the examination of large numbers of samples. However, the special features of AluScan data rendered difficult the calling of copy number variation (CNV) directly using the calling algorithms designed for whole genome sequencing (WGS) or exome sequencing.ResultsIn this study, an AluScanCNV package has been assembled for efficient CNV calling from AluScan sequencing data employing a Geary-Hinkley transformation (GHT) of read-depth ratios between either paired test-control samples, or between test samples and a reference template constructed from reference samples, to call the localized CNVs, followed by use of a GISTIC-like algorithm to identify recurrent CNVs and circular binary segmentation (CBS) to reveal large extended CNVs. To evaluate the utility of CNVs called from AluScan data, the AluScans from 23 non-cancer and 38 cancer genomes were analyzed in this study. The glioma samples analyzed yielded the familiar extended copy-number losses on chromosomes 1p and 9. Also, the recurrent somatic CNVs identified from liver cancer samples were similar to those reported for liver cancer WGS with respect to a striking enrichment of copy-number gains in chromosomes 1q and 8q. When localized or recurrent CNV-features capable of distinguishing between liver and non-liver cancer samples were selected by correlation-based machine learning, a highly accurate separation of the liver and non-liver cancer classes was attained.ConclusionsThe results obtained from non-cancer and cancerous tissues indicated that the AluScanCNV package can be employed to call localized, recurrent and extended CNVs from AluScan sequences. Moreover, both the localized and recurrent CNVs identified by this method could be subjected to machine-learning selection to yield distinguishing CNV-features that were capable of separating between liver cancers and other types of cancers. Since the method is applicable to any human DNA sample with or without the availability of a paired control, it can also be employed to analyze the constitutional CNVs of individuals.Electronic supplementary materialThe online version of this article (doi:10.1186/s13336-014-0015-z) contains supplementary material, which is available to authorized users.

High-resolution α-amylase assay combined with high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy for expedited identification of α-amylase inhibitors: proof of concept and α-amylase inhibitor in cinnamon.

Type 2 diabetes affects millions of people worldwide, and new improved drugs or functional foods containing selective α-amylase inhibitors are needed for improved management of blood glucose. In this article the development of a microplate-based high-resolution α-amylase inhibition assay with direct photometric measurement of α-amylase activity is described. The inhibition assay is based on porcine pancreatic α-amylase with 2-chloro-4-nitrophenyl-α-D-maltotriose as substrate, which this gives a stable, sensitive, and cheap inhibition assay as requested for high-resolution purposes. In combination with HPLC-HRMS-SPE-NMR, this provides an analytical platform that allows simultaneous chemical and biological profiling of α-amylase inhibitors in plant extracts. Proof-of-concept with an artificial mixture of six compounds-of which three are known α-amylase inhibitors-showed that the high-resolution α-amylase inhibition profiles allowed detection of sub-microgram amounts of the α-amylase inhibitors. Furthermore, the high-resolution α-amylase inhibition assay/HPLC-HRMS-SPE-NMR platform allowed identification of cinnamaldehyde as the α-amylase inhibitor in cinnamon (Cinnamomum verum Presl.).

Localized photovoltaic investigations on organic semiconductors and bulk heterojunction solar cells

Newly synthesized organic electronics materials are often available in submicrogram amounts only. Photoelectrochemical scanning droplet cell microscopy is a powerful method that allows a comprehensive characterisation of such small amounts including oxidation, reduction potentials, doping, determination of charge carriers, band gap, charge capacity, over-oxidation sensitivity and many more. Localized photoelectrochemical characterization of the poly[4,8-bis-substituted-benzo[1,2-b:4,5-b0]dithiophene-2,6-diyl-alt-4-substituted-thieno [3,4-b] thiophene-2,6-diyl] (PBDTTT-c) and PBDTTT-c:PCBM bulk heterojunction was performed using photoelectrochemical scanning droplet cell microscopy (PE-SDCM). The optical properties and the real and imaginary part of the dielectric function, of the polymer were determined using spectroscopic ellipsometry. The photoelectrochemical characterizations were performed in a three and two electrode configuration of PE-SDCM under laser and white light illumination. The effect of illumination was characterized using dark/illumination sequences. The stability of the photocurrent was studied using longer term (600 s) illumination. Finally the effect of cell configuration and illumination conditions on the photovoltage was studied.

Ultrasensitive proteome analysis using paramagnetic bead technology.

In order to obtain a systems-level understanding of a complex biological system, detailed proteome information is essential. Despite great progress in proteomics technologies, thorough interrogation of the proteome from quantity-limited biological samples is hampered by inefficiencies during processing. To address these challenges, here we introduce a novel protocol using paramagnetic beads, termed Single-Pot Solid-Phase-enhanced Sample Preparation (SP3). SP3 provides a rapid and unbiased means of proteomic sample preparation in a single tube that facilitates ultrasensitive analysis by outperforming existing protocols in terms of efficiency, scalability, speed, throughput, and flexibility. To illustrate these benefits, characterization of 1,000 HeLa cells and single Drosophila embryos is used to establish that SP3 provides an enhanced platform for profiling proteomes derived from sub-microgram amounts of material. These data present a first view of developmental stage-specific proteome dynamics in Drosophila at a single-embryo resolution, permitting characterization of inter-individual expression variation. Together, the findings of this work position SP3 as a superior protocol that facilitates exciting new directions in multiple areas of proteomics ranging from developmental biology to clinical applications.