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1. A High‐Throughput Absolute Quantification of Protein‐Bound Sulfur Amino Acids from Model and Crop Plant Seeds

   https://doi.org/10.1002/cpz1.861  

   Yobi, Abou; Ansaf, Huda; Angelovici, Ruthie (August 2023, Current Protocols)

   Abstract In this procedure, we describe a high‐throughput absolute quantification protocol for the protein‐bound sulfur amino acids, cysteine (Cys) and methionine (Met), from plant seeds. This procedure consists of performic acid oxidation that transforms bound Cys into cysteic acid (CysA) and bound Met into methionine sulfone (MetS) followed by acid hydrolysis. The absolute quantification step is performed by multiple reaction monitoring tandem mass spectrometry (LC‐MS/MS). The approach facilitates the analysis of a few hundred samples per week by using a 96‐well plate extraction setup. Importantly, the method uses only ∼4 mg of tissue per sample and uses the common acid hydrolysis protocol, followed by water extraction that includes DL‐Ser‐d3 and L‐Met‐d3 as internal standards to enable the quantification of the absolute levels of the protein‐bound Cys and Met with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples from Arabidopsis thaliana , Glycine max , and Zea mays but could be applied to other plant tissues. © 2023 Wiley Periodicals LLC. Basic Protocol : Analysis of protein‐bound cysteine and methionine from seeds 

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2. Detection and Quantification of RNA Phosphorothioate Modifications Using Mass Spectrometry

   https://doi.org/10.1002/cpnc.113  

   Wu, Ying; Zheng, Ya Ying; Lin, Qishan; Sheng, Jia (August 2020, Current Protocols in Nucleic Acid Chemistry)

   Abstract This article describes a protocol for detecting and quantifying RNA phosphorothioate modifications in cellular RNA samples. Starting from solid‐phase synthesis of phosphorothioate RNA dinucleotides, followed by purification with reversed‐phase HPLC, phosphorothioate RNA dinucleotide standards are prepared for UPLC‐MS and LC‐MS/MS methods. RNA samples are extracted from cells using TRIzol reagent, then digested with a nuclease mixture and analyzed by mass spectrometry. UPLC‐MS is employed first to identify RNA phosphorothioate modifications. An optimized LC‐MS/MS method is then employed to quantify the frequency of RNA phosphorothioate modifications in a series of model cells. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis, purification, and characterization of RNA phosphorothioate dinucleotides Basic Protocol 2: Digestion of RNA samples extracted from cells Basic Protocol 3: Detection and quantification of RNA phosphorothioate modifications by mass spectrometry 

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3. Membrane Ultrafiltration-Based Sample Preparation Method and Sheath-Flow CZE-MS/MS for Top-Down Proteomics

   Zhichang Yang, Liangliang Sun (June 2022, Methods in molecular biology)

   Mass spectrometry (MS)-based denaturing top-down proteomics (dTDP) identify proteoforms without pretreatment of enzyme proteolysis. A universal sample preparation method that can efficiently extract protein, reduce sample loss, maintain protein solubility, and be compatible with following up liquid-phase separation, MS, and tandem MS (MS/MS) is vital for large-scale proteoform characterization. Membrane ultrafiltration (MU) was employed here for buffer exchange to efficiently remove the sodium dodecyl sulfate (SDS) detergent in protein samples used for protein extraction and solubilization, followed by capillary zone electrophoresis (CZE)-MS/MS analysis. The MU method showed good protein recovery, minimum protein bias, and nice compatibility with CZE-MS/MS. Single-shot CZE-MS/MS analysis of an Escherichia coli sample prepared by the MU method identified over 800 proteoforms. 

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4. Codon-Dependent Transcriptional Changes in Response to Tryptophan Limitation in the Tryptophan Auxotrophic Pathogens Chlamydia trachomatis and Streptococcus pyogenes

   https://doi.org/10.1128/mSystems.01269-21  

   Ouellette, Scot P.; Hatch, Nathan D.; Wood, Nicholas A.; Herrera, Andrea L.; Chaussee, Michael S. (December 2021, mSystems)

   van Wezel, Gilles P. (Ed.)

   ABSTRACT Chlamydia trachomatis and Streptococcus pyogenes are among the most prevalent bacterial pathogens of humans. Interestingly, both pathogens are tryptophan (Trp) auxotrophs and must acquire this essential amino acid from their environment. For Chlamydia , an obligate intracellular bacterium, this means scavenging Trp from the host cell in which they reside. For Streptococcus , a primarily extracellular bacterium, this means scavenging Trp from the local environment. In the course of a natural immune response, both pathogens can be exposed to Trp-limiting conditions through the action of the interferon gamma-inducible IDO1 enzyme, which catabolizes Trp to N -formylkynurenine. How these pathogens respond to Trp starvation is incompletely understood. However, we have previously demonstrated that genes enriched in Trp codons were preferentially transcribed in C. pneumoniae during Trp limitation. Chlamydia , but not Streptococcus , lacks a stringent response, which is a global regulon activated by uncharged tRNAs binding in the A site of the ribosome. We hypothesized that the chlamydial response to Trp limitation is a consequence of lacking a stringent response. To test this, we compared global transcription profiles of C. trachomatis to both wild-type and stringent response mutant strains of Streptococcus during Trp starvation. We observed that both Trp auxotrophs respond with codon-dependent changes in their transcriptional profiles that correlate with Trp codon content but not transcript stability. Importantly, the stringent response had no impact on these transcriptional changes, suggesting an evolutionarily conserved adaptation to Trp starvation. Therefore, we have revealed a novel response of Trp auxotrophic pathogens in response to Trp starvation. IMPORTANCE Chlamydia trachomatis and Streptococcus pyogenes are important pathogens of humans. Interestingly, both are auxotrophic for tryptophan and acquire this essential amino acid from the host environment. However, part of the host defense against pathogens includes the degradation of tryptophan pools. Therefore, Chlamydia and Streptococcus are particularly susceptible to tryptophan starvation. Most model bacteria respond to amino acid starvation by using a global regulon called the stringent response. However, Chlamydia lacks a stringent response. Here, we investigated the chlamydial response to tryptophan starvation and compared it to both wild-type and stringent response mutant strains of S. pyogenes to determine what role a functional stringent response plays during tryptophan starvation in these pathogens. We determined that both of these pathogens respond to tryptophan starvation by increasing transcription of tryptophan codon-rich genes. This effect was not dependent on the stringent response and highlights a previously unrecognized and potentially evolutionarily conserved mechanism for surviving tryptophan starvation. 

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5. Quantitative analysis of microplastics in beach sand via low-temperature solvent extraction and thermal degradation: Effects of particle size and sample depth

   https://doi.org/10.1016/j.scitotenv.2024.176009  

   Sivaraman, Mythreyi; Fan, Lingfei; Yan, Weile (November 2024, Science of The Total Environment)

   Quantifying trace levels of microplastics in complex environmental media remains a challenge. In this study, an approach combining field collection of samples from different depths, sample size fractionation, and plastic quantification via pyrolysis-gas chromatography–mass spectrometry (Py-GC–MS) was employed to identify and quantify microplastics at two public beaches along the northeast coast of the U.S. (Salisbury beach, MA and Hampton beach, NH). A simple sampling tool was used to collect beach sand from depth intervals of 0–5 cm and 5–10 cm, respectively. The samples were sieved to give three size fractions: coarse (>1.2 mm), intermediate (100 μm–1.2 mm), and fine (1.2 μm–100 μm) particles. Following density separation and wet peroxide oxidation, a low-temperature solvent extraction protocol involving 2-chlorophenol was used to extract polyester (PET), polystyrene (PS), polyamide (PA), and polyvinyl chloride (PVC). The extract was analyzed using Py-GC–MS for the respective polymers, while the solid residue was pyrolyzed separately for polyethylene (PE) and polypropylene (PP). The one-step solvent extraction method significantly simplified the sample matrix and improved the sensitivity of analysis. Among the samples, PET was detected in greater quantities in the fine fraction than in the intermediate size fraction, and PET fine particles were located predominantly in the surface sand. Similar to PET, PS was detected at higher mass concentrations in the fine particles in most samples. These results underscore the importance of beach environment for plastic fragmentation, where a combination of factors including UV irradiation, mechanical abrasion, and water exposure promote plastic breakdown. Surface accumulation of fine plastic particles may also be attributed to transport of microplastics through wind and tides. The proposed sample treatment and analysis methods may allow sensitive and quantitative measurements of size or depth related distribution patterns of microplastics in complex environmental media. 

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