Total Protein Quantification Research Articles

The influence of proteoforms: assessing the accuracy of total vitamin D-binding protein quantification by proteolysis and LC-MS/MS.

Vitamin D-binding protein (VDBP), a serum transport protein for 25-hydroxyvitamin D [25(OH)D], has three common proteoforms which have co-localized amino acid variations and glycosylation. A monoclonal immunoassay was found to differentially detect VDBP proteoforms and methods using liquid chromatography-tandem mass spectrometry (LC-MS/MS) might be able to overcome this limitation. Previously developed multiple reaction monitoring LC-MS/MS methods for total VDBP quantification represent an opportunity to probe the potential effects of proteoforms on proteolysis, instrument response and quantification accuracy. VDBP was purified from homozygous human donors and quantified using proteolysis or acid hydrolysis and LC-MS/MS. An interlaboratory comparison was performed using pooled human plasma [Standard Reference Material® 1950 (SRM 1950) Metabolites in Frozen Human Plasma] and analyses with different LC-MS/MS methods in two laboratories. Several shared peptides from purified proteoforms were found to give reproducible concentrations [≤2.7% coefficient of variation (CV)] and linear instrument responses (R2≥0.9971) when added to human serum. Total VDBP concentrations from proteolysis or amino acid analysis (AAA) of purified proteoforms had≤1.92% CV. SRM 1950, containing multiple proteoforms, quantified in two laboratories resulted in total VDBP concentrations with 7.05% CV. VDBP proteoforms were not found to cause bias during quantification by LC-MS/MS, thus demonstrating that a family of proteins can be accurately quantified using shared peptides. A reference value was assigned for total VDBP in SRM 1950, which may be used to standardize methods and improve the accuracy of VDBP quantification in research and clinical samples.

Análisis de glioxalasa I (GLX-I) en maíces nativos de Oaxaca, México, infectados con Aspergillus flavus in vitro

The glyoxalase system plays an important role in various physiological processes in plants when they are subjected to different types of stress, whether physical, chemical or biological. Aspergillus flavus is an aflatoxin-producing fungus that contaminates dry grains, leading to a gradual deterioration of the grains and a significant reduction in their nutritional value. The objective of the present study was to evaluate the activity of the enzyme glyoxalase I (GLX-I) in maize coleoptiles from Oaxaca in response to infection caused by Aspergillus flavus. Nine maize samples from four different races were analyzed. The samples were inoculated with a suspension of Aspergillus flavus spores of known concentration and total protein extraction and quantification were performed on the coleoptiles, and GLX-I activity was determined by quantifying the amount of S-lactoylglutathione produced per minute. In addition, analysis of gene expression by reverse transcriptase polymerase chain reaction (RT-PCR) was performed. The inoculated maize coleoptiles showed symptoms of infection, color changes and wilting. The concentration of total proteins decreased significantly in the extracts of four samples in the presence of the fungus. In the GLX-I analysis, two samples had the highest enzymatic activity in the infected coleoptile extract with respect to the healthy one, in addition to presenting greater expression of the gene in the RT-PCR assay, this due to the response to Aspergillus flavus infection.

NMRpQuant: an automated software for large scale urinary total protein quantification by one-dimensional 1H NMR profiles.

Summary 1H nuclear magnetic resonance (NMR) spectroscopy is an established bioanalytical technology for metabolic profiling of biofluids in both clinical and large-scale population screening applications. Recently, urinary protein quantification has been demonstrated using the same 1D 1H NMR experimental data captured for metabolic profiling. Here, we introduce NMRpQuant, a freely available platform that builds on these findings with both novel and further optimized computational NMR approaches for rigorous, automated protein urine quantification. The results are validated by interlaboratory comparisons, demonstrating agreement with clinical/biochemical methodologies, pointing at a ready-to-use tool for routine protein urinalyses.Availability and implementation NMRpQuant was developed on MATLAB programming environment. Source code and Windows/macOS compiled applications are available at https://github.com/pantakis/NMRpQuant, and working examples are available at https://doi.org/10.6084/m9.figshare.18737189.v1.Supplementary information Supplementary data are available at Bioinformatics online.

The Effects of Nitrogen and Phosphorus on Colony Growth and Zoospore Characteristics of Soil Chytridiomycota.

The Chytridiomycota phylum contributes to nutrient cycling and the flow of energy between trophic levels in terrestrial and aquatic ecosystems yet remains poorly described or absent from publications discussing fungal communities in these environments. This study contributes to the understanding of three species of soil chytrids in vitro—Gaertneriomyces semiglobifer, Spizellomyces sp. and Rhizophlyctis rosea—in the presence of elevated concentrations of nitrogen and phosphorus and with different sources of nitrogen. Colony growth was measured after 4 weeks as dry weight and total protein. To determine the impacts on zoospore reproduction, motility, lipid content, and attachment to organic substrates, 4- and 8-week incubation times were investigated. Whilst all isolates were able to assimilate ammonium as a sole source of nitrogen, nitrate was less preferred or even unsuitable as a nutrient source for G. semiglobifer and R. rosea, respectively. Increasing phosphate concentrations led to diverse responses between isolates. Zoospore production was also variable between isolates, and the parameters for zoospore motility appeared only to be influenced by the phosphate concentration for Spizellomyces sp. and R. rosea. Attachment rates increased for G. semiglobifer in the absence of an inorganic nitrogen source. These findings highlight variability between the adaptive responses utilised by chytrids to persist in a range of environments and provide new techniques to study soil chytrid biomass and zoospore motility by total protein quantification and fluorescent imaging respectively.

Implementation of a Practical Teaching Course on Protein Engineering.

Simple SummaryProteins are the workhorses of the cell. With different combinations of the 20 common amino acids and some modifications of these amino acids, proteins have evolved with a staggering array of new functions and capabilities due to Protein Engineering techniques. The practical course presented was offered to undergraduate bioengineering and chemical students at the Faculty of Engineering of the University of Porto (Portugal) and consists of sequential laboratory sessions to learn the basic skills related to the expression and purification of recombinant proteins in bacterial hosts. These experiments were successfully applied by students as all working groups were able to isolate a model recombinant protein (the enhanced green fluorescent protein) from a cell lysate containing a mixture of proteins and other biomolecules produced by an Escherichia coli strain and evaluate the performance of the extraction and purification procedures they learned.Protein Engineering is a highly evolved field of engineering aimed at developing proteins for specific industrial, medical, and research applications. Here, we present a practical teaching course to demonstrate fundamental techniques used to express, purify and analyze a recombinant protein produced in Escherichia coli—the enhanced green fluorescent protein (eGFP). The methodologies used for eGFP production were introduced sequentially over six laboratory sessions and included (i) bacterial growth, (ii) sonication (for cell lysis), (iii) affinity chromatography and dialysis (for eGFP purification), (iv) bicinchoninic acid (BCA) and fluorometry assays for total protein and eGFP quantification, respectively, and (v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for qualitative analysis. All groups were able to isolate the eGFP from the cell lysate with purity levels up to 72%. Additionally, a mass balance analysis performed by the students showed that eGFP yields up to 46% were achieved at the end of the purification process following the adopted procedures. A sensitivity analysis was performed to pinpoint the most critical steps of the downstream processing.

Quantification of Soy-Derived Ingredients in Model Bread and Frankfurter Matrices with an Optimized Liquid Chromatography–Tandem Mass Spectrometry External Standard Calibration Workflow

The detection and quantification of soy protein is important for food allergen management and identifying the presence of undeclared soy proteins. Heat processing and matrix interactions can affect the accuracy of allergen detection methods. The sensitivity of enzyme-linked immunosorbent assay methods can be compromised if protein epitopes are modified during processing. Therefore, a mass spectrometry (MS)-based method was evaluated for the recovery of total soy protein in incurred matrices. MS-based quantification of total soy protein was assessed by using a combination of external and internal standards. The reproducibility of the standard curves was investigated by comparing within-day and among-day variation. Incurred samples were prepared using bread and frankfurters as model food matrices. Several soy-derived ingredients were used to prepare the matrices with varying levels of soy protein (1, 10, 50, or 100 ppm of total soy protein). A pooled standard curve was used to estimate the total soy protein concentration of the incurred food matrices and the percent total protein recovery. The variation of replicate standard curves between days and among all days was not significant. The differences in slopes obtained from replicate standards run on different days were minimal. The most influential factor on the quantitative protein recovery in incurred samples was the effect of the physical matrix structure on protein extraction. The lowest percent protein recoveries, less than 50%, were calculated for uncooked matrices. The cooked matrices had percentage recoveries between 50 and 150% for all total soy protein levels. Other factors, such as type of ingredient, were determined to be not as impactful on recovery. The MS method described in this study was able to provide sensitive detection and accurate quantification of total soy protein from various soy-derived ingredients present in processed food matrices.

Production and Isolation of the Candida Species Biofilm Extracellular Matrix.

The extracellular matrix (ECM) is a dynamic structure comprising of all four classes of macromolecules. In the biofilm setting, this matrix is key to the survival of microbial communities by conferring to biofilms both structural integrity and protection against diverse environmental insults. In Candida spp., this matrix contributes to pathogenesis by conferring to biofilms both drug resistance and protection against immune attack. Understanding the biochemical nature of the matrix and its individual components is critical to the development of novel diagnostics and antifungal strategies against persistent Candida biofilm infections. Therefore, efficient methods for ECM isolation are required. The two matrix isolation protocols described herein are adapted for both small- and large-scale isolation of biofilm matrix. Both procedures involve seeding of biofilms in either 6-well plates or large-surface-area roller bottles, followed by cell adhesion and biofilm maturation for 2days with continuous motion. In both cases, the matrix is separated from the biomass via sonication, a step which gently and effectively removes the matrix without disturbing the fungal cell wall. The large-scale protocol includes additional filtration, lyophilization, and dialysis steps to yield purified matrix material sufficient for numerous biochemical, structural, and functional assays. Small-scale isolation yields enough matrix for gas chromatography (GC), total carbohydrate quantification via the phenol-sulfuric acid method, and total protein quantification via the bicinchoninic acid (BCA) assay. Large-scale isolation yields enough matrix to perform NMR spectroscopy, liquid chromatography, mass spectrometry, and nucleic acid sequencing. These protocols have been adapted for use in Candida species but may be adapted for other biofilm-forming fungal species and bacteria.

Immunomodulatory properties of dilute drugs in human monocyte/macrophages cell line THP-1 as a model of dose-response effects

Immunomodulatory properties of dilute drugs in human monocyte/macrophages cell line THP-1 as a model of dose-response effects

Narrow Precursor Mass Range for DIA-MS Enhances Protein Identification and Quantification in Arabidopsis.

Data independent acquisition–mass spectrometry (DIA–MS) is becoming widely utilised for robust and accurate quantification of samples in quantitative proteomics. Here, we describe the systematic evaluation of the effects of DIA precursor mass range on total protein identification and quantification. We show that a narrow mass range of precursors (~250 m/z) for DIA–MS enables a higher number of protein identifications. Subsequent application of DIA with narrow precursor range (from 400 to 650 m/z) on an Arabidopsis sample with spike-in known proteins identified 34.7% more proteins than in conventional DIA (cDIA) with a wide precursor range of 400–1200 m/z. When combining several DIA–MS analyses with narrow precursor ranges (i.e., 400–650, 650–900 and 900–1200 m/z), we were able to quantify 10,099 protein groups with a median coefficient of variation of <6%. These findings represent a 54.7% increase in the number of proteins quantified than with cDIA analysis. This is particularly important for low abundance proteins, as exemplified by the six-protein mix spike-in. In cDIA only five out of the six-protein mix were quantified while our approach allowed accurate quantitation of all six proteins.

Cytochrome P450 3A4 (CYP3A4) protein quantification using capillary western blot technology and total protein normalization

The western blot (WB) is the predominate method for protein quantification, frequently used in pharmacological and toxicological studies. To control for technical variation, WB signals are normalized through immunodetection of an internal standard “house-keeping” gene or total protein quantification via staining of the same blot or a duplicate, sister blot. Increasing evidence suggests that house-keeping genes are subject to change after drug treatment or under disease states, causing protein quantification errors in WB. Recent advances in automated capillary-based WB technologies enable measurement of the protein of interest, internal standards, and total protein in a single capillary. Using this approach, we quantified cytochrome P450 3A4 (CYP3A4) across 179 liver samples and compared normalization by both β-actin and total protein to determine which better functions as an internal standard. CYP3A4 is responsible for metabolism of a wide array of xenobiotics and is known to exhibit large inter-person variation, making it a good candidate to evaluate protein quantification. We observed significant differences in β-actin protein levels between liver samples (~20-fold) and found better correlation between CYP3A4 protein and mRNA using total protein normalization than β-actin, indicating total protein normalization to be less error prone for estimation of CYP3A4. Furthermore, by using total protein normalization, we confirmed significant association between CYP3A4 protein expression and the functional CYP3A4 variant CYP3A4*22, which contains two linked SNPs rs35599367 and rs62471956. Our results indicate that the automatic capillary WB instrument combined with total protein normalization provides a high throughput and robust approach for protein quantification.

ATR-FTIR spectroscopy for the routine quality control of exosome isolations

Exosomes are nanosized vesicles containing specific cargos of DNA, RNA, proteins, metabolites, and intracellular and membrane lipids. Exosome isolation needs to be optimized carefully depending on the type of biofluid and tissue and the retrieved exosomes need to be characterized. The main objective of this study was to determine the feasibility of a multimodal analysis of Attenuated Total Reflectance – Fourier Transform Infrared (ATR-FTIR) spectroscopy and UPLC–QqTOF-MSMS for the development of a routine quality control tool of isolated exosomes and the rapid characterization of their lipid profiles and total protein content. Using human milk as model example, exosomes were isolated by multi-stage ultracentrifugation. After single-phase extraction, lipidomic analysis was carried out by UPLC–QqTOF-MSMS with automated MSMS-based annotation using HMDB, METLIN, LipidBlast and MSDIAL databases. The classes with the largest number of annotated features were glycerophospholipids, sphingolipids, and glycerolipids. Then, dry films of 2 ​μL exosomes were directly analyzed by ATR-FTIR. Multivariate analysis showed significant associations between ATR-FTIR specific regions and the concentrations of different lipid classes. Principal component analysis and Hierarchical Cluster Analysis of IR and lipidomic data showed that ATR−FTIR renders valuable qualitative descriptors of the lipid content of isolated exosomes. Total LC-MS lipid and total protein contents could also be quantified by using the area of CHs and CO stretching bands as well as the amide I band. As a conclusion, results obtained show that multimodal analysis of ATR-FTIR and UPLC-MS data is a useful tool for the development of spectroscopic methods. ATR-FTIR provided both, qualitative and quantitative chemical descriptors of isolated exosomes, enabling a fast and direct quantification of total protein and lipid contents.

Analysis of permeability and biological properties of dentin treated with experimental bioactive glasses

ObjectivesTo evaluate obliterating capability and biological performance of desensitizing agents. Methods50 dentin blocks were distributed according to the desensitizing agent used (n = 10): Control (Artificial saliva); Ultra EZ (Ultradent); Desensibilize Nano P (FGM); T5-OH Bioactive Glass (Experimental solution); F18 Bioactive Glass (Experimental solution). Desensitizing treatments were performed for 15 days. In addition, specimens were subjected to acid challenge to simulate oral environment demineralizing conditions. Samples were subjected to permeability analysis before and after desensitizing procedures and acid challenge. Cytotoxicity analysis was performed by using Alamar Blue assay and complemented by total protein quantification by Pierce Bicinchoninic Acid assay at 15 min, 24-h and 48-h time points. Scanning electron microscopy and energy dispersion X-ray spectroscopy were performed for qualitative analysis. Data of dentin permeability was analyzed by two-way repeated measures ANOVA and Tukey's test. For cytotoxicity, Kruskal-Wallis and Newman-Keuls tests. Resultsfor dentin permeability there was no significant difference among desensitizing agents after treatment, but control group presented highest values (0.131 ± 0.076 Lp). After acid challenge, control group maintained highest values (0.044 ± 0.014 Lp) with significant difference to other groups, except for Desensibilize Nano P (0.037 ± 0.019 Lp). For cytotoxicity, there were no significant differences among groups. ConclusionBioglass-based desensitizers caused similar effects to commercially available products, regarding permeability and dentin biological properties. Clinical significanceThere is no gold standard protocol for dentin sensitivity. The study of novel desensitizing agents that can obliterate dentinal tubules in a faster-acting and long-lasting way may help meet this clinical need.

Osteogenesis and biofilms formation on titanium surfaces submitted to oxygen plasma immersion ion implantation

The objectives of this study were to characterize titanium (Ti) surfaces treated by ion implantation by immersion in oxygen plasma (O-PIII) at different temperatures, correlating these implanted layers with therapeutic effects and with osteogenesis, as well as the formation of monotypic biofilms microbial. The groups were divided into: a) Ti (pre-treatment) b) Ti O-PIII at 400 ° C. c) Ti O-PIII at 500 ° C. d) Ti O-PIII at 600 ° C. The properties and surface characteristics were evaluated according to the roughness, texture, corrosion resistance, formation of new phases and the identification of chemical compounds present. Cellular analyzes investigated cell interaction, viability, total protein content, alkaline phosphatase and quantification of mineralized nodules using MG-63 cells. The formation of monotypic microbial biofilms, including P. aeruginosa, S. aureus, S. mutans and C. albicans were evaluated. The increase in surface roughness, corrosion resistance and oxygen content, leading to the formation of TiO2-rutile with more intense peaks and in greater numbers according to the increase in the substrate temperature, ionic implanted Ti samples was observed. There was also a significant increase in cell viability, total protein production, alkaline phosphatase activity and formation of mineralization nodules for the group treated with O-PIII at 600ºC compared to other groups, in addition to a reduction of microorganisms in the groups treated with O- PIII. Therefore, treatment with O-PIII at 600ºC in Ti grade IV showed favorable results for its use.

COMPOSITIONAL CHARACTERISTICS OF RUDRAKSHA (ELAEOCARPUS GANITRUS Roxb.)

Rudraksha bead or Elaeocarpus ganitrus (Roxb.) is a reverent plant based therapeutic agent with spiritual and holistic importance in traditional system of medicine since time immemorial. While multifarious pharmacological properties of Rudraksha beads have been reported against a range of chronic disorders, evidences about its compositional characteristics are highly superficial. The present study aims to quantify the biochemical, phytochemicals, functional groups, and elemental characteristics of three, four, and five Mukhi Rudraksha (3MR, 4MR, and 5MR) beads. Biochemical analysis encompasses quantification of total carbohydrates, total proteins, and crude fat content. Phytochemical analysis was performed to quantify alkaloids, saponins, flavonoids, phenolics, anthocyanins, ascorbic acid, tannins, and terpenoids. Identification of functional groups was carried out through Fourier transform infrared (FTIR) spectrophotometer and UV spectrophotometer. Elemental analysis was performed using CHNSO analyzer. One Way ANOVA with Posthoc Multiple Tukeys test was used for statistical analysis. Significant differences were obtained in all the biochemical, phytochemicals, functional groups, and elemental components among 3MR, 4MR, and 5MR. Future studies are warranted to identify the active biomarkers in different Mukhi Rudraksha that will add new therapeutic candidates in the ever-growing number

Systematic stability testing of insulins as representative biopharmaceuticals using ATR FTIR-spectroscopy with focus on quality assurance.

.Significance: Bioactive proteins represent the most important component class in biopharmaceutical products for therapeutic applications. Their production is most often biotechnologically realized by genetically engineered microorganisms. For the quality assurance of insulins as representatives of life-saving pharmaceuticals, analytical methods are required that allow more than total protein quantification in vials or batches. Chemical and physical factors such as unstable temperatures or shear rate exposure under storage can lead to misfolding, nucleation, and subsequent fibril forming of the insulins. The assumption is valid that these processes go parallel with a decrease in bioactivity.Aim: Infrared (IR) spectroscopy has been successfully utilized for secondary structure analysis in cases of protein misfolding and fibril formation.Approach: A reliable method for the quantification of the secondary structure changes has been developed using insulin dry-film Fourier-transform IR spectroscopy in combination with the attenuated total reflection (ATR) technique and subsequent data analyses such as band-shift determination, spectral band deconvolution, and principal component analysis.Results: A systematic study of insulin spectra was carried out on model insulin specimens, available either as original formulations or as hormones purified by ultrafiltration. Insulin specimens were stored at different temperatures, i.e., 0°C, 20°C, and 37°C, respectively, for up to three months. Weekly ATR-measurements allowed the monitoring of hormone secondary structure changes, which are supposed to be negatively correlated with insulin bioactivity.Conclusions: It could be shown that IR-ATR spectroscopy offers a fast and reliable analytical method for the determination of secondary structural changes within insulin molecules, as available in pharmaceutical insulin formulations and therefore challenges internationally established measurement techniques for quality control regarding time, costs, and effort of analysis.

Molecular neural crest cell markers enable discrimination of organophosphates in the murine cardiac embryonic stem cell test.

The cardiac embryonic stem cell test (ESTc) originally used the differentiation of beating cardiomyocytes for embryotoxicity screenings of compounds. However, the ESTc consists of a heterogeneous cell population, including neural crest (NC) cells, which are important contributors to heart development in vivo. Molecular markers for NC cells were investigated to explore if this approach improved discrimination between structurally related chemicals, using the three organophosphates (OP): chlorpyrifos (CPF), malathion (MLT), and triphenyl phosphate (TPP). To decrease the test duration and to improve the objective quantification of the assay read-out, gene transcript biomarkers were measured on study day 4 instead of the traditional cardiomyocyte beating assessment at day 10. Gene expression profiling and immunocytochemistry were performed using markers for pluripotency, proliferation and cardiomyocyte and NC differentiation. Cell proliferation was also assessed by measurements of embryoid body (EB) size and total protein quantification (day 7). Exposure to the OPs resulted in similar patterns of inhibition of beating cardiomyocyte differentiation and of myosin protein expression on day 10. However, these three chemically related compounds induced distinctive effects on NC cell differentiation, indicated by changes in expression levels of the NC precursor (Msx2), NC marker (Ap2α), and epithelial to mesenchymal transition (EMT; Snai2) gene transcripts. This study shows that investigating NC markers can provide added value for ESTc outcome profiling and may enhance the applicability of this assay for the screening of structurally related test chemicals.

Western Blotting Using In-Gel Protein Labeling as a Normalization Control: Advantages of Stain-Free Technology.

Western blotting is one of the most used techniques in research laboratories. It is popular because it is an easy way of semiquantifying protein amounts in different samples. In Western blotting, the most commonly used method for controlling the differences in the amount of protein loaded is to independently quantify housekeeping proteins (typically actin, GAPDH or tubulin). Another less commonly used method is total protein normalization using stains, such as Ponceau S or Coomassie Brilliant Blue, which stains all the proteins on the blots. A less commonly used but powerful total protein staining technique is stain-free normalization. The stain-free technology is able to detect total protein in a large linear dynamic range and has the advantage of allowing protein detection on the gel before transblotting. This chapter discusses the theory, advantages, and method used to do total protein quantification using stain-free gels for normalization of Western blots.

The sequential effects of a multifactorial detergent based decellularization process on bovine pericardium

Decellularization is a promising method for obtaining extracellular matrix scaffolds (ECM) to be used as replacement material in reconstructive procedures. The effectiveness of decellularization and the alterations to the ECM vary, depending on several factors, including the tissue source, composition and density. With an optimized decellularization process, decellularized scaffolds can preserve the spatial and temporal ECM microenvironment, which play an integral role in modulating cell migration, proliferation and differentiation. The exploration of a variety of decellularization protocols has led to mixed outcomes and comparisons between decellularization protocols could not attribute these differences to any single step in a multiple-step process. This study aimed to characterize the effects of each step of a multifactorial decellularization method on the scaffold structure and mechanical integrity of bovine pericardium. Each step of the decellularization process and the effect on the tissue was assessed using hematoxylin and eosin staining, electron microscopy, total protein, ECM protein and triglyceride quantification. The biomechanical properties were assessed using uniaxial tensile strength testing. Cell lysis occurred mainly during the detergent and alcohol steps. Collagen structural damage occurred during the detergent and alcohol steps, with no significant decreased in collagen concentration. No significant damage to elastin could be shown throughout the process, however glycosaminoglycans were significantly removed by detergent treatment. Triglycerides were removed mostly by the alcohol treatment. The strength of the pericardium decreased somewhat after each step of the protocol. It is important to characterize each decellularization protocol with regards to the decellularization efficiency and the effect on the ECM proteins structure and function to accurately evaluate in vivo outcomes.

Lysine and Arginine Protein Post-translational Modifications by Enhanced DIA Libraries: Quantification in Murine Liver Disease.

Proteoforms containing post-translational modifications (PTMs) represent a degree of functional diversity only harnessed through analytically precise simultaneous quantification of multiple PTMs. Here we present a method to accurately differentiate an unmodified peptide from its PTM-containing counterpart through data-independent acquisition-mass spectrometry, leveraging small precursor mass windows to physically separate modified peptidoforms from each other during MS2 acquisition. We utilize a lysine and arginine PTM-enriched peptide assay library and site localization algorithm to simultaneously localize and quantify seven PTMs including mono-, di-, and trimethylation, acetylation, and succinylation in addition to total protein quantification in a single MS run without the need to enrich experimental samples. To evaluate biological relevance, this method was applied to liver lysate from differentially methylated nonalcoholic steatohepatitis (NASH) mouse models. We report that altered methylation and acetylation together with total protein changes drive the novel hypothesis of a regulatory function of PTMs in protein synthesis and mRNA stability in NASH.

Effects of magnesium sulfate and dioctyl sodium sulfosuccinate on fecal hydration, output and systemic hydration in healthy horses

ABSTRACT Magnesium sulphate (MS) and dioctyl sodium sulphosuccinate (DSS) are laxative drugs frequently used for the treatment of impactions. The aim of this study was to compare the effects of MS and DSS in fecal hydration, output and systemic hydration in healthy horses. Five healthy horses received 3 treatments with a 21-day interval. Treatment 1 was performed with administration of 4 liters of warm water; treatment 2: administration of 4 liters of warm water associated with 1g/kg of MS; and treatment 3: administration of 4 liters of warm water associated with 20mg/kg DSS. General and specific physical examination of the digestive system were performed, alongside with packed cell volume and total plasma protein measurement, abdominal ultrasonography and quantification of the amount and hydration of feces, before and 6, 12, 24, 36 and 48 hours after the treatments. No adverse effects were observed. The administration of laxatives promoted greater fecal output and hydration without resulting in systemic dehydration, yet no differences were observed between treatments. The absence of adverse effects of DSS demonstrates the safety of its use as a laxative drug at a dose of 20mg/kg. Studies comparing the effects of the laxative drugs in horses with large colon impaction are needed.