Identification of Phytochemicals from the Water Extract of Eurycoma longifolia Roots using Solid-Liquid and Liquid-Liquid Extraction Based Fractionation Techniques

Phytochemicals in the water extract of Eurycoma longofolia roots were identified using both solid-liquid and liquid-liquid extraction based fractionation techniques. A reversed phase C18 solid phase extraction (SPE) was used as solid-liquid extraction, whereas solvent partition was applied as liquid-liquid extraction. Total saponin was increased after fractionation. A few known quassinoids; eurycomanone, 13α(21)-epoxyeurycomanone, pasakbumin D, 13β,18-dihydroeurycomanol and 13β,21-dihydroxyeurycomanol were identified from the 40% and 60% methanol fractions of SPE. Solvent partition extract using ethyl acetate was found to have the highest saponin content compared to butanol and chloroform fractions. Subsequent acetone precipitation of the organic fractions recovered a formylated hexose trimer and other saccharide-containing compounds. Ethyl acetate effectively recovered saponins from E. longofolia water extract using liquid-liquid extraction followed by acetone precipitation.

Trichloroacetic Acid/Acetone Precipitation Method to Optimize Canine Synovial Fluid for One and Two-Dimensional Gel Electrophoresis Studies

The challenge associated with the use of synovial fluid for osteoarthritic proteome studies is the optimization step, which involves the depletion of high abundant proteins from the samples. The objective of this study was to develop a cost efficient and effective method to remove albumin from canine synovial fluid for proteome studies. Pooled synovial fluid samples were obtained from clinically healthy dogs (n=5), with no radiographic features of osteoarthritis. The acetone precipitation method and 10% w/v of trichloroacetic acid/acetone were chosen to deplete the albumin from canine synovial fluid and the outcome from the different methods were compared using one dimensional and two-dimensional gel electrophoresis studies. The results showed that the 10% w/v TCA/acetone precipitation method removed highly abundant proteins from synovial fluid for gel electrophoresis studies compared to the acetone precipitation method. The 10% w/v TCA/acetone precipitation method provides an effective method to remove albumin from the synovial fluid using gel electrophoresis, especially two-dimensional gel electrophoresis. The accessibility and cost of TCA and acetone make this method of precipitation a simple and cost-effective technique in preparing a canine synovial fluid for two-dimensional gel electrophoresis analysis.

Comparative Analysis of Universal Protein Extraction Methodologies for Screening of Lipase Activity from Agricultural Products

Protein extraction techniques are absolutely required for the research of biological catalysts. The present study compared four universal protein extraction methodologies (ammonium sulfate precipitation, TCA/acetone precipitation, and two commercial kits) to provide practical information on protein extraction in order to discover a novel lipase in agricultural products. Yields of protein extraction from 24 domestic agricultural products and their specific activities were evaluated and compared with each other. TCA/acetone precipitation showed a relatively higher extraction yield (on average, 3.41 ± 1.08 mg protein/0.1 g sample) in crude protein extraction, whereas the Pierce™ Plant Total Protein Extraction Kit showed the highest specific lipase activity on average in both spectrophotometric (266.61 ± 235.78 μU/mg protein) and fluorometric (41.52 ± 32.63 μU/mg protein) assays. Our results suggest that commercial kits for the rapid extraction of soluble functional proteins would be a better choice than conventional precipitation techniques to perform the high-throughput screening of enzyme activity from plant sources. Finally, several agricultural products such as cordyceps, pepper, bracken, and hemp, all of which exhibited an excellent specific lipase activity, were proposed as promising candidates for a source of novel lipases.

Preparation of mare's colostrum and milk sample for 2-DE separation without acetone precipitation

Before electrophoretic separation is performed, the samples must be dissolved in a lysis buffer (necessary to keep proteins dissolved and unbound during proteomic analyses during for a separation of proteins on polyacrylamide gels). The first step in preparing samples for proteomic analyses is their precipitation using e.g. acetone. The aim of precipitation is to obtain proteins from the sample and to remove the compounds interfering with 2-D electrophoresis. Due to difficulties in dissolving some colostrum and mare's milk samples in buffer lysis electrophoretic separation of this biological material was performed without acetone precipitation of proteins. To assess the effectiveness of the applied method, after two-dimensional separation of proteins (2-DE), the obtained gels were stained and archived. The preparation of mare's colostrum and milk samples for proteomic analyses, consisting of defatting, then precipitation of caseins and separation 2-DE, which was not preceded by precipitation of the samples with acetone, resulted in the loss of many protein spots which made it impossible to identify them later using the mass spectrometer.

Evaluation of female Aedes aegypti proteome via LC-ESI-MS/MS using two protein extraction methods

Background Proteomic analyses have broadened the horizons of vector control measures by identifying proteins associated with different biological and physiological processes and give further insight into the mosquitoes’ biology, mechanism of insecticide resistance and pathogens-mosquitoes interaction. Female Ae. aegypti ingests human blood to acquire the requisite nutrients to make eggs. During blood ingestion, female mosquitoes transmit different pathogens. Therefore, this study aimed to determine the best protein extraction method for mass spectrometry analysis which will allow a better proteome profiling for female mosquitoes. Methods In this present study, two protein extractions methods were performed to analyze female Ae. aegyti proteome, via TCA acetone precipitation extraction method and a commercial protein extraction reagent CytoBusterTM. Then, protein identification was performed by LC-ESI-MS/MS and followed by functional protein annotation analysis. Results The CytoBusterTM reagent gave the highest protein yield with a mean of 475.90 µg compared to TCA acetone precipitation extraction showed 283.15 µg mean of protein. LC-ESI-MS/MS identified 1,290 and 890 proteins from the CytoBusterTM reagent and TCA acetone precipitation, respectively. When comparing the protein class categories in both methods, there were three additional categories for proteins identified using CytoBusterTM reagent. The proteins were related to scaffold/adaptor protein (PC00226), protein binding activity modulator (PC00095) and intercellular signal molecule (PC00207). In conclusion, the CytoBusterTM protein extraction reagent showed a better performance for the extraction of proteins in term of the protein yield, proteome coverage and extraction speed.

Phytochemical profile of Orthosiphon aristatus samples prepared by acetone precipitation.

Background: Orthosiphon aristatus is a popular medicinal herb because of its pharmacological significance. Therefore, a lot of products formulated from the plant extract are available in the market. However, many phytochemicals in the plant extract are still remained to be unknown. Objective: The objective of this study was to profile phytochemicals from the plant extracts prepared using water and ethanol in a reflux system for comparison. Method: The commonly used analytical techniques of acetone precipitation (gravimetry) and vanillin assay (colorimetry) were used to estimate total saponin in the plant extracts. Low dielectric constant of acetone (20.7) was used to precipitate saponins. The phytochemicals in the plant extracts and their respective precipitates were then identified by LC-MS/MS. Results: Higher amount of precipitate was formed from ethanol extract than water extract. The results of LC-MS/MS revealed that not only saponins, but other phytochemicals such as phenolic acids, flavonoids, terpenoids and aliphatic acids were also detected in the precipitates. In particular, ethanol extract and its precipitate were found to have higher flavonoids. Acetone precipitation could significantly increase the content of sagerinic acid and rosmarinic acid, as well as polyhydroxylated and polymethoxylated flavones from the water extract. Conclusion: Acetone precipitation is not specific for saponin, but also any phytochemicals which have lower solubility due to the lack of hydrogen bonding in high dipole moment medium of acetone. Gravimetric acetone precipitation had successfully profiled the phytochemicals in both highly complex water and ethanol based crude extracts.

Mass Spectrometric Identification of SARS-CoV-2 Proteins from Gargle Solution Samples of COVID-19 Patients

Mass spectrometry (MS) can deliver valuable diagnostic data that complements genomic information and allows us to increase our current knowledge of the COVID-19 disease caused by the SARS-CoV-2 virus. We developed a simple, MS-based method to specifically detect SARS-CoV-2 proteins from gargle solution samples of COVID-19 patients. Our protocol consists of an acetone precipitation and tryptic digestion of proteins contained within the gargle solution, followed by a targeted MS analysis. Our methodology identifies unique peptides originating from SARS-CoV-2 nucleoprotein. Building on these promising initial results, faster MS protocols can now be developed as routine diagnostic tools for COVID-19 patients.Graphical AbstractImage credit (left): Gerd Altmann, Pixabay License, https://pixabay.com/illustrations/corona-coronavirus-virus-covid-19-4959447