Protein Extraction Methods Research Articles

Evaluation of early detection of mucorales by rapid one step off- plate protein extraction using maldi -tof ms

Aim: To validate a simple one step method of protein extraction of moulds for routine early detection of fungi using automated instrument like MALDI- TOF MS. Methodology: A total of 123 clinical mould isolated were tested in Department of Microbiology, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India during period of 3 months (August 2021-October 2021) with the routine Laboratory and culture investigations. Out of these specimens, approximately 50 specimens were suspected of Mucorales based on their growth timing, colony morphology, history of patient, character of tissue or sample, and corresponding KOH mount findings having hyphae in tissues or samples were included in study. Samples were inoculated on routine fungal culture media like SDA. The plates were examined at earliest for appearance of any growth and the process of protein extraction was done to be analysed in MALDI- TOF MS and compared with KOH preparation findings of sample. The culture were again incubated further for appearance of mature colony and later on isolates were identified by LPCB mount examination. Results: Around 70% growth of mucorales could be identified by this method and 30% could not be identified.

Dry fractionation to produce functional fractions from mung bean, yellow pea and cowpea flour

Dry fractionation of mung bean, yellow pea, and cowpea was performed to investigate the compositions and functional properties of the obtained fractions. Dry fractionation consisted of milling and subsequent air classification using three air classifier wheel speeds. Water holding capacity, gelation, and rheological properties of all fractions were studied. Separation between protein and starch granules was successfully performed by air classifying, as visualized with SEM images. Protein contents in fine fractions ranged from 42% to 58% (w/w), which was dependent on classifier wheel speed and material source. The correlation between water holding capacity and protein content was dependent on material source. Gelation of fine fractions occurred at lower concentrations than flours and coarse fractions. Fine fractions of all materials formed gels with higher elastic modulus (G') than the flour and coarse fractions, while the flour and coarse fractions formed more solid-like gels (lower tan δ). Industrial relevanceThe need for functional plant protein ingredients for food applications is growing continuously. It is thus important to study sustainable methods for plant protein extraction. Dry fractionation is often considered an efficient alternative to the conventional wet fractionation process, that preserves the native functionality of the proteins in addition. For the application of ingredients produced from dry fractionation in food products, it is important to understand their functional properties. Therefore, the functional properties of dry fractionated ingredients produced from three different legumes are evaluated in this manuscript.

Deep eutectic solvents and alkaline extraction of protein from seabuckthorn seed meal: a comparison study

Seabuckthorn seed meal (SSM) is a waste of oil extraction industry that rich in protein. In order to seek suitable protein extraction method, three different deep eutectic solvents (DESs) (including choline chloride-glycerol, choline chloride-oxalic acid and choline chloride-urea) were developed for extracting protein from SSM and compared with alkaline. Result indicated that alkaline could effectively extract 56.9% protein from SSM and its protein content was 73.1%, higher than DES at 31.0%41.4% and 64.3%67.5%, respectively. However, compared to alkali, DES led to a product with less β-sheet, more β-turn, more essential amino acids, higher total amino acid content, especially choline chloride-urea which extracted protein showing an integrated and similar protein weight distribution compared to SSM. Also, this protein extracted chloride-urea showed a highest digestibility in vitro (by pepsin) (54.2%). These results indicated that choline chloride-urea extraction is better than alkaline extraction for SSM.

Objective discrimination of fur type based on electrophoresis optimized for fur-hair proteins.

The accurate identification of animal species used for fur is important for conserving endangered animals, stopping illegal fur distribution, and addressing consumer concerns. Animal species used for fur are currently differentiated by observing species-specific morphological fur-hair features through a microscope. Although this method is simple, the results may differ among inspectors owing to its subjective nature. To develop an objective approach for differentiating animal species based on fur, we utilized the electrophoretic patterns of fur-hair proteins. First, we optimized protein extraction methods to produce clear electrophoretic patterns from fur-hair proteins. Then, we obtained 324 electrophoretic patterns from 54 fur samples belonging to 24 different animals; 216 of the 324 patterns were used for the construction of a discrimination model using two-way orthogonal partial least squares discriminant analysis. The model correctly discriminated between all the remaining 108 patterns without any false negatives or positives. Moreover, this model could discriminate between fur samples from closely related species that are difficult to distinguish using conventional microscopic identification because of the visual similarity of the fur hairs.

Mathematical Models and Methods for Research and Optimization of Protein Extraction Processes from Chickpea and Curd Whey Solutions by Electroflotation Coagulation Method

The development of mathematical models and efficient technologies for the processing of protein-containing dairy and vegetable raw materials and the production of food and feed concentrates with controlled functional properties is one of the most promising areas within the agricultural industry. In this work, the suitability of the electroflotation coagulation method for the combined extraction of vegetable and milk proteins was established by changing the electrolysis parameters and directed regulation of the isoelectric state of proteins. The research methodology is based on modern achievements of leading domestic and foreign researchers in the field of electrolysis of solutions and the creation of reagentless technologies for extracting proteins, as well as on the use of guest methods of physicochemical analysis, pH-metry, potentiometric and organoleptic analysis, methods of cyclic chronovoltammetry and currentless chronopotentiometry. The paper presents technological schemes for the extraction of vegetable and milk proteins, based on the combination of electroflotation and electrocoagulation processes. We carried out technological tests, which made it possible to determine the optimal conditions that ensure the highest yield of the product and its quality indicators. Ready-made isolates and concentrates of chickpea proteins and curd whey were obtained.

Collection, Storage and Protein Extraction Method of Gingival Crevicular Fluid for Proteomic Analysis

Gingival crevicular fluid (GCF) may reflect the events associated with orthodontic tooth movement. Attempts have been conducted to identify biomarkers reflecting optimum orthodontic force, unwanted sequallea (i.e. root resorption) and accelerated tooth movement. The aim of the present study is to find out a standardized GCF collection, storage and total protein extraction method from apparently healthy gingival sites with orthodontics that is compatible with further high-throughput proteomics. Eighteen patients who required extractions of both maxillary first premolars were recruited in this study. These teeth were randomly assigned to either heavy (225g) or light force (25g), and their site specific GCF was collected at baseline and after 1hr, 1day, 7days, 14days, 21days and 28days post force application. Periostrips were used for GCF collection and subsequent phosphate buffered saline (PBS) was used for immediate protein elution with centrifugal speed of 10000rpm for 5min and stored at -80°C. Protein concentration was estimated using Bradford colorimetric assay. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to resolve the purity of proteins in the collected samples and the method of collection was validated by western immuno-blotting of alpha amylase salivary enzyme. The current collection, storage and protein extraction protocol showed the best protein recovery and purity with validated collection free of salivary contamination. In conclusion, tiny GCF volume from healthy sites and evaporation issues of such promising non-invasive fluid motivate us to investigate a standardized protocol enabling optimal preservation of GCF sample and the currently followed protocol may serve as a reference for future proteomic studies searching for GCF biomarkers in diagnosing and monitoring orthodontic tooth movement.

Perspective on the Effect of Protein Extraction Method on the Antinutritional Factor (ANF) Content in Seeds

Plants contain antinutritional factors (ANFs) with negative impact on protein digestibility and nutritional quality. We aim to highlight the importance of extraction method on the coextraction of different ANFs to put focus on this challenge regarding the usability of plant-protein ingredients. We mainly focus on the research behind the established, industrial techniques, wet extraction and dry fractionation, in relation to ANFs. These methods give protein powders differing considerably in protein concentrations and ANF contents. Wet extraction offers the highest protein concentration (typically >85%) and lowest ANF content (reduction between 75% and 96%, depending on the type of ANF). Usage of chemicals and energy has pushed the development of new extraction techniques; hence dry fractionation is explored. However, it was typically found that dry fractionation resulted in an accumulation of some ANFs in the resultant protein concentrates. Innovative separation methods, like selective electrodialysis, forward osmosis, immobilized metal affinity, and zinc reductants, are suggested to be effective processes for concentration, purification, and selection of proteins. In general, more research is needed, and strategies to sophisticate extraction methods, for example by combining with pre- and postprocessing, and ensure high quality of the protein fractions are mandatory.

Membrane extraction with styrene-maleic acid copolymer results in insulin receptor autophosphorylation in the absence of ligand

Extraction of integral membrane proteins with poly(styrene-co-maleic acid) provides a promising alternative to detergent extraction. A major advantage of extraction using copolymers rather than detergent is the retention of the lipid bilayer around the proteins. Here we report the first functional investigation of the mammalian insulin receptor which was extracted from cell membranes using poly(styrene-co-maleic acid). We found that the copolymer efficiently extracted the insulin receptor from 3T3L1 fibroblast membranes. Surprisingly, activation of the insulin receptor and proximal downstream signalling was detected upon copolymer extraction even in the absence of insulin stimulation. Insulin receptor and IRS1 phosphorylations were above levels measured in the control extracts made with detergents. However, more distal signalling events in the insulin signalling cascade, such as the phosphorylation of Akt were not observed. Following copolymer extraction, in vitro addition of insulin had no further effect on insulin receptor or IRS1 phosphorylation. Therefore, under our experimental conditions, the insulin receptor is not functionally responsive to insulin. This study is the first to investigate receptor tyrosine kinases extracted from mammalian cells using a styrene-maleic acid copolymer and highlights the importance of thorough functional characterisation when using this method of protein extraction.

Nutritional, Techno-Functional and Structural Properties of Black Soldier Fly (Hermetia illucens) Larvae Flours and Protein Concentrates.

Due to their protein content and balanced amino acid profile, edible insects have been described as an excellent alternative protein source to combat malnutrition. As the global population continues to grow, edible insects such as the black soldier fly larvae (BSFL) may contribute to food security. The effect of different protein extraction methods, i.e., alkaline solution and acid precipitation (BSFL-PC1) and extraction with an alkali (BSFL-PC2), on the nutritional, techno-functional, and structural properties of BSFL flours and protein concentrates were studied. The highest protein content (73.35%) was obtained under alkaline and acid precipitation extraction (BSFL-PC1). The sum of essential amino acids significantly increased (p < 0.05) from 24.98% to 38.20% due to the defatting process during extraction. Protein solubility was significantly higher in protein concentrates (85–97%) than flours (30–35%) at pH 2. The emulsion capacity (EC) was significantly higher (p < 0.05) in the protein concentrates (BSFL-PC1 and BSFL-PC2) compared to the freeze-dried and defatted BSFL flours, while the emulsion stability (ES) was significantly (p < 0.05) higher in BSFL-PC1 (100%) compared with BSFL-PC2 (49.8%). No significant differences (p > 0.05) were observed in foaming stability (FS) between freeze-dried and defatted BSFL flours. Fourier transform infrared spectroscopy (FT-IR) analysis revealed distinct structural differences between BSFL flours and protein concentrates. This was supported by surface morphology through scanning electron microscopy (SEM) images, which showed that the protein extraction method influenced the structural properties of the protein concentrates. Therefore, based on the nutritional and techno-functional properties, BSFL flour fractions and protein concentrates show promise as novel functional ingredients for use in food applications.

Macroalgal Proteins: A Review.

Population growth is the driving change in the search for new, alternative sources of protein. Macroalgae (otherwise known as seaweeds) do not compete with other food sources for space and resources as they can be sustainably cultivated without the need for arable land. Macroalgae are significantly rich in protein and amino acid content compared to other plant-derived proteins. Herein, physical and chemical protein extraction methods as well as novel techniques including enzyme hydrolysis, microwave-assisted extraction and ultrasound sonication are discussed as strategies for protein extraction with this resource. The generation of high-value, economically important ingredients such as bioactive peptides is explored as well as the application of macroalgal proteins in human foods and animal feed. These bioactive peptides that have been shown to inhibit enzymes such as renin, angiotensin-I-converting enzyme (ACE-1), cyclooxygenases (COX), α-amylase and α-glucosidase associated with hypertensive, diabetic, and inflammation-related activities are explored. This paper discusses the significant uses of seaweeds, which range from utilising their anthelmintic and anti-methane properties in feed additives, to food techno-functional ingredients in the formulation of human foods such as ice creams, to utilising their health beneficial ingredients to reduce high blood pressure and prevent inflammation. This information was collated following a review of 206 publications on the use of seaweeds as foods and feeds and processing methods to extract seaweed proteins.

Optimization of proteomics sample preparation for identification of host and bacterial proteins in mouse feces.

Bottom-up proteomics is a powerful method for the functional characterization of mouse gut microbiota. To date, most of the bottom-up proteomics studies of the mouse gut rely on limited amounts of fecal samples. With mass-limited samples, the performance of such analyses is highly dependent on the protein extraction protocols and contaminant removal strategies. Here, protein extraction protocols (using different lysis buffers) and contaminant removal strategies (using different types of filters and beads) were systematically evaluated to maximize quantitative reproducibility and the number of identified proteins. Overall, our results recommend a protein extraction method using a combination of sodium dodecyl sulfate (SDS) and urea in Tris-HCl to yield the greatest number of protein identifications. These conditions led to an increase in the number of proteins identified from gram-positive bacteria, such as Firmicutes and Actinobacteria, which is a challenging task. Our analysis further confirmed these conditions led to the extraction of non-abundant bacterial phyla such as Proteobacteria. In addition, we found that, when coupled to our optimized extraction method, suspension trap (S-Trap) outperforms other contaminant removal methods by providing the most reproducible method while producing the greatest number of protein identifications. Overall, our optimized sample preparation workflow is straightforward and fast, and requires minimal sample handling. Furthermore, our approach does not require high amounts of fecal samples, a vital consideration in proteomics studies where mice produce smaller amounts of feces due to a particular physiological condition. Our final method provides efficient digestion of mouse fecal material, is reproducible, and leads to high proteomic coverage for both host and microbiome proteins.

Current Progress in the Extraction, Functional Properties, Interaction with Polyphenols, and Application of Legume Protein.

Legume protein can replace animal-derived protein because of its high protein content, low price, lack of cholesterol, complete amino acids, and requirements of vegetarianism. Legume protein has not only superior functional properties but also high biological activities. Therefore, it is widely used in the food industry. However, there are few studies on the comprehensive overview of legume protein. In this review, the extraction, functional properties, interaction with polyphenols, application of legume protein, and activities of their peptides were comprehensively reviewed. Legume proteins are mainly composed of globulin and albumin. The methods of protein extraction from legumes mainly include wet separation (alkali solution and acid precipitation, salt extraction, enzyme extraction, and ultrasonic-assisted extraction) and dry separation (electrostatic separation). Besides, various factors (heat, pH, and concentration) could significantly affect the functional properties of legume protein. Some potential modification technologies could further improve the functionality and quality of these proteins. Moreover, the application of legume protein and the effects of polyphenols on structural properties of legume-derived protein were concluded. Furthermore, the bioactivities of peptides from legume proteins were discussed. To improve the bioactivity, bioavailability, and commercial availability of legume-derived protein and peptides, future studies need to further explore new preparation methods and potential new activities of legume-derived proteins and active peptides. This review provides a real-time reference for further research on the application of legume protein in the food industry. In addition, this review provides a new reference for the development of legume-derived protein functional foods and potential therapeutic agents.

Complementary protein extraction methods increase the identification of the Park Grass Experiment metaproteome

Although the Park Grass Experiment is an important international reference soil for temperate grasslands, it still lacks the direct extraction of its metaproteome. The identification of these proteins can be crucial to our understanding of soil ecology and major biogeochemical processes. However, the extraction of protein from soil is a technically fraught process due to difficulties with co-extraction of humic material and lack of compatible databases to identify proteins. To address these issues, we combined two protein extraction techniques on Park Grass experiment soil, one based on humic acid removal, namely a modified freeze-dry, heat/thaw/phenol/chloroform (HTPC) method and another which co-extracts humic material, namely an established surfactant method. A broad range of proteins were identified by matching the mass spectra of extracted soil proteins against a tailored Park Grass proteome database. These were mainly in the categories of “protein metabolism”, “membrane transport”, “carbohydrate metabolism”, “respiration” “ribosomal and nitrogen cycle” proteins, enabling reconstitution of specific processes in grassland soil. Protein annotation using NCBI and EBI databases inferred that the Park Grass soil is dominated by Proteobacteria, Actinobacteria, Acidobacteria and Firmicutes at phylum level and Bradyrhizobium, Rhizobium, Acidobacteria, Streptomyces and Pseudolabrys at genus level. Further functional enrichment analysis enabled us to connect protein identities to regulatory and signalling networks of key biogeochemical cycles, notably the nitrogen cycle. The newly identified Park Grass metaproteome thus provides a baseline on which future targeted studies of important soil processes and their control can be built.

Activity- and Enrichment-Based Metaproteomics Insights into Active Urease from the Rumen Microbiota of Cattle.

Regulation of microbial urease activity plays a crucial role in improving the utilization efficiency of urea and reducing nitrogen emissions to the environment for ruminant animals. Dealing with the diversity of microbial urease and identifying highly active urease as the target is the key for future regulation. However, the identification of active urease in the rumen is currently limited due to large numbers of uncultured microorganisms. In the present study, we describe an activity- and enrichment-based metaproteomic analysis as an approach for the discovery of highly active urease from the rumen microbiota of cattle. We conducted an optimization method of protein extraction and purification to obtain higher urease activity protein. Cryomilling was the best choice among the six applied protein extraction methods (ultrasonication, bead beating, cryomilling, high-pressure press, freeze-thawing, and protein extraction kit) for obtaining protein with high urease activity. The extracted protein by cryomilling was further enriched through gel filtration chromatography to obtain the fraction with the highest urease activity. Then, by using SDS-PAGE, the gel band including urease was excised and analyzed using LC-MS/MS, searching against a metagenome-derived protein database. Finally, we identified six microbial active ureases from 2225 rumen proteins, and the identified ureases were homologous to those of Fibrobacter and Treponema. Moreover, by comparing the 3D protein structures of the identified ureases and known ureases, we found that the residues in the β-turn of flap regions were nonconserved, which might be crucial in influencing the flexibility of flap regions and urease activity. In conclusion, the active urease from rumen microbes was identified by the approach of activity- and enrichment-based metaproteomics, which provides the target for designing a novel efficient urease inhibitor to regulate rumen microbial urease activity.

Simple Scalable Protein Expression and Extraction Using Two-stage Autoinducible Cell Autolysis and DNA/RNA Autohydrolysis in Escherichia coli.

Recombinant protein expression is extensively used in biological research. Despite this, current protein expression and extraction methods are not readily scalable or amenable for high-throughput applications. Optimization of protein expression conditions using traditional methods, reliant on growth-associated induction, is non-trivial. Similarly, protein extraction methods are predominantly restricted to chemical methods, and mechanical methods reliant on expensive specialized equipment more tuned for large-scale applications. In this article, we outline detailed protocols for the use of an engineered autolysis/autohydrolysis E. coli strain, in two-stage fermentations in shake-flasks. This two-stage fermentation protocol does not require optimization of expression conditions and results in high protein titers. Cell lysis in an engineered strain is tightly controlled and only triggered post-culture by addition of a 0.1% detergent solution. Upon cell lysis, a nuclease digests contaminating host oligonucleotides, which facilitates sample handling. This method has been validated for use at different scales, from microtiter plates to instrumented bioreactors. Graphic abstract: Two-stage protein expression, cell autolysis and DNA/RNA autohydrolysis. Reprinted with permission from Menacho-Melgar et al. (2020a). Copyright 2020 John Wiley and Sons.

Protein Extraction and Sample Preparation Methods for Shotgun Proteomics with Central Nervous System Cells and Brain Tissue.

Shotgun proteomics based in mass spectrometry has been extensively utilized to investigate biological samples for basic and applied research in the clinical field to elucidate changes in the molecular mechanisms caused by diseases. There is still a great lack of information about the molecular mechanisms and the origins of most brain disorders, which makes shotgun proteomics an interesting tool in the study of these diseases. A wide range of samples can be used to study such diseases, such as cerebrospinal fluid, central nervous system cells, and brain tissue via postmortem analysis. As such, different protein extraction methods must be applied to achieve the best results for each sample type. The lysis buffer, digestion protocol, and peptide purification steps chosen prior to liquid chromatography-mass spectrometry analyses are essential to obtain reliable proteomic datasets. For this reason, the availability of a list with a variety of methods and a description of the pros and cons for each one has been compiled and elaborated upon. This review presents several methods for protein extraction, protein digestion, and sample cleanup with a focus on shotgun proteomics via mass spectrometry and a further focus on studying brain disorders.

The optimization of PpSP15 purification from salivary glands in Iranian wild Phlebotomus papatasi (Diptera: Psychodidae).

Sand fly saliva contains proteins that modulate the host immune system and it plays an important role in both blood feeding and the outcome of Leishmania infections. The profile of the salivary proteins was examined and analyzed from an endemic focus of zoonotic cutaneous leishmaniasis by wild P. papatasi to find local and suitable antigens as potential proteins for developing Leishmania vaccine alongside the development of a new extraction technique. Specimens were caught from Bojnord, using funnel and CDC traps. Different methods of protein extraction were employed and a new technique was developed. The proteins were extracted from the salivary glands tissues with a lysis buffer. Purification was performed using RP-HPLC, with a linear gradient protocol from 0-60 % of acetonitrile. PpSP15 was characterized by SDS-PAGE. The concentration of extracted protein content was 0.5 and 0.03 μg/μl in chemical and physical methods, respectively. PpSP15 was isolated at a weight of 15kDa in 80-85 min of run time. SDS-PAGE was able to characterize PpSP15. The crude extract of the chemical method, revealed 15 separated bands, ranging from 11-100 KDa. Tajima D index was positive. PpSP15 was characterized from Iranian specimens; it is a very highly hydrophobic protein of salivary glands among SP15- like proteins. The chemical method of extraction was found to be more effective than physical methods (P < 0.05). For developing a vaccine against leishmaniasis, depending on the location, choosing suitable proteins should be considered and an efficient extraction method should be used.

Purification and Phosphoproteomic Analysis of Plasma-Derived Extracellular Vesicles.

A successful phosphoproteomics analysis of extracellular vesicles (EVs) requires a unique approach, fine-tuned to address the challenges that have plagued plasma-based biomarker discovery. Here, I detail a procedure, which combines EVtrap-based high-recovery EV isolation, phase-transfer surfactant method for protein extraction, and PolyMAC-based enrichment of phosphopeptides. The combination of these methods provides a highly effective strategy for EV-based phosphoproteome analysis and leads to the discovery of novel phospho-markers previously undetectable.

Detection of Damage-Activated Metacaspase Activity by Western Blot in Plants.

Metacaspases are cysteine proteases that are present in plants, protists, fungi, and bacteria. Previously, we found that physical damage, e.g., pinching with forceps or grinding on liquid nitrogen of plant tissues, activates Arabidopsis thaliana METACASPASE 4 (AtMCA4). AtMCA4 subsequently cleaves PROPEP1, the precursor pro-protein of the plant elicitor peptide 1 (Pep1). Here, we describe a protein extraction method to detect activation of AtMCA4 by Western blot with antibodies against endogenous AtMCA4 and a PROPEP1-YFP fusion protein. It is important to (1) keep plant tissues at all times on liquid nitrogen prior to protein extraction, and (2) denature the protein lysate as fast as possible, as metacaspase activation ensues quasi immediately because of tissue damage inherent to protein extraction. In theory, this method can serve to detect damage-induced alterations of any protein-of-interest in any organism for which antibodies or fusion proteins are available, and hence, will greatly aid the study of rapid damage-activated proteolysis in the future.

Extracting protein from microalgae (Tetraselmis chuii) for proteome analysis

Microalgae have high potential as a resource for sustainable and green protein for food or bioactive molecules. Nonetheless, despite the high protein content of microalgae (40 - 70% dry weight) progress in the characterization of their protein composition remains challenging. This is due to the highly variable chemical composition of microalgae strains and factors such as their rigid thick cell wall, polysaccharide content, protein stability, pH. The method described herein was developed to optimize protein extraction for proteome analysis of microalgae (Tetraselmis chuii) biomass. The effects on protein solubility of solvent type (organic, denaturing, and non-denaturing) combined with three customized microalgae disruption methods were investigated. The proteome targeted high quality protein extracts were for hydro-soluble proteins recovered by cell disruption using bead milling coupled to centrifugation (protein yield ≈ 13%). The developed method is inexpensive, efficient (yielding high-quality protein extracts with a low content of interfering compounds) and from an industrial perspective easily scalable and compatible with other applications. To add value to the end product we additionally propose the use of stabilizing agents to maintain protein solubility during refrigerated storage and a method targeting the fractionation of low molecular weight proteins.• An inexpensive easy-to-do 5 step protocol for microalgae protein extracts.• A protein extraction method free from dangerous or highly polluting chemicals.• Production of high yield aqueous protein extracts suitable for proteomics.