Protein Extraction Methods Research Articles

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.

Effect of cold plasma on breaking activated sludge and the output dominance of protein

ABSTRACT Upon contacting with water, cold plasma should produce numerous ozone molecules and free electrons at room temperature. In this study, a cold plasma generator was used to break the walls of residual activated sludge obtained from domestic sewage. The impact was mainly influenced by the ozone generated. With 800 W power, sludge wastewater pH of 12.0, and under continuous treatment for 10 h, the system’s reduction efficiency for the dry sludge was ≈90%. Furthermore, the organic matter content (especially protein) of the upper layer of the sludge solution increased a lot after the sludge digestion. This observation proved the reduction of sludge from both sides. Moreover, when the cold plasma technique was compared with thermal acid hydrolysis, thermal alkali hydrolysis, and ultrasonication for extracting protein from activated sludge, cold plasma wall-breaking sludge exhibited the highest efficiency, reaching 38.2% under ambient temperature. After the analysis, the toxic metal content in the extracted protein was near zero, which is a level other protein extraction methods via sludge breaking have not achieved to date, we attribute this efficiency to free electrons the cold plasma produce. These species promote the transformation of metal ions into atomic metals, thereby facilitating their removal.

Protein extraction from cold-pressed hempseed press cake: From laboratory to pilot scale.

During the production of industrial hempseed oil, a press cake is formed as a byproduct, which is often used as animal feed although it contains a high amount of protein that could be used for human consumption. Extracting this valuable protein would reduce food waste and increase the availability of plant-based protein. A protein extraction process based on the pH-shift method was adapted to improve the protein extraction yield from industrial hempseed press cake (HPC). Parameters such as alkali extraction pH, time, and temperature, as well as isoelectric precipitation pH, were investigated in laboratory scale and were thereafter carried out in a pilot trial to explore the suitability for future scale up. The phytic acid content of the extracted protein isolate was also analyzed to investigate any potential inhibitory effect on mineral absorption. A final protein yield of 60.6%, with a precipitated protein content of 90.3% (dw), was obtained using a constant alkali extraction pH of 10.5 for 1 h at room temperature, followed by precipitation at pH 5.5. The pilot trial showed promising results for the future production of industrial hemp protein precipitate on a larger scale, showing a protein yield of 57.0% and protein content of 90.8% (dw). The amount of phytic acid in the protein isolate produced in the optimal laboratory experiment and in the pilot trial was 0.595 and 0.557g phytic acid/100g dw, respectively, which is 83%-88% less than in the HPC. This is in the range of other plant-based protein sources (tofu, kidney beans, peas, etc.). PRACTICAL APPLICATION: Industrial hempseed press cake is a byproduct in the production of industrial hempseed oil, which is mostly used as animal feed, but has the potential to become an additional source of plant-based protein for human consumption with a suitable protein extraction method. The extracted hemp protein could be used to develop new plant-based dairy or meat analog products.

Dry bean proteins: Extraction methods, functionality, and application in products for human consumption

AbstractBackground and objectivesLegume proteins, such as those from dried beans, are extracted by methods that can be divided into aqueous or dry fractionation processes. Several techniques have been studied to improve protein extraction in legumes and their functional properties. This article provides relevant information on available extraction methods, their advantages and disadvantages, and the functionality and application of dried bean protein in the manufacture of food products.FindingsIt is possible to obtain yields of up to 93% in dry bean protein extraction by the aqueous fractionation method, resulting in concentrates with a protein content above 70%. The combination of other treatments (thermal or not), applied before or after extraction, can improve the extraction yield and the functional properties of proteins, respectively. Dry fractionation allows yields of up to 30%, which can be improved with electrostatic separation. Bean proteins have equal or superior performance to soybeans in terms of emulsifying properties, high hydrophobicity, and foaming.ConclusionsDry fractionation is a more sustainable method of protein extraction, as it uses few resources and preserves the native properties of proteins. Aqueous fractionation guarantees a higher protein yield. Dried beans are a raw material with enormous potential for protein extraction and application of its protein in the manufacture of vegetable‐based food products with added value and high nutritional and health benefits, such as vegetable meats, for example, which are part of the trend of this decade.Significance and noveltyThis review provides insights into the different bean protein extraction methods and their combinations and how the functional properties can be influenced by them, providing the reader with an available resource to select the most appropriate extraction technique(s) for its application in food.

What You Extract Is What You Get: Different Methods of Protein Extraction from Hemp Seeds

Cannabis sativa L. seeds are rich in essential polyunsaturated fatty acids and highly digestible proteins, with a good nutritional value. Proteomics studies on hempseed reported so far have mainly been conducted on processed seeds and, to our knowledge, no optimization of protein extraction from hemp seeds has been performed. This study investigates the SDS-PAGE profile of hempseed proteins comparing different methods of extraction, (Osborne sequential extraction, TCA/acetone, MTBE/methanol, direct protein solubilization of defatted hempseed flour), two conditions to keep low temperature during seed grinding (liquid nitrogen or ice) and two solubilization buffers (urea-based or Laemmli buffer). Among the tested conditions, the combination of liquid nitrogen + TCA/acetone + Laemmli buffer was not compatible with SDS-PAGE of proteins. On the other hand, urea-based buffer achieved more reproducible results if combined with all the other conditions. TCA/acetone, MTBE/methanol, and direct protein solubilization of defatted hempseed flour demonstrated a good overview of protein content, but less abundant proteins were poorly represented. The Osborne sequential separation was helpful in diluting abundant proteins thus enhancing the method sensitivity.

Screening for Anti-Diabetic Peptides from Moringa oleifera leaves

Moringa oleifera commonly called as Drumstick tree is a multipurpose tree and it is widely present in the places like India, Asia, Africa, etc. Its leaves are emetic and their juices are used for medicinal purposes. Diabetes mellitus is a clinical syndrome with insufficient insulin secretion and abnormal glucose tolerance. It is a group of diseases which results in too much sugar in the blood. Leaves from Moringa oleifera were collected, analysed for their anti- diabetic activity. First the proteins are extracted using protein extraction methods, estimated using Lowry’s method, and then anti- diabetic activity was checked using Glucose uptake assay by yeast cells, where the plant extract (small proteins) was able to uptake glucose. SDS-PAGE was carried out to check the protein’s size and molecular weight. The characterization of Moringa oleifera conducted in this study shows that the small peptides from the leaves of this plant can contribute significantly in the daily recommended intake of it, since it serves as a rich source of all minerals, vitamins etc. This study has confirmed that the small peptides from the leaves of M.oleifera can fight against diabetics and the result indicated that Murungai (Moringa) leaves are suitable source of green leaf to reduce the diabetic complications in diabetic patients.

Effects of Various Extraction Factors on Protein Yield of Haliotis discus hannai (Abalone)

Abalone (Haliotis discus hannai) is a widely consumed seafood in Asian countries. Rich in protein, abalone is consumed for refreshment, pregnancy care, and vitality. Although many studies have found that abalone protein has beneficial effects, the efficiency of the protein extraction method for abalone has rarely been studied. Therefore, this study aimed to examine the effects of various factors of abalone protein extraction, including extraction buffer, sonication, salt (NaCl) concentration, surfactant, and heating. Phosphate buffer showed higher protein yield compared with Tris-HCl buffer. In addition, the highest protein yield for each factor was observed at 60 s of sonication (84.44 µg/mg dw), 0.6 M NaCl (141.9 µg/mg dw), and 16 mM sodium dodecyl sulfate (SDS) (253.15 µg/mg dw). However, a combined effect was not observed. Lower protein extraction efficiency was observed for sous vide-cooked abalone. The electrophoresis assay revealed myofibrillar proteins, including paramyosin, actin, and tropomyosin. Overall, our results demonstrate that various extract conditions affect the protein extraction of abalone.

Proteomic Analysis of Common Bean (Phaseolus vulgaris L.) by Two-Dimensional Gel Electrophoresis and Mass Spectrometry

The modern cultivated common bean (Phaseolus vulgaris) has evolved from wild common beans distributed in Central America, Mexico and the Andean region of South America. It has been reported that wild common bean accessions have higher levels of protein content than the domesticated dry bean cultivars. However, there is limited proteomic analysis of wild or domesticated common beans. In this investigation, we reported a proteomic analysis of a wild bean from Mexico. We utilized the TCA/Acetone method for protein extraction, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein separation, and subsequent mass spectrometry (MS) for protein identification. Proteins were separated in the first dimension using pH range from 4-7. A total of 237 protein spots from the second dimension were isolated, digested with trypsin, and analyzed by MALDI/TOF/TOF mass spectrometry. We identified 141 protein spots by searching NCBI non redundant databases using the Mascot search engine and found a total of 43 unique proteins. Gene Ontology (GO) analysis was employed to understand the molecular processes in which the identified common bean proteins are involved. The majority of proteins are involved in binding (41.5%) and catalytic activity (35.8%), followed by nutrient reservoir activity (7.5%), antioxidant activity (1.9%), transporter activity (3.8%), enzyme regulator activity (3.8%), structural molecule activity (1.9%), and electron carrier activity (3.8%). The results indicate that TCA/Acetone extraction and 2D-PAGE is efficient in separating common bean proteins. Further identification of these proteins by MS demonstrates the quality of this protein extraction method. These results will be useful for the development of value added common beans by alteration of protein components.

An efficient protein extraction method applied to mangrove plant Kandelia obovata leaves for proteomic analysis

BackgroundMangroves plants, an important wetland system in the intertidal shores, play a vital role in estuarine ecosystems. However, there is a lack of a very effective method for extracting protein from mangrove plants for proteomic analysis. Here, we evaluated the efficiency of three different protein extraction methods for proteomic analysis of total proteins obtained from mangrove plant Kandelia obovata leaves.ResultsThe protein yield of the phenol-based (Phe-B) method (4.47 mg/g) was significantly higher than the yields of the traditional phenol (Phe) method (2.38 mg/g) and trichloroacetic acid-acetone (TCA-A) method (1.15 mg/g). The Phe-B method produced better two-dimensional electrophoresis (2-DE) protein patterns with high reproducibility regarding the number, abundance and coverage of protein spots. The 2-DE gels showed that 847, 650 and 213 unique protein spots were separated from the total K. obovata leaf proteins extracted by the Phe-B, Phe and TCA-A methods, respectively. Fourteen pairs of protein spots were randomly selected from 2-DE gels of Phe- and Phe-B- extracted proteins for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) technique, and the results of three pairs were consistent. Further, oxygen evolving enhancer protein and elongation factor Tu could be observed in the 2-DE gels of Phe and Phe-B methods, but could only be detected in the results of the Phe-B methods, showing that Phe-B method might be the optimized choice for proteomic analysis.ConclusionOur data provides an improved Phe-B method for protein extraction of K. obovata and other mangrove plant tissues which is rich in polysaccharides and polyphenols. This study might be expected to be used for proteomic analysis in other recalcitrant plants.

Mass spectrometry determination of potential species-specific peptide markers in commercial seasoning cubes

In recent years, issues related to food product authenticity have been alarming, particularly in meat-based products. Proteomic techniques in food authentication and their applications in determining quality and safety issues of ingredients have been progressively developed by researchers. The complexity of matrices in commercial seasoning cubes is one of the main concerns in developing a robust authentication method. Hence, the present study focuses on the development of a protein extraction method for commercial seasoning cubes and to determine potential species-specific peptide markers in commercial seasoning cubes. The efficacy of solvent extraction was determined using Bradford assays, while the electrophoretic profiles of commercial seasoning cubes were visualized via Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The determination of potential species-specific peptide markers in commercial seasoning cubes was carried out via Liquid Chromatography (LC)-Orbitrap Fusion-Mass spectrometry (MS/MS) analysis (Thermo Fisher Scientific). In this study, the urea-thiourea solvent was ideal in extracting protein from commercial seasoning cubes as it produces a homogenous solution upon mixing, suggesting the diffusibility of solutes that is essential for continuing analysis. Moreover, a bovine-specific peptide marker ‘VGGHAAEYGAEALER’ and two porcine-specific peptide markers; ‘VGGQAGAHGAEALER’ and ‘EAVLGLWGK’ were discovered. The methodology developed in this study was successful in identifying species-specific peptide markers in seasoning cubes and can be used as a reference for validating the authenticity of food products, especially commercial seasonings.