TMUB1 is an endoplasmic reticulum-resident escortase that promotes the p97-mediated extraction of membrane proteins for degradation.

Membrane protein clients of endoplasmic reticulum (ER)-associated degradation must be retrotranslocated from the ER membrane by the AAA-ATPase p97 for proteasomal degradation. Before direct engagement with p97, client transmembrane domains (TMDs) that have partially or fully crossed the membrane must be constantly shielded to avoid non-native interactions. How client TMDs are seamlessly escorted from the membrane to p97 is unknown. Here, we identified ER-anchored TMUB1 as a TMD-specific escortase. TMUB1 interacts with the TMD of clients within the membrane and holds ∼10-14 residues of a hydrophobic sequence that is exposed out of membrane, using its transmembrane and cytosolic regions, respectively. The ubiquitin-like domain of TMUB1 recruits p97, which can pull client TMDs from bound TMUB1 into the cytosol. The disruption of TMUB1 escortase activity impairs retrotranslocation and stabilizes retrotranslocating intermediates of client proteins within the ER membrane. Thus, TMUB1 promotes TMD segregation by safeguarding the TMD movement from the membrane to p97.

Comparing and integrating TMT-SPS-MS3 and label-free quantitative approaches for proteomics scrutiny in recalcitrant Mango (Mangifera indica L.) peel tissue during postharvest period.

Despite substantial advances in the use of proteomic technologies, their widespread application in fruit tissues of non-model and recalcitrant species remains limited. This hampers the understanding of critical molecular events during the postharvest period of fleshy tropical fruits. Therefore, we evaluated label-free quantitation (LFQ) and TMT-SPS-MS3 (TMT) approaches to analyse changes in the protein profile of mango peels during postharvest period. We compared two extraction methods (phenol and chloroform/methanol) and two peptide fractionation schemes (SCX and HPRP). We accurately identified 3065 proteins, of which, 1492 were differentially accumulated over at 6 days after harvesting (DAH). Both LFQ and TMT approaches share 210 differential proteins including cell wall proteins associated with fruit softening, as well as aroma and flavour-related proteins, which were increased during postharvest period. The phenolic protein extraction and the high-pH reverse-phase peptide fractionation was the most effective pipeline for relative quantification. Nevertheless, the information provided by the other tested strategies was significantly complementary. Besides, LFQ spectra allowed us to track down intact N-glycopeptides corroborating N-glycosylations on the surface of a desiccation-related protein. This work represents the largest proteomic comparison of mango peels during postharvest period made so far, shedding light on the molecular foundation of edible fruit during ripening.

Bone Proteomics Method Optimization for Forensic Investigations.

The application of proteomic analysis to forensic skeletal remains has gained significant interest in improving biological and chronological estimations in medico-legal investigations. To enhance the applicability of these analyses to forensic casework, it is crucial to maximize throughput and proteome recovery while minimizing interoperator variability and laboratory-induced post-translational protein modifications (PTMs). This work compared different workflows for extracting, purifying, and analyzing bone proteins using liquid chromatography with tandem mass spectrometry (LC-MS)/MS including an in-StageTip protocol previously optimized for forensic applications and two protocols using novel suspension-trap technology (S-Trap) and different lysis solutions. This study also compared data-dependent acquisition (DDA) with data-independent acquisition (DIA). By testing all of the workflows on 30 human cortical tibiae samples, S-Trap workflows resulted in increased proteome recovery with both lysis solutions tested and in decreased levels of induced deamidations, and the DIA mode resulted in greater sensitivity and window of identification for the identification of lower-abundance proteins, especially when open-source software was utilized for data processing in both modes. The newly developed S-Trap protocol is, therefore, suitable for forensic bone proteomic workflows and, particularly when paired with DIA mode, can offer improved proteomic outcomes and increased reproducibility, showcasing its potential in forensic proteomics and contributing to achieving standardization in bone proteomic analyses for forensic applications.

Trends in extracting protein from microalgae Spirulina platensis, using innovative extraction techniques: mechanisms, potentials, and limitations.

Microalgal, species are recognized for their high protein content, positioning them as a promising source of this macronutrient. Spirulina platensis, in particular, is noteworthy for its rich protein levels (70 g/100 g dw), which are higher than those of meat and legumes. Incorporating this microalgae into food can provide various benefits to human health due to its diverse chemical composition, encompassing high amount of protein and elevated levels of minerals, phenolics, essential fatty acids, and pigments. Conventional techniques employed for protein extraction from S. platensis have several drawbacks, prompting the exploration of innovative extraction techniques (IETs) to overcome these limitations. Recent advancements in extraction methods include ultrasound-assisted extraction, microwave-assisted extraction, high-pressure-assisted extraction, supercritical fluid extraction, pulse-electric field assisted extraction, ionic liquids assisted extraction, and pressurized liquid extraction. These IETs have demonstrated efficiency in enhancing protein yield of high quality while maximizing biomass utilization. This comprehensive review delves into the mechanisms, applications, and drawbacks associated with implementing IETs in protein extraction from S. platensis. Notably, these innovative methods offer advantages such as increased extractability, minimized protein denaturation, reduced solvent consumption, and lower energy consumption. However, safety considerations and the synergistic effects of combined extraction methods warrant further exploration and investigation of their underlying mechanisms.

A comprehensive review on pulse protein fractionation and extraction: processes, functionality, and food applications.

The increasing world population requires the production of nutrient-rich foods. Protein is an essential macronutrient for healthy individuals. Interest in using plant proteins in foods has increased in recent years due to their sustainability and nutritional benefits. Dry and wet protein fractionation methods have been developed to increase protein yield, purity, and functional and nutritional qualities. This review explores the recent developments in pretreatments and fractionation processes used for producing pulse protein concentrates and isolates. Functionality differences between pulse proteins obtained from different fractionation methods and the use of fractionated pulse proteins in different food applications are also critically reviewed. Pretreatment methods improve the de-hulling efficiency of seeds prior to fractionation. Research on wet fractionation methods focuses on improving sustainability and functionality of proteins while studies on dry methods focus on increasing protein yield and purity. Hybrid methods produced fractionated proteins with higher yield and purity while also improving protein functionality and process sustainability. Dry and hybrid fractionated proteins have comparable or superior functionalities relative to wet fractionated proteins. Pulse protein ingredients are successfully incorporated into various food formulations with notable changes in their sensory properties. Future studies could focus on optimizing the fractionation process, improving protein concentrate palatability, and optimizing formulations using pulse proteins.

A single step and rapid protein extraction protocol developed for cell lines and tissues: Compatible for gel based and gel free proteomic approaches.

Proteins are crucial research molecules in modern biology. Almost every biological research area needs protein-based assays to answer the research questions. The study of the total protein content of a biological sample known as Proteomics, is one of the highly rated qualitative and quantitative approach to address numerous biological problems including clinical research. The key step to successfully generate high quality proteomics data is the efficient extraction of proteins from biological samples. Although different methods are in use for protein extraction from a wide variety of samples, however, because of their prolonged protocol and multiple steps involved, final protein yield is sacrificed. Here, we have shown the development of a simple single step method for extraction of proteins from mammalian cell lines as well as tissue samples in an effective and reproducible manner. This method is based on lysis of samples directly in a modified lysis buffer without CHAPS (7 M Urea, 2 M Thiourea, and 10 mM Tris-Cl; pH 8.5) that is compatible with gel based and gel free approaches. This developed protocol is reliable and should be useful for a wide range of proteomic studies involving various biological samples.

Variability of BMP-2 content in DBM products derived from different long bone.

Demineralized bone matrix (DBM) has been regarded as an ideal bone substitute as a native carrier of bone morphogenetic proteins (BMPs) and other growth factors. However, the osteoinductive properties diverse in different DBM products. We speculate that the harvest origin further contributing to variability of BMPs contents in DBM products besides the process technology. In the study, the cortical bone of femur, tibia, humerus, and ulna from a signal donor were prepared and followed demineralizd into DBM products. Proteins in bone martix were extracted using guanidine-HCl and collagenase, respectively, and BMP-2 content was detected by sandwich enzyme-linked immunosorbent assay (ELISA). Variability of BMP-2 content was found in 4 different DBM products. By guanidine-HCl extraction, the average concentration in DBMs harvested from ulna, humerus, tibia, and femur were 0.613 ± 0.053, 0.848 ± 0.051, 3.293 ± 0.268, and 21.763 ± 0.344, respectively (p < 0.05), while using collagenase, the levels were 0.089 ± 0.004, 0.097 ± 0.004, 0.330 ± 0.012, and 1.562 ± 0.008, respectively (p < 0.05). In general, the content of BMP-2 in long bones of Lower limb was higher than that in long bones of upper limb, and GuHCl had remarkably superior extracted efficiency for BMP-2 compared to collagenase. The results suggest that the origin of cortical bones harvested to fabricate DBM products contribute to the variability of native BMP-2 content, while the protein extracted method only changes the measured values of BMP-2.

Effects of Alkaline Extraction pH on Amino Acid Compositions, Protein Secondary Structures, Thermal Stability, and Functionalities of Brewer's Spent Grain Proteins.

A high alkaline pH was previously demonstrated to enhance the extraction yield of brewer's spent grains (BSG) proteins. The effects of extraction pH beyond the extraction yield, however, has not been investigated before. The present work examined the effects of extraction pH (pH 8-12) on BSG proteins' (1) amino acid compositions, (2) secondary structures, (3) thermal stability, and (4) functionalities (i.e., water/oil holding capacity, emulsifying, and foaming properties). The ideal extraction temperature (60 °C) and BSG-to-solvent ratio (1:20 w/v) for maximizing the extraction yield were first determined to set the conditions for the pH effect study. The results showed that a higher extraction pH led to more balanced compositions between hydrophilic and hydrophobic amino acids and higher proportions of random coils structures indicating increased protein unfolding. This led to superior emulsifying properties of the extracted proteins with more than twofold improvement between pH 8 and a pH larger than 10. The extraction pH, nevertheless, had minimal impact on the water/oil holding capacity, foaming properties, and thermal denaturation propensity of the proteins. The present work demonstrated that a high alkaline pH at pH 11-12 was indeed ideal for both maximizing the extraction yield (37-46 wt.%) and proteins' functionalities.

Ultrasound as Green Technology for the Valorization of Pumpkin Leaves: Intensification of Protein Recovery.

The recovery of valuable nutritional compounds, like proteins, from waste streams and by-products is a key strategy for enhancing production sustainability and opening up new market potential. This research aimed to use high-intensity ultrasound as an innovative technique to extract the soluble proteins from the pumpkin leaves. The impact of various sonication amplitudes and duration periods on protein yield, functional properties, antioxidant qualities, and structural characteristics, were studied. Utilization of ultrasound technology significantly increased the yield of pumpkin leaf protein by up to 40%-six times higher than maceration. The ultrasound extraction provided a RuBisCO-rich protein fraction with high radical scavenging and chelating activities, especially at 40% amplitude. Cavitation modified the tertiary and secondary structures of leaf proteins: the amount of α-helix changed based on amplitude (12.3-37.7%), the amount of random coil increased to 20.4%, and the amount of ß-turn reduced from 31 to 18.6%. The alteration of the protein fluorescence spectrum (blue shift in spectrum) provides further evidence that ultrasound alters the proteins' molecular structure in comparation with maceration; the maximum tryptophan fluorescence intensity decreased from 22.000 to 17.096. The hydrophobicity values of 76.8-101.5 were substantially higher than the maceration value of 53.4, indicating that ultrasound improved the hydrophobicity of protein surfaces. Ultrasound resulted in a significant increase in solubility in an acidic environment with the increase in sonication amplitude. A 2.4-fold increase in solubility at pH 2 becomes apparent (20% amplitude; 43.1%) versus maceration (18.2%). The emulsifying ability decreases from 6.62 to 5.13 m2/g once the sonication amplitude increases by 20-70%. By combining the ultrasound periods and amplitudes, it is possible to create high-value protein leaf extracts with improved properties which can find real application as food additives and dietary supplements.

Effects of ultrasound-assisted intermittent tumbling on the quality of cooked ham through modifying muscle structure and protein extraction.

BACKGROUND: Tumbling treatment is widely used in the production of cooked ham. However, traditional intermittent tumbling (IT) treatment is time-consuming. To enhance the tumbling efficiency, high-intensity ultrasound was used to assist IT treatment (UIT). RESULTS: UIT treatment reduced the tumbling time and significantly improved the water holding capacity, tenderness, sliceability and texture of cooked ham compared to IT treatment. Furthermore, more violent destruction of meat tissue was exhibited in the UIT treatment. This change facilitated extraction of more salt-soluble protein, which in turn welded meat pieces tightly and improved the quality of the cooked ham. CONCLUSION: UIT treatment could accelerate the tumbling process and enhance the quality of cooked ham. These results may provide guidance on effective strategies for a high-quality meat production process. © 2023 Society of Chemical Industry.

Evaluation of protein extraction methods to improve meta-proteomics analysis of treated wastewater biofilms.

Metaproteomics can be used to study functionally active biofilm-based bacterial populations in reclaimed water distribution systems, which in turn result in bacterial regrowth that impacts the water quality. However, existing protein extraction methods have differences in their protein recovery and have not been evaluated for their efficacies in reclaimed water biofilm samples. In this study, we first evaluated six different protein extraction methods with diverse chemical and physical properties on a mixture of bacterial cell culture. Based on a weighting scores-based evaluation, the extraction protocols in order of decreasing performance are listed as B-PER > RIPA > PreOmics > SDS > AllPrep > Urea. The highest four optimal methods on cell culture were further tested against treated wastewater non-chlorinated and chlorinated effluent biofilms. In terms of protein yield, our findings showed that RIPA performed the best; however, the highest number of proteins were extracted from SDS and PreOmics. Furthermore, SDS and PreOmics worked best to rupture gram-positive and gram-negative bacterial cell walls. Considering the five evaluation factors, PreOmics obtained highest weighted score, indicating its potential effectiveness in extracting proteins from biofilms. This study provides the first insight into evaluating protein extraction methods to facilitate metaproteomics for complex reclaimed water matrices.

A Rapid and Efficient Method for the Extraction of Histone Proteins.

Current protocols used to extract and purify histones are notoriously tedious, especially when using yeast cells. Here, we describe the use of a simple filter-aided sample preparation approach enabling histone extraction from yeast and mammalian cells using acidified ethanol, which not only improves extraction but also inactivates histone-modifying enzymes. We show that our improved method prevents N-terminal clipping of H3, an artifact frequently observed in yeast cells using standard histone extraction protocols. Our method is scalable and provides efficient recovery of histones when extracts are prepared from as few as two million yeast cells. We further demonstrate the application of this approach for the analysis of histone modifications in fungal clinical isolates available in a limited quantity. Compared with standard protocols, our method enables the study of histones and their modifications in a faster, simpler, and more robust manner.

Utilizing proteomics to identify and optimize microalgae strains for high-quality dietary protein: a review.

Algae-derived protein has immense potential to provide high-quality protein foods for the expanding human population. To meet its potential, a broad range of scientific tools are required to identify optimal algal strains from the hundreds of thousands available and identify ideal growing conditions for strains that produce high-quality protein with functional benefits. A research pipeline that includes proteomics can provide a deeper interpretation of microalgal composition and biochemistry in the pursuit of these goals. To date, proteomic investigations have largely focused on pathways that involve lipid production in selected microalgae species. Herein, we report the current state of microalgal proteome measurement and discuss promising approaches for the development of protein-containing food products derived from algae.

Unlocking the nutritional and functional potential of legume waste to produce protein ingredients.

Worldwide, many production supply chains generate a considerable amount of legume by-products (e.g., leaves, husks, broken seeds, defatted cakes). These wastes can be revalorized to develop sustainable protein ingredients, with positive economic and environmental effects. To separate protein from legume by-products, a broad spectrum of conventional (e.g., alkaline solubilization, isoelectric precipitation, membrane filtration) and novel methodologies (e.g., ultrasound, high-pressure homogenization, enzymatic approaches) have been studied. In this review, these techniques and their efficiency are discussed in detail. The present paper also provides an overview of the nutritional and functional characteristics of proteins extracted from legume by-products. Moreover, existing challenges and limitations associated with the valorization of by-product proteins are highlighted, and future perspectives are proposed.

Technological advances in protein extraction, structure improvement and assembly, digestibility and bioavailability of plant-based foods.

Plant-based foods are being considered seriously to replace traditional animal-origin foods for various reasons. It is well known that animals release large amounts of greenhouse gases into the environment during feeding, and eating animal-origin foods may also cause some health problems. Moreover, animal resources will likely be in short supply as the world population grows. It is highly likely that serious health problems ascribed to insufficient protein intake in some areas of the world will occur. Studies have shown that environmentally friendly, abundant, and customizable plant-based foods can be an effective alternative to animal-based foods. However, currently, available plant-based foods lack nutrients unique to animal-based foods. Innovative processing technologies are needed to improve the nutritional value and functionality of plant-based foods and make them acceptable to a wider range of consumers. Therefore, protein extraction technologies (e.g., high-pressure extraction, ultrasound extraction, enzyme extraction, etc.), structure improvement and assembly technologies (3D printing, micro-encapsulation, etc.), and technologies to improve digestibility and utilization of bioactive substances (microbial fermentation, physical, etc.) in the field of plant-based foods processing are reviewed. The challenges of plant-based food processing technologies are summarized. The advanced technologies aim to help the food industry solve production problems using efficient, environmentally friendly, and economical processing technologies and to guide the development of plant-based foods in the future.

Exploration on bioactive properties of quinoa protein hydrolysate and peptides: a review.

Quinoa is an excellent source of nutritional and bioactive components. Protein is considered a key nutritional advantage of quinoa grain, and many studies have highlighted the nutritional and physicochemical properties of quinoa protein. In addition, quinoa protein is a good precursor of bioactive peptides. This review focused on the biological properties of quinoa protein hydrolysate and peptides, and gave a summary of the preparation and functional test of quinoa protein hydrolysate and peptides. A combination of milling fractionation and solvent extraction is recommended for the efficient production of quinoa protein. The biological functionalities of quinoa protein hydrolysate, including antidiabetic, antihypertensive, anti-inflammatory, antioxidant activities, and so on, have been extensively investigated based on in vitro studies and limited animal models. Additionally, bioinformatics analysis, including proteolysis simulation, virtual screening, and molecular docking, provides an alternative or assistive approach for exploring the potential bioactivity of quinoa protein and peptides. Nevertheless, further research is required for industrial production of bioactive quinoa peptides, verification of health benefits in humans, and mechanism interpretation of observed effects.

A comparative study for optimization of MALDI-TOF MS identification of filamentous fungi.

PURPOSE: To evaluate and compare the performance of three commercial culture media, two filamentous fungi libraries, and two different protein extraction procedures in MALDI-TOF MS fungal identification. METHODS: A total of 21 quality control samples were cultured on Sabouraud dextrose agar (SDA), ID fungi plate medium (IDFP), and Sabouraud gentamicin chloramphenicol 2 agar (SGC2). For four consecutive days, fungal growths were inoculated on a MALDI target plate both by using a direct transfer technique (DT) and by using a formic acid-ethanol protein extraction procedure (EEP). The MALDI-TOF MS-generated spectra were identified by the MBT Bruker library and the MSI database. RESULTS: Selective culture media (IDFP and SGC2) significantly outperformed the non-selective SDA medium. IDFP was superior to the SGC2 medium for dermatophyte identification. The EEP only demonstrated a benefit over DT in the underperforming SDA medium. The MBT Bruker library outperformed the MSI database in Aspergillus identification while the MSI database outperformed the MBT library in dermatophyte identification. For non-Aspergillus fungi, the libraries performed comparably. CONCLUSION: The results of our study show the necessity of using selective culture media (IDFP and SGC2) for fungal identification with MALDI-TOF MS and demonstrate no significant benefit of the formic acid-ethanol protein extraction technique in these media. Given the relative strengths and weaknesses of the MBT library and the MSI database, it might currently be beneficial to consider these libraries as complementary and employ both databases to achieve optimal fungal identification.

Development of an Efficient Extraction Methodology to Analyse Potential Inflammatory Biomarkers from Sebum.

INTRODUCTION: Proteins, such as cytokines and chemokines, are present in varying concentrations in a range of biofluids, with an important signalling role in maintaining homeostasis. Commercial tapes have been employed to non-invasively collect these potential biomarkers in sebum from the skin surface to examine their concentrations in conditions including acne, atopic dermatitis, and pressure ulcers. However, the identification of robust biomarker candidates is limited by the low abundance of specific proteins extracted by current methodologies. Therefore, this study was designed to develop an optimized extraction method for potential inflammatory biomarkers in sebum collected with Sebutapes. METHODS: Commercial tapes (Sebutapes) coated with synthetic sebum were used to systematically evaluate the effects of chemical and mechanical stimuli on extraction efficiency. Varying concentrations of high- and low-abundance biomarkers (IL-1α, IL-6, IL-8, INF-γ, TNF-α, and IL-1RA) were used to spike the synthetic sebum samples. Methodological variables included different surfactants, mechanical stimuli, and buffer volume. Extraction efficiency was estimated using immunoassay kits from the extracted buffer. RESULTS: The results revealed that the use of a surfactant, i.e., ß-dodecyl maltoside, in addition to the mechanical stimuli, namely, sonication and centrifugation, resulted in an increased recovery of cytokines, ranging from 80% for high-abundant cytokines, such as IL-1α and IL-1RA, and up to 50% for low-abundance cytokines, including TNF-α, IL-6 and IL-8. Compared to previous methods, the new extraction protocol resulted in between a 1.5-2.0-fold increase in extraction efficiency. CONCLUSION: The study revealed that there was a high degree of variability in the extraction efficiency of different cytokines. However, improved efficiency was achieved across all cytokines with selective surfactants and mechanical stimuli. The optimised protocol will provide means to detect low levels of potential biomarkers from skin surface, enabling the evaluation of local changes in pro- and anti-inflammatory cytokines present in different skin conditions.

Extraction of Heat-Stabilised Defatted Rice Bran Protein by Solid-State Fermentation Using Heterofermentative Microbes from Traditional Asian Starters.

Research background: Heat-stabilised defatted rice bran (HSDRB) is a primary by-product of rice bran oil extraction industry and a nutritious source of protein. However, despite the unique nutritional profile of rice bran protein, the protein-rich by-product, HSDRB is underutilised as a low-value animal feed. Research on protein extraction from HSDRB by enzymatic hydrolysis has attracted the attention of numerous scientists. However, a cost-effective extraction method is required to mitigate the high costs associated with the use of enzymes. Therefore, we have presented an alternative economical and natural approach for protein extraction from HSDRB by solid-state fermentation (SSF) with heterofermentative microbes. Experimental approach: SSF of HSDRB with two types of traditional Asian fermentation starters, namely loog-pang and koji, were evaluated for enzyme production and their efficacy in extracting proteins from HSDRB. For this purpose, HSDRB fermentation was carried out for 0, 12, 24, 48, 72 and 96 h followed by 24-hour hydrolysis to evaluate the extracted rice bran protein. In addition, microbiome diversity in the fermentation starters was also determined by metagenomic sequencing of 16S rRNA and internal transcribed spacer to identify bacteria and fungi, respectively. Results and conclusions: The microbial community in the fermentation starters showed the dominance of lactic acid bacteria (LAB) such as Bacillus subtilis in loog-pang and Streptococcus lutetiensis, Bacillus pumilus, Lactococcus cremoris, Lactococcus garvieae and Pediococcus pentosaceus in koji, while yeast species Saccharomycopsis fibuligera and Saccharomyces cerevisiae dominated the fungal diversity in loog-pang and koji starters, respectively. The results suggest that loog-pang and koji can produce cellulase, neutral and acid proteases during fermentation. Despite the discrepancy in their microbial diversity and the enzyme activity during SSF, both starters could effectively increase protein extraction from HSDRB. A positive relationship between the SSF duration and extracted protein was observed. During SSF with loog-pang and koji after 72 h followed by 24-hour hydrolysis, 65.66 and 66.67 % protein was extracted from HSDRB, respectively. The amino acid analysis of the protein hydrolysate produced by the non-fermented and fermented methods showed no difference and had an abundance of glutamic and aspartic acids, leucine, arginine, alanine and glycine amino acids, which accounted for approx. 58 % of the total amino acids. Novelty and scientific contribution: Loog-pang and koji (traditional Thai and Japanese fermentation starters, respectively) were found to be effective in extracting proteins from HSDRB by SSF although they are inexpensive microbial enzyme sources. Future research aimed at scaling up HSDRB protein extraction for usage in industrial applications can draw on our results.

Plant leaf proteins for food applications: Opportunities and challenges.

Plant-based proteins are gaining a lot of attention for their health benefits and are considered as an alternative to animal proteins for developing sustainable food systems. Against the backdrop, ensuring a healthy diet supplemented with good quality protein will be a massive responsibility of governments across the globe. Increasing the yield of food crops has its limitations, including low acceptance of genetically modified crops, land availability for cultivation, and the need for large quantities of agrochemicals. It necessitates the sensible use of existing resources and farm output to derive the proteins. On average, the protein content of plant leaves is similar to that of milk, which can be efficiently tapped for food applications across the globe. There has been limited research on utilizing plant leaf proteins for food product development over the years, which has not been fruitful. However, the current global food production scenario has pushed some leading economies to reconsider the scope of plant leaf proteins with dedicated efforts. It is evident from installing pilot-scale demonstration plants for protein extraction from agro-food residues to cater to the protein demand with product formulation. The present study thoroughly reviews the opportunities and challenges linked to the production of plant leaf proteins, including its nutritional aspects, extraction and purification strategies, anti-nutritional factors, functional and sensory properties in food product development, and finally, its impact on the environment. Practical Application: Plant leaf proteins are one of the sustainable and alternative source of proteins. It can be produced in most of the agroclimatic conditions without requiring much agricultural inputs. It's functional properties are unique and finds application in novel food product formulations.