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.

Secure Food-Allergen Determination by Combining Smartphone-Based Raw Image Analyses and Liquid Chromatography-Mass Spectrometry for the Quantification of Proteins Contained in Lateral Flow Assays.

The current food safety testing system, based on laboratory-based quantification, is difficult to scale up in line with the growth in the export market and does not enable traceability through the nodes of the food supply system. Screening assays, for example, lateral flow assays (LFAs), can improve traceability but often lack the required reliability to guarantee compliance. Here, we present an alternative pipeline for secure on-site compliance testing, using allergens as a case study. The pipeline features smartphone-driven LFA quantification and an liquid chromatography-mass spectrometry (LC-MS) method enabling direct quantification of the allergens contained in the LFA. The system enables swift and objective screening and provides a control measure to verify LFA assay reliability. For the smartphone assay, 8-bit RGB and grayscale colorimetric channels were compared with 16-bit raw intensity values. The latter outperformed RGB and grayscale channels in sensitivity, repeatability, and precision, while ratiometric ambient light correction resulted in excellent robustness for light-intensity variation. Calibration curves for peanut determination using two commercial LFAs featured excellent analytical parameters (R2 = 0.97-0.99; RSD 7-1%; LOD 3-7 ppm). Gluten determination with a third commercial LFA was equally established. A prediction error of 13 ± 11% was achieved for the best performing assay. Good performance-calibration curves (R2 = 0.93-0.99) and CVs (<15%)- were observed for the analyte quantification from the LFA by LC-MS. The LOD for the LC-MS assay was 0.5 ppm, well below the LODs reported for the LFAs. This method creates a digital, fast, and secure food safety compliance testing paradigm that can benefit the industry and consumer alike.

Quantitative mass spectrometry-based analysis of proteins related to cattle and their products - Focus on cows' milk beta-casein proteoforms.

Modern mass spectrometers can accurately measure thousands of compounds in complex mixtures over a given liquid chromatograph method, depending on desired outcome and method duration. This stream of analytical chemistry has wide ranging application across food, pharma, environmental, forensics, clinical and research. With consistent pressure on both the ruminant production and product industries to face new and substantial challenges, liquid chromatography-mass spectrometry (LC-MS) is an ideal tool to identify, detect and quantify markers of breeding, production and adaption to support both research and industry to overcome these challenges. Herein, we provide a description of the theoretical basis and framework for LC-MS as a rapidly developing technique and highlight its application in measuring cattle and cattle product traits through protein quantitation with specific focus on beta-casein proteoforms.

Biomarkers and biosensors for the diagnosis of noncompliant pH, dark cutting beef predisposition, and welfare in cattle.

Meat quality can be affected by stress, exhaustion, feed composition, and other physical and environmental conditions. These stressors can alter the pH in postmortem muscle, leading to high pH and low-quality dark cutting (DC) beef, resulting in considerable economic loss. Moreover, the dark cutting prediction may equally provide a measure for animal welfare since it is directly related to animal stress. There are two needs to advance on-site detection of dark cutters: (1) a clear indication that biomarker (signature compounds) levels in cattle correlate with stress and DC outcome; and (2) measuring these biomarkers rapidly and accurately on-farm or the abattoir, depending on the objectives. This critical review assesses which small molecules and proteins have been identified as potential biomarkers of stress and dark cutting in cattle. We discuss the potential of promising small molecule biomarkers, including catecholamine/cortisol metabolites, lactate, succinate, inosine, glucose, and ß-hydroxybutyrate, and we identify a clear research gap for proteomic biomarker discovery in live cattle. We also explore the potential of chemical-sensing and biosensing technologies, including direct electrochemical detection improved through nanotechnology (e.g., carbon and gold nanostructures), surface-enhanced Raman spectroscopy in combination with chemometrics, and commercial hand-held devices for small molecule detection. No current strategy exists to rapidly detect predictive meat quality biomarkers due to the need to further validate biomarkers and the fact that different biosensor types are needed to optimally detect different molecules. Nonetheless, several biomarker/biosensor combinations reported herein show excellent potential to enable the measurement of DC potential in live cattle.

Identification and Quantitation of Amylase Trypsin Inhibitors Across Cultivars Representing the Diversity of Bread Wheat.

α-Amylase/trypsin inhibitors (ATIs) may have a role in nonceliac wheat sensitivity (NCWS) and celiac disease (CD), but the ATI content and diversity across a range of wheat cultivars are not well characterized. Discovery proteomics was used to detect ATIs across two wheat cultivars: Chara and Magenta. Comprehensive mapping of detected ATIs with the ATIs from the recently published wheat genome RefSeq v1.0 shows the presence of three major subclasses: monomeric (9%), dimeric (61%), and chloroform-methanol (CM) type (30%). Subsequently, the level of 18 ATI isoforms (63 peptides) grouped into four subtypes was monitored across 15 commercial wheat cultivars and the eight parental lines from a multiparent advanced-generation intercross (MAGIC) population using liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS). The ATI content of wheat cultivars Janz, Sunvale, Diamond Bird, and Longreach Scout was significantly lower than that of other wheat cultivars. The MAGIC parental cultivars Baxter and Xiaoyan 54 contain higher levels (∼115% relative to the average wheat ATI content), whereas cultivar Pastor contained the lowest levels (∼87%). Comprehensive sequence analysis, annotation, chromosomal locations, and epitope mapping enabled us to build an LC-MRM-MS method to monitor and quantify the immunostimulatory ATI proteins potentially related to NCWS, autoimmune diseases, and metabolic disorders. This provides an opportunity to select wheat cultivars with significantly lower levels of ATIs.

Comparative label-free mass spectrometric analysis of temporal changes in the skeletal muscle proteome after impact trauma in rats.

Proteomics offers the opportunity to identify and quantify many proteins and to explore how they correlate and interact with each other in biological networks. This study aimed to characterize changes in the muscle proteome during the destruction, repair, and early-remodeling phases after impact trauma in male Wistar rats. Muscle tissue was collected from uninjured control rats and rats that were euthanized between 6 h and 14 days after impact injury. Muscle tissue was analyzed using unbiased, data-independent acquisition LC-MS/MS. We identified 770 reviewed proteins in the muscle tissue, 296 of which were differentially abundant between the control and injury groups (P ≤ 0.05). Around 50 proteins showed large differences (≥10-fold) or a distinct pattern of abundance after injury. These included proteins that have not been identified previously in injured muscle, such as ferritin light chain 1, fibrinogen γ-chain, fibrinogen ß-chain, osteolectin, murinoglobulin-1, T-kininogen 2, calcium-regulated heat-stable protein 1, macrophage-capping protein, retinoid-inducible serine carboxypeptidase, ADP-ribosylation factor 4, Thy-1 membrane glycoprotein, and ADP-ribosylation factor-like protein 1. Some proteins increased between 6 h and 14 days, whereas other proteins increased in a more delayed pattern at 7 days after injury. Bioinformatic analysis revealed that various biological processes, including regulation of blood coagulation, fibrinolysis, regulation of wound healing, tissue regeneration, acute inflammatory response, and negative regulation of the immune effector process, were enriched in injured muscle tissue. This study advances the understanding of early muscle healing after muscle injury and lays a foundation for future mechanistic studies on interventions to treat muscle injury.

Multi-Omics Strategies for Decoding Smoke-Assisted Germination Pathways and Seed Vigour.

The success of seed germination and the successful establishment of seedlings across diverse environmental conditions depends on seed vigour, which is of both economic and ecologic importance. The smoke-derived exogenous compound karrikins (KARs) and the endogenous plant hormone strigolactone (SL) are two classes of butanolide-containing molecules that follow highly similar signalling pathways to control diverse biological activities in plants. Unravelling the precise mode-of-action of these two classes of molecules in model species has been a key research objective. However, the specific and dynamic expression of biomolecules upon stimulation by these signalling molecules remains largely unknown. Genomic and post-genomic profiling approaches have enabled mining and association studies across the vast genetic diversity and phenotypic plasticity. Here, we review the background of smoke-assisted germination and vigour and the current knowledge of how plants perceive KAR and SL signalling and initiate the crosstalk with the germination-associated hormone pathways. The recent advancement of 'multi-omics' applications are discussed in the context of KAR signalling and with relevance to their adoption for superior agronomic trait development. The remaining challenges and future opportunities for integrating multi-omics datasets associated with their application in KAR-dependent seed germination and abiotic stress tolerance are also discussed.

Deep Sequencing MicroRNAs from Extracellular Membrane Vesicles Revealed the Association of the Vesicle Cargo with Cellular Origin.

Extracellular membrane vesicles (EVs) have emerged as potential candidates for diagnostics and therapeutics. We have previously reported that keratinocytes release three types of EVs into the extracellular environment. Importantly, those EVs contain a large number of microRNAs (miRNAs) as cargo. In this study, we examined the expression level of keratinocyte-derived EV miRNAs, their target genes and potential functions. Next generation sequencing results showed that over one hundred miRNAs in each EV subtype exhibited greater than 100 reads per million (RPM), indicating a relatively high abundance. Analysis of the miRNAs with the highest abundance revealed associations with different keratinocyte cell sources. For instance, hsa-miR-205 was associated with the HaCaT cells whereas hsa-miR-21, hsa-miR-203, hsa-miR-22 and hsa-miR-143 were associated with human primary dermal keratinocytes (PKCs). Additionally, functional annotation analysis of genes regulated by those miRNAs, especially with regard to biological processes, also revealed cell-type-specific associations with either HaCaTs or PKCs. Indeed, EV functional effects were related to their parental cellular origin; specifically, PKC-derived EVs influenced fibroblast migration whereas HaCaT-derived EVs did not. In addition, the data in this current study indicates that keratinocyte-derived EVs and/or their cargoes have potential applications for wound healing.

Characterization of the Blister Fluid Proteome for Pediatric Burn Classification.

Blister fluid (BF) is a novel and viable research matrix for burn injury study, which can reflect both systemic and local microenvironmental responses. The protein abundance in BF from different burn severities were initially observed using a 2D SDS-PAGE approach. Subsequently, a quantitative data independent acquisition (DIA) method, SWATH, was employed to characterize the proteome of pediatric burn blister fluid. More than 600 proteins were quantitatively profiled in 87 BF samples from different pediatric burn patients. These data were correlated with clinically assessed burn depth and time until complete wound re-epithelialization through several different statistical analyses. Several proteins from these analyses exhibited significant abundance change between different burn depth or re-epithelialization groups, and can be considered as potential biomarker candidates. Further gene ontology (GO) enrichment analysis of the significant proteins revealed the most significant burn related biological processes (BP) that are altered with burn depth, including homeostasis and oxygen transport. However, for wounds with re-epithelialization times more or less than 21 days, the significant GO annotations were related to enzyme activity. This quantitative proteomics investigation of burn BF may enable objective classification of burn wound severity and assist with clinical decision-making. Data are available via ProteomeXchange with identifier PXD011102.

Pan-proteomics, a concept for unifying quantitative proteome measurements when comparing closely-related bacterial strains.

The comparison of proteomes between genetically heterogeneous bacterial strains may offer valuable insights into physiological diversity and function, particularly where such variation aids in the survival and virulence of clinically-relevant strains. However, reports of such comparisons frequently fail to account for underlying genetic variance. As a consequence, the current knowledge regarding bacterial physiological diversity at the protein level may be incomplete or inaccurate. To address this, greater consideration must be given to the impact of genetic heterogeneity on proteome comparisons. This may be possible through the use of pan-proteomics, an analytical concept that permits the ability to qualitatively and quantitatively compare the proteomes of genetically heterogeneous organisms. Limited examples of this emerging technology highlight currently unmet analytical challenges. In this article we define pan-proteomics, where its value lies in microbiology, and discuss the technical considerations critical to its successful execution and potential future application.

The biochemistry of blister fluid from pediatric burn injuries: proteomics and metabolomics aspects.

Burn injury is a prevalent and traumatic event for pediatric patients. At present, the diagnosis of burn injury severity is subjective and lacks a clinically relevant quantitative measure. This is due in part to a lack of knowledge surrounding the biochemistry of burn injuries and that of blister fluid. A more complete understanding of the blister fluid biochemistry may open new avenues for diagnostic and prognostic development. Burn insult induces a highly complex network of signaling processes and numerous changes within various biochemical systems, which can ultimately be examined using proteome and metabolome measurements. This review reports on the current understanding of burn wound biochemistry and outlines a technical approach for 'omics' profiling of blister fluid from burn wounds of differing severity.

A pre-clinical functional assessment of an acellular scaffold intended for the treatment of hard-to-heal wounds.

The majority of the population experience successful wound-healing outcomes; however, 1-3% of those aged over 65 years experience delayed wound healing and wound perpetuation. These hard-to-heal wounds contain degraded and dysfunctional extracellular matrix (ECM); yet, the integrity of this structure is critical in the processes of normal wound healing. Here, we evaluated a novel synthetic matrix protein for its ability to act as an acellular scaffold that could replace dysfunctional ECM. In this regard, the synthetic protein was subjected to adsorption and diffusion assays using collagen and human dermal tissues; evaluated for its ability to influence keratinocyte and fibroblast attachment, migration and proliferation and assessed for its ability to influence in vivo wound healing in a porcine model. Critically, these experiments demonstrate that the matrix protein adsorbed to collagen and human dermal tissue but did not diffuse through human dermal tissue within a 24-hour observation period, and facilitated cell attachment, migration and proliferation. In a porcine wound-healing model, significantly smaller wound areas were observed in the test group compared with the control group following the third treatment. These data provide evidence that the synthetic matrix protein has the ability to function as an acellular scaffold for wound-healing purposes.

Metabolic and hormonal responses to isoenergetic high-intensity interval exercise and continuous moderate-intensity exercise.

This study investigated the effects of high-intensity interval training (HIIT) vs. work-matched moderate-intensity continuous exercise (MOD) on metabolism and counterregulatory stress hormones. In a randomized and counterbalanced order, 10 well-trained male cyclists and triathletes completed a HIIT session [81.6 ± 3.7% maximum oxygen consumption (V̇o2 max); 72.0 ± 3.2% peak power output; 792 ± 95 kJ] and a MOD session (66.7 ± 3.5% V̇o2 max; 48.5 ± 3.1% peak power output; 797 ± 95 kJ). Blood samples were collected before, immediately after, and 1 and 2 h postexercise. Carbohydrate oxidation was higher (P = 0.037; 20%), whereas fat oxidation was lower (P = 0.037; -47%) during HIIT vs. MOD. Immediately after exercise, plasma glucose (P = 0.024; 20%) and lactate (P < 0.01; 5.4×) were higher in HIIT vs. MOD, whereas total serum free fatty acid concentration was not significantly different (P = 0.33). Targeted gas chromatography-mass spectromtery metabolomics analysis identified and quantified 49 metabolites in plasma, among which 11 changed after both HIIT and MOD, 13 changed only after HIIT, and 5 changed only after MOD. Notable changes included substantial increases in tricarboxylic acid intermediates and monounsaturated fatty acids after HIIT and marked decreases in amino acids during recovery from both trials. Plasma adrenocorticotrophic hormone (P = 0.019), cortisol (P < 0.01), and growth hormone (P < 0.01) were all higher immediately after HIIT. Plasma norepinephrine (P = 0.11) and interleukin-6 (P = 0.20) immediately after exercise were not significantly different between trials. Plasma insulin decreased during recovery from both HIIT and MOD (P < 0.01). These data indicate distinct differences in specific metabolites and counterregulatory hormones following HIIT vs. MOD and highlight the value of targeted metabolomic analysis to provide more detailed insights into the metabolic demands of exercise.

Urinary biomarkers of physical activity: candidates and clinical utility.

Chronic physical inactivity is a major risk factor for a number of important lifestyle diseases, while inappropriate exposure to high physical demands is a risk factor for musculoskeletal injury and fatigue. Proteomic and metabolomic investigations of the physical activity continuum - extreme sedentariness to extremes in physical performance - offer increasing insight into the biological impacts of physical activity. Moreover, biomarkers, revealed in such studies, may have utility in the monitoring of metabolic and musculoskeletal health or recovery following injury. As a diagnostic matrix, urine is non-invasive to collect and it contains many biomolecules, which reflect both positive and negative adaptations to physical activity exposure. This review examines the utility and landscape of biomarkers of physical activity with particular reference to those found in urine.

The Allergen Profile of Two Edible Insect Species-Acheta domesticus and Hermetia illucens.

SCOPE: Edible insect proteins are increasingly introduced as an alternative sustainable food source to address the world's need to feed the growing population. Tropomyosin is the main insect allergen; however, additional potential allergens are not well characterized and the impact of extraction procedures on immunological reactivity is unknown. METHODS AND RESULTS: Proteins from different commercial food products derived from cricket (Acheta domesticus) and black soldier fly (BSF) (Hermetia illucens) are extracted using five different extraction buffers. The proteins are analyzed by SDS-PAGE and immunoblotting using allergen-specific antibodies and crustacean allergic patient sera. IgE binding bands are analyzed by mass spectrometry as well as the complete allergen profile of all 30 extracts. Urea-based buffers are most efficient in extracting insect allergens. Shrimp-specific antibody cross-reactivity to tropomyosin from cricket and BSF indicates high sequence and structural similarity between shrimp and insects. Additional unique allergens are identified in both species, including hemocyanin, vitellogenin, HSP20, apolipophorin-III, and chitin-binding protein. CONCLUSIONS: Identifying potential allergenic proteins and their isoforms in cricket and BSF requires specific extraction approaches using urea-based methods. While tropomyosin is the most abundant and immunoreactive allergen, seven unique allergens are identified, highlighting the need for insect species-specific allergen detection in food products.

Metabolic disruptions and impaired reproductive fitness in wild-caught freshwater turtles (Emydura macquarii macquarii) exposed to elevated per- and polyfluoroalkyl substances (PFAS).

Per- and poly-fluoroalkyl substances (PFAS) pose a threat to organisms and ecosystems due to their persistent nature. Ecotoxicology endpoints used in regulatory guidelines may not reflect multiple, low-level but persistent stressors. This study examines the biological effects of PFAS on Eastern short-necked turtles in Queensland, Australia. In this study, blood samples were collected and analysed for PFAS, hormone levels, and functional omics endpoints. High levels of PFAS were found in turtles at the impacted site, with PFOS being the dominant constituent. The PFAS profiles of males and females differed, with males having higher PFAS concentrations. Hormone concentrations differed between impacted and reference sites in male turtles, with elevated testosterone and corticosterone indicative of stress. Further, energy utilisation, nucleotide synthesis, nitrogen metabolism, and amino acid synthesis were altered in both male and female turtles from PFAS-impacted sites. Both sexes show similar metabolic responses to environmental stressors from the PFAS-contaminated site, which may adversely affect their reproductive fitness. Purine metabolism, caffeine metabolism, and ferroptosis pathway changes in turtles can cause gout, cell death, and overall health problems. Further, the study showed that prolonged exposure to elevated PFAS levels in the wild could compromise turtle reproductive fitness by disrupting reproductive steroids and metabolic pathways.

Multi-omics eco-surveillance of complex legacy contamination with a locally adapted estuarine invertebrate.

Complex legacy contamination from human use is a major issue for estuaries globally. In particular, contamination of water and sediments with bioavailable metals/metalloids, in addition to other industrial contaminants, such as hydrocarbons. Yet, understanding of complex toxicity and local adaptation in field exposed, non-model, invertebrate communities is limited. Herein, we apply multi-omics (metabolomics, lipidomics, proteomics) coupled to traditional sediment quality analyses, to better characterise molecular and cellular responses necessary for application to monitoring, as an eco-surveillance tool. Using these approaches, we characterise functional phenotypes of a sediment associated invertebrate (sipunculid), from an estuary exposed to complex legacy contamination (metals: Zn, Hg, Cd, Pb, Cu, As; and polycyclic aromatic hydrocarbons, PAHs). We sampled individuals at a range of exposure sites, highly (NTB5), moderately (NTB13), and lesser-influenced reference sites. Size differences were observed in sampled individuals between sites, with smaller individuals collected from NTB13. Analysis of environmental variables that correlated with change in the metabolite data revealed that the metabolism of smaller individuals at medium exposure NTB13 was highly differentiated by sediment concentrations of Hg, despite higher concentrations at more exposed NTB5. Functional phenotypes of these smaller individuals were characterised by sulphur and aromatic amino acid metabolism, increases in oxidised intermediates, upregulation of protein responses to oxidative stress, and melanin synthesis, and saturation of membrane and storage of lipids; in addition to the metabolism of naphthalene (PAH). Such widespread change was not observed in the metabolite and lipid profiles of larger individuals at high exposure NTB5, suggesting possible differences in effects between sites may also be associated with size (developmental stage, or age) and/or PAH exposure. This study serves to further understanding of differing modes of toxicity and local adaptation to multiple contaminants, and drivers of functional change in a complex estuary environment.

Nutrition, allergenicity and physicochemical qualities of food-grade protein extracts from Nannochloropsis oculata.

Microalgae offer an opportunity to act as a sustainable source of dietary protein. This study aimed to evaluate the impact of different protein extraction methods on the nutritional and physicochemical properties of Nannochloropsis oculata. Food-grade protein extracts were obtained by hypotonic osmotic shock using milli-Q water. Food grade (FG) and non-food grade (NFG) extraction buffers were compared along with three cell disruption methods including bead beating, probe sonication and a combination of both methods for protein extraction. Mass spectrometry was used for protein and putative allergen identification in FG extracts. Bead beating led to a slightly higher number of identifiable proteins in FG extracts compared to control condition. Putative allergenic proteins were identified in FG extracts of N. oculata using different in-silico methods. These findings support the need to further evaluate the potential allergenic proteins in microalgae including N. oculata such as immunoglobulin E (IgE) binding tests.

Conventional solid-state fermentation impacts the white lupin proteome reducing the abundance of allergenic peptides.

The demand for high-quality and sustainable protein sources is on the rise. Lupin is an emerging plant-based source of protein with health-enhancing properties; however, the allergenic potential of lupins limits their widespread adoption in food products. A combination of discovery and targeted quantitative proteome measurements was used to investigate the impact of solid-state fermentation induced by Rhizopus oligosporus on the proteome composition and allergenic protein abundances of white lupin seed. In total, 1,241 proteins were uniquely identified in the fermented sample. Moreover, the effectiveness of the solid-state fermentation in reducing the abundance of the tryptic peptides derived from white lupin allergens was demonstrated. Comparably, a greater decrease was noted for the major white lupin allergen based on ß-conglutin peptide abundances. Hence, conventional solid-state fermentation processing can be beneficial for reducing the potential allergenicity of lupin-based foods. This finding will open new avenues for unlocking the potential of this under-utilised legume.

Unpacking the Proteome and Metaproteome of the Black Soldier Fly Larvae: Efficacy and Complementarity of Multiple Protein Extraction Protocols.

The larvae of the black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), have demonstrated the ability to efficiently bioconvert organic waste into a sustainable source of food and feed, but fundamental biology remains to be discovered to exploit their full biodegradative potential. Herein, LC-MS/MS was used to assess the efficiency of eight differing extraction protocols to build foundational knowledge regarding the proteome landscape of both the BSF larvae body and gut. Each protocol yielded complementary information to improve BSF proteome coverage. Protocol 8 (liquid nitrogen, defatting, and urea/thiourea/chaps) was better than all other protocols for the protein extraction from larvae gut samples, and the exclusion of defatting steps yielded the highest number of proteins for the larval body samples. Protocol-specific functional annotation using protein level information has shown that the selection of extraction buffer can affect protein detection and their associated functional classes within the measured BSF larval gut proteome. A targeted LC-MRM-MS experiment was performed on the selected enzyme subclasses to assess the influence of protocol composition using peptide abundance measurements. Metaproteome analysis of the BSF larvae gut has uncovered the prevalence of two bacterial phyla: actinobacteria and proteobacteria. We envisage that using complementary extraction protocols and investigating the proteome from the BSF body and gut separately will expand the fundamental knowledge of the BSF proteome and thereby provide translational opportunities for future research to enhance their efficiency for waste degradation and contribution to the circular economy.