Peptidomics analysis of in vitro digested wheat breads: Effect of genotype and environment on protein digestibility and release of celiac disease and wheat allergy related epitopes.

Wheat proteins can trigger immunogenic reactions due to their resistance to digestion and immunostimulatory epitopes. Here, we investigated the peptidomic map of partially digested bread samples and the fingerprint of epitope diversity from 16 wheat genotypes grown in two environmental conditions. Flour protein content and composition were characterized; gastric and jejunal peptides were quantified using LC-MS/MS, and genotypes were classified into high or low bread protein digestibility. Differences in flour protein content and peptide composition distinguish high from low digestibility genotypes in both growing environments. No common peptide signature was found between high- and low-digestible genotypes; however, the celiac or allergen epitopes were noted not to be higher in low-digestible genotypes. Overall, this study established a peptidomic and epitope diversity map of digested wheat bread and provided new insights and correlations between weather conditions, genotypes, digestibility and wheat sensitivities such as celiac disease and wheat allergy.

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.

Low Gluten Beers Contain Variable Gluten and Immunogenic Epitope Content.

Gluten content labels inform food choice and people practicing a gluten-free diet rely upon them to avoid illness. The regulations differ between jurisdictions, especially concerning fermented foodstuffs such as beer. Gluten abundance is typically measured using ELISAs, which have come into question when testing fermented or hydrolysed foodstuffs such as beer. Mass spectrometry can be used to directly identify gluten peptides and reveal false negatives recorded by ELISA. In this survey of gluten in control and gluten-free beers, gluten protein fragments that contain known immunogenic epitopes were detected using liquid chromatography-mass spectrometry in multiple beers that claim to be gluten-free and have sufficiently low gluten content, as measured by ELISA, to qualify as being gluten-free in some jurisdictions. In fact, several purportedly gluten-free beers showed equivalent or higher hordein content than some of the untreated, control beers. The shortcomings of ELISAs for beer gluten testing are summarised, the mismatch between ELISA and mass spectrometry results are explored, and the suitability of existing regulations as they pertain to the gluten content in fermented foods in different jurisdictions are discussed.

Proteome Changes Resulting from Malting in Hordein-Reduced Barley Lines.

Hordeum vulgare L., commonly known as barley, is primarily used for animal feed and malting. The major storage proteins in barley are hordeins, known triggers of celiac disease (CD). Here, sequential window acquisition of all theoretical mass spectra (SWATH)-MS proteomics was employed to investigate the proteome profile of grain and malt samples from the malting barley cultivar Sloop and single-, double-, and triple hordein-reduced lines bred in a Sloop background. Using a discovery proteomics approach, 2688 and 3034 proteins were detected from the grain and malt samples, respectively. By utilizing label-free relative quantitation through SWATH-MS, a total of 2654 proteins have been quantified from grain and malt. The comparative analyses between the barley grain and malt samples revealed that the C-hordein-reduced lines have a more significant impact on proteome level changes due to malting than B- and D-hordein-reduced lines. Upregulated proteins in C-hordein-reduced lines were primarily involved in the tricarboxylic acid cycle and fatty acid peroxidation processes to provide more energy for seed germination during malting. By applying proteomics approaches after malting in hordein-reduced barley lines, we uncovered additional changes in the proteome driven by the genetic background that were not apparent in the sound grain. Our findings offer valuable insights for barley breeders and maltsters seeking to understand and optimize the performance of gluten-free grains in malt products.

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.

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.

Safe food through better labelling; a robust method for the rapid determination of caprine and bovine milk allergens.

Accidental milk cross-contamination is one of the most common causes for costly food recalls. Yet, quantifying trace-levels of allergen is time-consuming and current methods are not adapted for routine analyses making quality control for trace-level allergen content impractical. This perpetuates voluntary "may-contain" statements that are unhelpful for people suffering from food allergies. Here, we developed a rapid LC-MS method enabling milk allergen quantification by comparing all tryptic-peptides of major milk allergens. The bovine-specific αS-2 casein peptide and allergen-epitope NAVPITPTLNR provided excellent performance in sensitivity (LOD 1 mg.kg-1; LOQ 2 mg.kg-1) across various dairy products, good recovery rates in baked croissants (77% with a 10% inter-day RSD) and a linear range of 2-2,000 mg.kg-1. The method can be used for routine determination of trace-contamination with bovine milk allergen and the adulteration of high-value caprine dairy products with lower-value bovine milk products, protecting consumer trust and the growing population suffering from food allergies.

Structure and proteomic analysis of the crown-of-thorns starfish (Acanthaster sp.) radial nerve cord.

The nervous system of the Asteroidea (starfish or seastar) consists of radial nerve cords (RNCs) that interconnect with a ring nerve. Despite its relative simplicity, it facilitates the movement of multiple arms and numerous tube feet, as well as regeneration of damaged limbs. Here, we investigated the RNC ultrastructure and its molecular components within the of Pacific crown-of-thorns starfish (COTS; Acanthaster sp.), a well-known coral predator that in high-density outbreaks has major ecological impacts on coral reefs. We describe the presence of an array of unique small bulbous bulbs (40-100 µm diameter) that project from the ectoneural region of the adult RNC. Each comprise large secretory-like cells and prominent cilia. In contrast, juvenile COTS and its congener Acanthaster brevispinus lack these features, both of which are non-corallivorous. Proteomic analysis of the RNC (and isolated neural bulbs) provides the first comprehensive echinoderm protein database for neural tissue, including numerous secreted proteins associated with signalling, transport and defence. The neural bulbs contained several neuropeptides (e.g., bombyxin-type, starfish myorelaxant peptide, secretogranin 7B2-like, Ap15a-like, and ApNp35) and Deleted in Malignant Brain Tumor 1-like proteins. In summary, this study provides a new insight into the novel traits of COTS, a major pest on coral reefs, and a proteomics resource that can be used to develop (bio)control strategies and understand molecular mechanisms of regeneration.

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.

Proteome analysis of Campylobacter jejuni poultry strain 2704 survival during 45 min exposure to peracetic acid.

Peracetic acid (PAA) applied to whole poultry carcasses can reduce the number of Campylobacter, a leading cause of human gastroenteritis. However, previous modelling experiments indicated that Campylobacter survived in greater numbers when pre-treated with a thermal stress equivalent to poultry processing scalding prior to chilling with PAA than when subject to chilling with PAA only. To better understand how Campylobacter responds to PAA, proteomes of C. jejuni poultry strain 2704 were measured after exposure to PAA (60 ppm, pH 4.0) for 45 min under laboratory ambient conditions (approximately 23 °C) to establish a foundational map of survival mechanism before combining with other stresses. Analysis of 580 quantified proteins did not indicate a triggered "peroxide shock" response, nor were common heat shock responses detected. Thioredoxin, iron homeostatic, peroxiredoxins and cytochrome c peroxidases became more abundant suggesting that PAA disturbed cytoplasmic redox homeostasis resulting in antioxidant activation and increased prioritisation of iron homeostasis. The PAA treatment led to responses that included an increased priority for oxidative phosphorylation and a simultaneous decrease in central metabolism associated protein abundances. Lon protease was induced suggesting it has a role in maintaining homeostasis during non-thermal stress. Proteins in flagella and chemotaxis became more abundant though whether PAA has a chemorepellent effect requires further investigation. Overall, the proteome data suggests there was a rapid cellular response to applied PAA stress in the first 15 min with the adaptation to the stress completing between 30 and 45 min. The findings will help guide PAA implementation in commercial poultry processing in terms of processing location and length of application.

Comparison of protein extraction protocols and allergen mapping from black soldier fly Hermetia illucens.

Exploration of important insect proteins - including allergens - and proteomes can be limited by protein extraction buffer selection and the complexity of the proteome. Herein, LC-MS/MS-based proteomics experiments were used to assess the protein extraction efficiencies for a suite of extraction buffers and the effect of ingredient processing on proteome and allergen detection. Discovery proteomics revealed that SDS-based buffer yields the maximum number of protein groups from three types of BSF samples. Bioinformatic analysis revealed that buffer composition and ingredient processing could influence allergen detection. Upon applying multi-level filtering criteria, 33 putative allergens were detected by comparing the detected BSF proteins to sequences from public allergen protein databases. A targeted LC-MRM-MS assay was developed for the pan-allergen tropomyosin and used to assess the influence of buffer composition and ingredient processing using peptide abundance measurements. SIGNIFICANCE: We demonstrated that the selection of protein extraction buffer and the processing method could influence protein yield and cross-reactive allergen detection from processed and un-processed black soldier fly (BSF) samples. In total, 33 putative allergens were detected by comparing the detected BSF proteins to sequences from public allergen protein databases. An LC-MRM-MS assay was developed for tropomyosin, indicating the importance of buffer selection and processing conditions to reduce BSF samples' allergenicity.

Proteome Phenotypes Discriminate the Growing Location and Malting Traits in Field-Grown Barley.

Barley is one of the key cereal grains for malting and brewing industries. However, climate variability and unprecedented weather events can impact barley yield and end-product quality. The genetic background and environmental conditions are key factors in defining the barley proteome content and malting characteristics. Here, we measure the barley proteome and malting characteristics of three barley lines grown in Western Australia, differing in genetic background and growing location, by applying liquid chromatography-mass spectrometry (LC-MS). Using data-dependent acquisition LC-MS, 1571 proteins were detected with high confidence. Quantitative data acquired using sequential window acquisition of all theoretical (SWATH) MS on barley samples resulted in quantitation of 920 proteins. Multivariate analyses revealed that the barley lines' genetics and their growing locations are strongly correlated between proteins and desired traits such as the malt yield. Linking meteorological data with proteomic measurements revealed how high-temperature stress in northern regions affects seed temperature tolerance during malting, resulting in a higher malt yield. Our results show the impact of environmental conditions on the barley proteome and malt characteristics; these findings have the potential to expedite breeding programs and malt quality prediction.

Perturbation of the gut microbiome in wild-caught freshwater turtles (Emydura macquarii macquarii) exposed to elevated PFAS levels.

Per- and polyfluoroalkyl substances (PFAS) are environmentally persistent and pervasive. Understanding the toxicity of PFAS to wildlife is difficult, both due to the complexity of biotic and abiotic perturbations in the taxa under study and the practical and ethical problems associated with studying the impacts of environmental pollutants on free living wildlife. One avenue of inquiry into the effects of environmental pollutants, such as PFAS, is assessing the impact on the host gut microbiome. Here we show the microbial composition and biochemical functional outputs from the gut microbiome of sampled faeces from euthanised and necropsied wild-caught freshwater turtles (Emydura macquarii macquarii) exposed to elevated PFAS levels. The microbial community composition was profiled by 16S rRNA gene sequencing using a Nanopore MinION and the biochemical functional outputs of the gut microbiome were profiled using a combination of targeted central carbon metabolism metabolomics using liquid chromatography coupled to a triple quadrupole mass spectrometer (LC-QqQ-MS) and untargeted metabolomics using liquid chromatography coupled to a quadrupole time of flight mass spectrometer (LC-QToF-MS). Total PFAS was measured in the turtle serum using standard methods. These preliminary data demonstrated a 60-fold PFAS increase in impacted turtles compared to the sampled aquatic environment. The microbiome community was also impacted in the PFAS exposed turtles, with the ratio of Firmicutes-to-Bacteroidetes rising from 1.4 at the reference site to 5.5 at the PFAS impacted site. This ratio increase is indicative of host stress and dysfunction of the gut microbiome that was correlated with the biochemical metabolic function data, metabolites observed that are indications of stress and inflammation in the gut microbiome. Utilising the gut microbiome of sampled faeces collected from freshwater turtles provides a non-destructive avenue for investigating the impacts of PFAS in native wildlife, and provides an avenue to explore other contaminants in higher-order taxa within the environment.

The mosaic oat genome gives insights into a uniquely healthy cereal crop.

Cultivated oat (Avena sativa L.) is an allohexaploid (AACCDD, 2n = 6x = 42) thought to have been domesticated more than 3,000 years ago while growing as a weed in wheat, emmer and barley fields in Anatolia1,2. Oat has a low carbon footprint, substantial health benefits and the potential to replace animal-based food products. However, the lack of a fully annotated reference genome has hampered efforts to deconvolute its complex evolutionary history and functional gene dynamics. Here we present a high-quality reference genome of A. sativa and close relatives of its diploid (Avena longiglumis, AA, 2n = 14) and tetraploid (Avena insularis, CCDD, 2n = 4x = 28) progenitors. We reveal the mosaic structure of the oat genome, trace large-scale genomic reorganizations in the polyploidization history of oat and illustrate a breeding barrier associated with the genome architecture of oat. We showcase detailed analyses of gene families implicated in human health and nutrition, which adds to the evidence supporting oat safety in gluten-free diets, and we perform mapping-by-sequencing of an agronomic trait related to water-use efficiency. This resource for the Avena genus will help to leverage knowledge from other cereal genomes, improve understanding of basic oat biology and accelerate genomics-assisted breeding and reanalysis of quantitative trait studies.

Targeted proteomics for rapid and robust peanut allergen quantification.

This study improves LC-MS-based trace level peanut allergen quantification in processed food by refining method robustness, total analysis time and method sensitivity. Extraction buffer (six compared) and peptide choice were optimised and found to profoundly affect method robustness. A rapid extraction and in-solution digestion method was developed omitting subsequent reduction, alkylation and sample clean-up steps effectively reducing total analysis time from the previously reported ∼5.5-20 h to ∼2.5 h. For the three best performing peptides, accurate quantification (CVs < 15%) with matrix-matched calibration curves (R2 = 0.99-0.97) was achieved for peanut muffin and ice-cream with excellent linearity (0.25-1000 mg kg-1). The best performing peptide enabled excellent recovery rates in ice-cream (106.0 ± 15.1%) and peanut muffin (72.7 ± 13.4%). Sensitivity (LOD = 0.25-0.5 mg kg-1; LOQ = 0.5-1.0 mg kg-1) was 2- to 20-fold improved compared to previous methods depending on the peptide. These methodological improvements contribute to robust peanut detection in food and can be translated to additional food-borne allergens.

Omics-based ecosurveillance for the assessment of ecosystem function, health, and resilience.

Current environmental monitoring efforts often focus on known, regulated contaminants ignoring the potential effects of unmeasured compounds and/or environmental factors. These specific, targeted approaches lack broader environmental information and understanding, hindering effective environmental management and policy. Switching to comprehensive, untargeted monitoring of contaminants, organism health, and environmental factors, such as nutrients, temperature, and pH, would provide more effective monitoring with a likely concomitant increase in environmental health. However, even this method would not capture subtle biochemical changes in organisms induced by chronic toxicant exposure. Ecosurveillance is the systematic collection, analysis, and interpretation of ecosystem health-related data that can address this knowledge gap and provide much-needed additional lines of evidence to environmental monitoring programs. Its use would therefore be of great benefit to environmental management and assessment. Unfortunately, the science of 'ecosurveillance', especially omics-based ecosurveillance is not well known. Here, we give an overview of this emerging area and show how it has been beneficially applied in a range of systems. We anticipate this review to be a starting point for further efforts to improve environmental monitoring via the integration of comprehensive chemical assessments and molecular biology-based approaches. Bringing multiple levels of omics technology-based assessment together into a systems-wide ecosurveillance approach will bring a greater understanding of the environment, particularly the microbial communities upon which we ultimately rely to remediate perturbed ecosystems.

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.

Bioaccumulation and impact of maternal PFAS offloading on egg biochemistry from wild-caught freshwater turtles (Emydura macquarii macquarii).

Per- and polyfluoroalkyl substances (PFAS) are persistent synthetic contaminants that are pervasive in the environment. Toxicity resulting from elevated PFAS concentrations in wildlife has been studied, yet evidence of their accumulation, developmental toxicity and maternal offloading in egg-laying species is limited. Here we show the maternal offloading of PFAS in freshwater short-necked turtles (Emydura macquarii macquarii) exposed to elevated PFAS and the resulting biological impact on oviducal eggs. Total PFAS concentrations were determined in serum from adult females and harvested oviducal eggs collected from euthanised turtles exposed to low and high levels of PFAS and compared against turtle serum and eggs collected from a suitable reference site. Multi-omics assays were utilised to explore the biochemical impact of elevated PFAS on egg albumen, yolk and eggshell using a range of metabolomics, lipidomics, and proteomics techniques. Eggshells were also screened for metals by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Analysis of the serum collected from adult female turtles and their oviducal eggs demonstrated PFAS offloading and transference that is 1.6 and 5.3 times higher in the low and high PFAS impacted eggs, respectively, compared to maternal serum concentrations. Oviducal egg yolk comprised >90% of the bioaccumulated PFAS load. Multi-omic analysis of the dissected egg fractions illustrated PFAS impacted eggs are significantly elevated in purine metabolism metabolites, which are tied to potential biological dysfunctional processes. The yolks were significantly depleted in lipids and lipid quality tied to growth and development. The high PFAS impacted oviducal eggshells were lower in calcium, important developmental and immune response proteins, and higher in glycerophosphoethanolamines (PE) lipids and histidine metabolism metabolites that are tied to a weakened physical structure. Further investigation is needed to establish the rate of PFAS offloading and quantify the developmental impact on hatchling and hatchling success to fully demonstrate PFAS-developmental toxicity linkages.

Evaluation of protein extraction methods for in-depth proteome analysis of narrow-leafed lupin (Lupinus angustifolius) seeds.

Lupin is slated as a potential contributor towards future food security. Lupin possesses several nutritional and nutraceutical attributes, many linked to seed proteins. For in-depth characterisation of the lupin proteome, liquid chromatography-tandem mass spectrometry was used to evaluate four protein extraction procedures. The proteomes of three narrow-leafed lupin were qualitatively evaluated using protein/peptide identifications and further quantitatively assessed by data-independent proteome measurement. Each extraction buffer led to unique protein identifications; altogether yielding 2,760 protein identifications from lupin varieties. The analysis of protein abundance data highlighted distinct differences between Tris-HCl and urea extracted proteomes, while also revealing variation amongst the cultivar proteomes with the wild accession (P27255) distinctly different from the domesticated cultivars (Tanjil, Unicrop). The extraction buffer used influenced the proteome coverage, downstream functional annotation results and consequently the biological interpretation demonstrating the need to optimise and understand the impact of protein extraction conditions.