Introduction:Q fever, caused by Coxiella burnetii, is a zoonotic disease of global relevance with domestic ruminants as the main reservoirs. Serological diagnosis, especially enzyme-linked immunosorbent assay (ELISA), often suffers from limited sensitivity and specificity due to antigenic variability and cross-reactivity. Scientific goal:In this study, a combined proteomic and literature research approach was used to identify immunoreactive proteins and predict linear B-cell epitopes as alternative diagnostic targets. Methods:Total protein extracts of a C. burnetii field isolate from sheep were separated by two-dimensional gel electrophoresis, and immunoreactive proteins were detected by Western blotting using pooled sheep sera obtained from various flocks with known Q fever status. Immunoreactive proteins were identified by LC-MS/MS and used for linear B-cell epitope prediction for peptide synthesis. Peptides (n = 30) were initially screened by fluorescent ELISA against nine field serum pools (90 individual sera), and the most promising peptides (n = 15) were individually tested with 79 single sera. Diagnostic performance was assessed by receiver operating characteristic (ROC) analysis and by a multi-peptide rule ("positive if ≥1 peptide reactive"). Results:A total of 156 seroreactive proteins, including 51 previously reported antigens, were detected, among others, Com1, CBU_0482, and Mip. Although the selected 15 peptides showed a specific reaction with pooled sera, they showed limited diagnostic performance with an area under the curve (AUC) of 0.5-0.7 when using single serum samples (n = 79). Multi-peptide combinations (6-8 peptides) increased sensitivity (Se) to 80% and specificity (Sp) to 75%. Discussion:Although single peptides lacked discriminatory power, multi-epitope combinations reached acceptable accuracy and may be used as a complementary tool for commercial ELISAs. However, larger bioinformatic approaches and validation studies are required to identify specific peptides of high diagnostic accuracy.

We tested the effects of galactoglucomannan oligosaccharides (GGMOs) and/or cadmium (Cd) on peroxidase activity and the proteome in maize (Zea mays L.) roots and leaves. Our previous work confirmed that GGMOs ameliorate the symptoms of Cd stress in seedlings. Here, the plants were hydroponically cultivated for 7 days, and the protein content and peroxidase activity were estimated in intracellular, neutral cell wall, and acidic cell wall protein fractions. The peroxidase activity varied between the plant organs as well as among the fractions and treatments. The GGMOs in the presence of Cd did not significantly influence content of peroxidases but modulated their activity, which implies posttranslational regulation. The changes in the content of various proteins (e.g., related to the defence reactions, cell wall structure/metabolism, and activation of plant hormones) caused by GGMOs and Cd indicate possible protective mechanisms that improve the vitality of maize seedlings exposed to metal stress. GGMOs partially reverted Cd-induced protein disbalance, which was a reoccurring phenomenon of mitigation in leaves.

The geographical authentication of green specialty coffee is an economically sensitive analytical task that is not yet fully resolved. We used an innovative combination of proteomic profiling with linear discriminant analysis for the authentication of the geographical origin of green specialty coffee beans from well-known harvesting regions in Central America, South America, Africa, and Asia. Out of 1596 identified proteins, we selected the top 30 target markers ranked by ANOVA. The model's prediction performance using leave-one-out cross-validation reached 85.3 %, with the lowest accuracy in the prediction rate for Asian samples. Besides, model performance and prediction sensitivity to random states were tested using 5-fold cross-validation. After 20 iterations, the model performance slightly decreased to 84.0 %. This research contributes to advancing traceability tools in the coffee industry, ensuring product authenticity, and promoting fair trade practices. Specificity and sensitivity confirmed that the model appears to be reliable at distinguishing Asian and African samples.

Euglena gracilis is a flagellate photosynthetic microalga that, thanks to its metabolic adaptability, can grow under both autotrophic and heterotrophic conditions. This adaptability makes euglena an interesting species for applied biotechnology. We focused on the proteome of E. gracilis cultivated in Cramer-Myers medium (supplemented with ethanol) in dark and light conditions. Cultures grown in the light showed a characteristic green coloration, while cultures incubated in the dark were bright yellow. When cultured in the dark, microalga showed reduced concentration of chlorophylls (a, b, and total) and carotenoids compared to cells cultured in the light. Conversely, there was an increase in proline content in the dark compared to light cultivation. Using proteomic approach, we revealed 162 differentially accumulated proteins in light- and dark-grown cells classified into 12 functional groups. Notably, alterations in the metabolism of fatty acids and amino acids, secondary metabolism, and accumulation of stress- and detoxification-related proteins in microalgal cells cultivated in darkness with ethanol as a carbon source may help euglena adapt to these conditions. Based on our results and literature, we hypothesize that vitamin B12 potentially plays an important role in light/dark metabolic switch, similarly as in bacteria.

Chronic ionizing radiation causes elevated levels of DNA damage and reactive oxygen species in plants. Aquatic ecosystems in Chornobyl zone, a major radiological disaster site, are contaminated by harmful radionuclides. We focused on explaining the biochemical mechanisms responsible for the susceptibility of a wild aquatic plant (common reed, Phragmites australis) grown in Chornobyl zone to biotic stress. The fungal infection assay indicated that life in a radionuclide-contaminated environment might compromise plant immunity. Proteome profiling identified 1,867 proteins and we selected several dozen proteins with consistently higher and lower abundance in the samples from the littoral of contaminated lakes by hierarchical clustering. Discordant expression of coding genes pointed to posttranscriptional regulation. Proteins that accumulated in reed upon chronic irradiation suggested a biochemically stable phenotype with effective protection against reactive carbonyls. Simultaneously, proteins that depleted in plants collected from the littoral of radiologically contaminated lakes indicated worse stress resilience and enhanced susceptibility to biotic agents. Furthermore, the quantification of antioxidant enzyme activities and carbonylated proteins rebutted the idea about substantial oxidative stress in chronically irradiated plants. We advocate the necessity to consider increased pathogen sensitivity while developing policies for the management of radionuclide-contaminated areas.

Plant synaptotagmins, especially SYT1, are involved in responses to different environmental stressors. We revealed that SYT1 abundance at the cell periphery in Arabidopsis roots and its turnover decelerated upon salt stress. Results of proteomic analysis of the Arabidopsis syt1-2 allele demonstrated that under salt stress, more proteins accumulated and were depleted in the mutant than in the wild-type seedlings' roots. In both genotypes, these were mostly proteins hitherto unknown to be affected by salt stress. Proteins associated with the cytoskeleton, water deprivation, proteasome activity, oxidative stress, glutathione, lipid transfer, and glucosinolates were the most affected. When we analyzed the consequence of the mutation, 10 proteins were differentially abundant in mutant versus wild type in standard conditions, and 24 proteins under sodium chloride stress. Since these were relatively small numbers, syt1-2 was subjected to thorough molecular analysis. Results indicated that the altered SYT1 transcript was produced in syt1-2 with 75% reduced abundance compared to the unchanged transcript in the wild type.

The concept of the ischaemic penumbra - stroke tissue with the potential to survive - has opened the door to a wide range of experimental strategies that could benefit the recovery of patients after a stroke. In this study, we used proteomic analysis to examine how remote ischaemic postconditioning (RIPC) mediates a shift from a vulnerable to a tolerant penumbra. We identified 450 differentially abundant proteins between the control group and the groups subjected to ischaemia via middle cerebral artery occlusion with or without RIPC during infarct expansion. The majority of proteins were downregulated following RIPC. Based on Gene Ontology enrichment analysis, we uncovered 24 gene sets significantly influenced during the reprogramming from a vulnerable to a tolerant penumbra. RIPC treatment positively impacted the synthesis of proteins enriched in the cytosol (GO:0005829) but inhibited the abundance of proteins belonging to the cytoskeleton (GO:0005874 microtubule) and the glutamatergic synapse (GO:0098978). The shift to a tolerant phenotype involved overexpression of aminopeptidases (GO:0004177) related to proteolysis (GO:0006508). RIPC also downregulated proteins involved in the tricarboxylic acid cycle (GO:0006099), adenosine triphosphate (ATP) binding (GO:0005524), and ATP hydrolysis (GO:0016887). We validated our proteomic findings by selecting two candidate genes (Map2 and Tubb3) for immunofluorescence. We identified the low-molecular-weight Map2 isoform as a potential marker of the shift from a vulnerable to a tolerant penumbra. In summary, our findings have revealed novel avenues for multimodal investigation of reprogramming the penumbra as part of recovery from stroke.

Mesenchymal stem cells (MSCs) are an essential tool in cell-based therapies. One of the most crucial factors for efficacy in regenerative medicine is the source of MSCs. Tissue origin has long been suggested as a potential determinant of MSC properties. Human dermal fibroblasts (HDFa) share similar characteristics with MSCs, and the question of whether HDFa are functionally equivalent to MSCs remains debated. The present work used proteomic and phenotypic analyses to compare HDFa, dental pulp stem cells (DPSCs), and adipose-derived mesenchymal stem cells (AD-MSCs). We observed similarities and/or differences in morphology, cell surface markers, differentiation, and proteomic profile. Proteome was profiled by nano liquid chromatography and comprehensively quantified by mass spectrometry. In fact, HDFa and MSCs shared similar surface markers, growth kinetics, and differentiation capacity. Proteomic analysis reproducibly identified and quantified 3,051 proteins, 86 of them were differentially abundant according to strict statistical criteria. We identified a set of proteins that determined signatures for each stem cell origin. Gene Ontology (GO) term enrichment of differentially accumulated proteins, and Gene Set Enrichment Analysis (GSEA) identified signaling pathways characteristic to individual cell types. Particularly, we highlighted signaling pathways involved in cell migration, adhesion, and Wnt signaling as downregulated in HDFa compared to DPSCs. Angiogenesis and vascularization were explicitly associated with AD-MSCs. The tissue repair process requires a well-coordinated integration of complex molecular events, including cell migration and proliferation, extracellular matrix deposition, angiogenesis, and remodeling. We propose that HDFa are an alternative to MSCs, but predict their worse behavior in defect repair models compared to DPSCs. Plausibly, AD-MSCs are more suitable candidates for angiogenesis models compared to DPSCs.

Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the linker disaccharide [rhamnosyl (Rha) - N-acetyl-glucosaminyl (GlcNAc) phosphate] on the decaprenyl-phosphate carrier. This decaprenyl-P-P-GlcNAc-Rha intermediate is extended by two bifunctional galactosyl transferases, GlfT1 and GlfT2, and then it is translocated to the periplasmic space by an ABC transporter Wzm-Wzt. The cell wall core synthesis is finalized by the action of an array of arabinosyl transferases, mycolyl transferases, and ligases that catalyze an attachment of the arabinogalactan polymer to peptidoglycan through the linker region. Based on visualization of the GlfT2 enzyme fused with fluorescent tags it was proposed that galactan polymerization takes place in a specific compartment of the mycobacterial cell envelope, the intracellular membrane domain, representing pure plasma membrane free of cell wall components (previously denoted as the "PMf" domain), which localizes to the polar region of mycobacteria. In this work, we examined the activity of the galactan-producing cellular machine in the cell-wall containing cell envelope fraction and in the cell wall-free plasma membrane fraction prepared from Mycobacterium smegmatis by the enzyme assays using radioactively labeled substrate UDP-[C-14]-galactose as a tracer. We found that despite a high abundance of GlfT2 in both of these fractions as confirmed by their thorough proteomic analyses, galactan is produced only in the reaction mixtures containing the cell wall components. Our findings open the discussion about the distribution of GlfT2 and the regulation of its activity in mycobacteria.

Purpose: Ionizing radiation is a harsh environmental factor that could induce plant senescence. We hypothesized that radiation-related senescence remodels proteome, particularly by triggering the accumulation of prion-like proteins in plant tissues. The object of this study, pea (Pisum sativum L.), is an agriculturally important legume. Research on the functional importance of amyloidogenic proteins was never performed on this species. Materials and methods: Pea seeds were irradiated in the dose range 5-50 Gy of X-rays. Afterward, Fourier-transform infrared spectroscopy (FTIR) was used to investigate changes in the secondary structure of proteins in germinated 3-day-old seedlings. Specifically, we evaluated the ratio between the amide I and II peaks. Next, we performed protein staining with Congo red to compare the presence of amyloids in the samples. In parallel, we profiled the detergent-resistant proteome fraction by ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS). Differentially accumulated proteins were functionally analyzed in MapMan software, and the PLAAC tool was used to predict putative prion-like proteins. Results: We showed a reduced germination rate but higher plant height and faster appearance of reproductive organs in the irradiated at dose of 50 Gy group compared with the control; furthermore, we demonstrated more beta-sheets and amyloid aggregates in the roots of stressed plants. We detected 531 proteins in detergent-resistant fraction extracted from roots, and 45 were annotated as putative prion-like proteins. Notably, 29 proteins were significantly differentially abundant between the irradiated and the control groups. These proteins belong to several functional categories: amino acid metabolism, carbohydrate metabolism, cytoskeleton organization, regulatory processes, protein biosynthesis, and RNA processing. Thus, the discovery proteomics provided deep data on novel aspects of plant stress biology. Conclusion: Our data hinted that protein accumulation stimulated seedlings' growth as well as accelerated ontogenesis and, eventually, senescence, primarily through translation and RNA processing. The increased abundance of primary metabolism-related proteins indicates more intensive metabolic processes triggered in germinating pea seeds upon X-ray exposure. The functional role of detected putative amyloidogenic proteins should be validated in overexpression or knockout follow-up studies. [Graphics] .

Nickel is an essential micronutrient for plant growth and development. However, in excessive amounts caused by accidental pollution of soils, this heavy metal is toxic to plants. Although silicon is a non-essential nutrient, it accumulates in most monocots, particularly the vital crop maize (corn, Zea mays). In fact, this metalloid mineral can alleviate the toxicity of heavy metals, though the mechanism is not entirely clear yet. Herein, we measured proteome, gene expression, enzyme activities, and selected sugars to investigate such effect thoroughly. Deep proteomic analysis revealed a minor impact of 100 µM Ni, 2.5 mM Si, or their combination on roots in 12-day-old hydroponically grown maize seedlings upon 9 days of exposure. Nonetheless, we suggested plausible mechanisms of Si mitigation of excessive Ni: Chelation by metallothioneins and phytochelatins, detoxification by glycine betaine pathway, and restructuring of plasma membrane transporters. Higher activity of glutathione S-transferase confirmed its plausible involvement in reducing Ni toxicity in combined treatment. Accumulation of sucrose synthase and corresponding soluble sugars in Ni and combined treatment implied high energy requirements both during heavy metal stress and its mitigation. Expression analysis of genes coding a few differentially accumulated proteins failed to reveal concordant changes, indicating posttranscriptional regulation. Proposed mitigation mechanisms should be functionally validated in follow-up studies.

In nature, plants are simultaneously exposed to different abiotic (e.g., heat, drought, and salinity) and biotic (e.g., bacteria, fungi, and insects) stresses. Climate change and anthropogenic pressure are expected to intensify the frequency of stress factors. Although plants are well equipped with unique and common defense systems protecting against stressors, they may compromise their growth and development for survival in such challenging environments. Ionizing radiation is a peculiar stress factor capable of causing clustered damage. Radionuclides are both naturally present on the planet and produced by human activities. Natural and artificial radioactivity affects plants on molecular, biochemical, cellular, physiological, populational, and transgenerational levels. Moreover, the fitness of pests, pathogens, and symbionts is concomitantly challenged in radiologically contaminated areas. Plant responses to artificial acute ionizing radiation exposure and laboratory-simulated or field chronic exposure are often discordant. Acute or chronic ionizing radiation exposure may occasionally prime the defense system of plants to better tolerate the biotic stress or could often exhaust their metabolic reserves, making plants more susceptible to pests and pathogens. Currently, these alternatives are only marginally explored. Our review summarizes the available literature on the responses of host plants, biotic factors, and their interaction to ionizing radiation exposure. Such systematic analysis contributes to improved risk assessment in radiologically contaminated areas.

Spontaneous tumor regression is a recognized phenomenon across various cancer types. Recent research emphasizes the alterations in autoantibodies against carbonic anhydrase I (CA I) (anti-CA I) levels as potential prognostic markers for various malignancies. Particularly, autoantibodies targeting CA I and II appear to induce cellular damage by inhibiting their respective protein's catalytic functions. Our study illuminates the profound impact of anti-CA I autoantibodies from patient serum on the esterase activity of human CA I, exhibiting inhibitory effects akin to the acetazolamide inhibitor. Concurrently, our newly synthesized mouse monoclonal IgG antibody, mAb 2B8, against human CA I showcased a potent inhibitory action. An in-depth exploration into mAb 2B8 ' s binding dynamics with its target enzyme was undertaken. Leveraging epitope extraction and phage display library techniques, we identified the amino acid sequence DFWTYP (positions 191-196 of CA I) as crucial for mAb 2B8 ' s interaction. In 3-D structural analysis, this sequence is spatially adjacent to a previously identified epitope (DFWTYP) that interacts with patient-derived autoantibodies. Critically, mAb 2B8 demonstrated an ability to infiltrate eukaryotic cells, engaging specifically with its intracytoplasmic target. This positions mAb 2B8 as a promising model for future studies aimed at tumor cell eradication.

Little is known on the molecular mechanisms of deterioration in aging edible nuts. Proteomic differences between aging and germinating nuts may unravel these mechanisms. Walnut (Juglans regia) kernels have physiological dormancy which can be alleviated by cyanide accompanying with enhanced gluconeogenesis. Warm/moist conditions however, promote kernel deterioration marked by reduced germination associated with increased lipase activity. The hypotheses that aging i) brings about increased lipase activity and enhanced lipid mobilization through increased abundance of lipase protein, ii) compromises gluconeogenesis of lipid reserves, iii) enhances proteolysis and amino acid catabolism, and finally iv) leads to oxidative stress were tested in a proteomic study of kernels aged by controlled deterioration, and those dormant non-aged and cyanide-treated ones all with 15% moisture content; having low, medium and high germination potentials, respectively. This revealed 155 differentially abundant proteins out of 930 identified ones compared to dry (6% moisture content) kernels. In deteriorated nuts, proteins with increased abundance were mainly involved in protein folding, translation and degradation, stress response/detoxification and glycolysis, but those with decreased abundance were related to gluconeogenesis and amino acid metabolism. Contrastingly, in non-aged cyanide-treated kernels, the proteins belonged to gluconeogenesis and oxidative pentose phosphate pathway showed increased abundance. Unlike aging seeds of many other plants, the deteriorated kernels accumulated various chaperons and detoxification proteins. Thus, deteriorating kernels experienced perturbations in lipid gluconeogenesis and amino acid metabolism associated with enhanced respiration and oxidative stress. As no lipase protein increased during kernel aging, the aging-induced lipase activation is possibly regulated by post-translational mechanisms.

Ksg1 is an essential protein kinase of the fission yeast S. pombe that belongs to the AGC kinase family and is homologous to the mammalian PDPK1 kinase. Previous studies have shown that Ksg1 functions in the nutrient-sensing TOR signaling pathway and is involved in the phosphorylation and activation of other AGC kinases, thereby affecting various downstream targets related to metabolism, cell division, stress response, and gene expression. To date, the molecular function of Ksg1 has been analyzed using its temperature sensitive mutants or mutants expressing its truncated isoforms, which are not always suitable for functional studies of Ksg1 and the identification of its targets. To overcome these limitations, we employed a chemical genetic strategy and used a conditional ksg1as mutant sensitive to an ATP analog. Combining this mutant with quantitative phosphoproteomics analysis, we identified 1986 phosphosites that were differentially phosphorylated when Ksg1as kinase was inhibited by an ATP analog. We found that proteins whose phosphorylation was dysregulated after inhibition of Ksg1as kinase were mainly represented by those involved in the regulation of cytokinesis, contractile ring contraction, cell division, septation initiation signaling cascade, intracellular protein kinase cascade, barrier septum formation, protein phosphorylation, intracellular signal transduction, cytoskeleton organization, cellular response to stimulus, or in RNA, ncRNA and rRNA processing. Importantly, proteins with significantly down-regulated phosphorylation were specifically enriched for R-X-X-S and R-X-R-X-X-S motifs, which are typical consensus substrate sequences for phosphorylation by the AGC family of kinases. The results of this study provide a basis for further analysis of the role of the Ksg1 kinase and its targets in S. pombe and may also be useful for studying Ksg1 orthologs in other organisms.

In eukaryotes, pyruvate, a key metabolite produced by glycolysis, is converted by a tripartite mitochondrial pyruvate dehydrogenase (PDH) complex to acetyl-coenzyme A, which is fed into the tricarboxylic acid cycle. Two additional enzyme complexes with analogous composition catalyze similar oxidative decarboxylation reactions albeit using different substrates, the branched-chain ketoacid dehydrogenase (BCKDH) complex and the 2-oxo-glutarate dehydrogenase (OGDH) complex. Comparative transcriptome analyses of diplonemids, one of the most abundant and diverse groups of oceanic protists, indicate that the conventional E1, E2, and E3 subunits of the PDH complex are lacking. E1 was apparently replaced in the euglenozoan ancestor of diplonemids by an AceE protein of archaeal type, a substitution that we also document in dinoflagellates. Here, we demonstrate that the mitochondrion of the model diplonemid Paradiplonema papillatum displays pyruvate and 2-oxoglutarate dehy-drogenase activities. Protein mass spectrometry of mitochondria reveal that the AceE protein is as abundant as the E1 subunit of BCKDH. This corroborates the view that the AceE subunit is a functional component of the PDH complex. We hypothesize that by acquiring AceE, the diplonemid ancestor not only lost the eukaryotic-type E1, but also the E2 and E3 subunits of the PDH complex, which are present in other euglenozoans. We posit that the PDH activity in diplonemids seems to be carried out by a complex, in which the AceE protein partners with the E2 and E3 subunits from BCKDH and/or OGDH.

Drought is among the most limiting factors for sustainable agricultural production. Water shortage at the onset of flowering severely affects the quality and quantity of grain yield of bread wheat (Triticum aestivum). Herein, we measured oxidative stress and photosynthesis-related parameters upon applying transient drought on contrasting wheat cultivars at the flowering stage of ontogenesis. The sensitive cultivar (Darunok Podillia) showed ineffective water management and a more severe decline in photosynthesis. Apparently, the tolerant genotype (Odeska 267) used photorespiration to dissipate excessive light energy. The tolerant cultivar sooner induced superoxide dismutase and showed less inhibited photosynthesis. Such a protective effect resulted in less affected yield and spectrum of seed proteome. The tolerant cultivar had a more stable gluten profile, which defines bread-making quality, upon drought. Water deficit caused the accumulation of medically relevant proteins: (i) components of gluten in the sensitive cultivar and (ii) metabolic proteins in the tolerant cultivar. We propose specific proteins for further exploration as potential markers of drought tolerance for guiding efficient breeding: thaumatin-like protein, 14-3-3 protein, peroxiredoxins, peroxidase, FBD domain protein, and Ap2/ERF plus B3 domain protein.

Somatic embryogenesis is an efficient mean for rapid micropropagation and preservation of the germplasm of valuable coniferous trees. Little is known about how the composition of secretome tracks down the level of embryogenic capacity. Unlike embryogenic tissue on solid medium, suspension cell cultures enable the study of extracellular proteins secreted into a liquid cultivation medium, avoiding contamination from destructured cells. Here, we present proteomic data of the secretome of Pinus nigra cell lines with contrasting embryogenic capacity, accounting for variability between genotypes. Our results showed that cell wall-related and carbohydrate-acting proteins were the most differentially accumulated. Peroxidases, extensin, α-amylase, plant basic secretory family protein (BSP), and basic secretory protease (S) were more abundant in the medium from the lines with high embryogenic capacity. In contrast, the medium from the low embryogenic capacity cell lines contained a higher amount of polygalacturonases, hothead protein, and expansin, which are generally associated with cell wall loosening or softening. These results corroborated the microscopic findings in cell lines with low embryogenic capacity-long suspensor cells without proper assembly. Furthermore, proteomic data were subsequently validated by peroxidase and α-amylase activity assays, and hence, we conclude that both tested enzyme activities can be considered potential markers of high embryogenic capacity.

Honey possesses health-promoting properties; however, industrial processing and manipulation of raw honey can harm on its biological activities, including antibacterial activity. Therefore, this study aimed to compare the honey's antibacterial activity, its total protein content, and the abundance of the most dominant bee-derived proteins in honey between raw (n = 92) and supermarket (n = 17) samples. We showed that tested raw honey samples were much more effective in inhibiting the growth of Staphylococcus aureus, a model bacterium, at a median minimal inhibitory concentration (MIC) value of 4.5 % compared to supermarket honey samples ceasing bacterial growth at median MIC value of 36 %. Moreover, raw honey samples, tested in this study, contained significantly higher amounts of total protein as well as the content of particular bee-derived proteins (major royal jelly protein 1 (MRJP1), glucose oxidase (GOX), and alpha-glucosidase) in contrast to supermarket honey samples. These data hint that some supermarket honey samples exhibited strikingly low protein content. Furthermore, alpha-amylase (diastase) from Aspergillus oryzae was found in the supermarket sample characterised with the lowest protein content. Our findings highlight the burning need to refine and monitor the specific quality parameters, ensuring the authenticity of honey and maintaining its reputation as a functional food.