Application of transglutaminases in the modification of wool textiles

The use of the protein-crosslinking enzymes transglutaminases (EC 2.3.2.13), as biocatalysts in the processing of wool textiles offers a variety of exciting and realistic possibilities, which include reducing the propensity of wool fabric to shrink and maintaining or increasing fabric strength. Guinea pig liver (GPL) transglutaminase or the microbial transglutaminase isolated from Streptoverticilium mobaraense, when applied to wool either alone or following a protease treatment, resulted in an increase in wool yarn and fabric strength (up to a 25% increase compared to a control). This indicates that transglutaminases can remediate the negative effects of proteolytic treatments in terms of loss in fibre strength. Incubation of samples pretreated with different oxidative and reducing agents with both sources of transglutaminases led to significant increases in tensile strength for all samples tested, suggesting that yarn strength lost following chemical treatments can also be recovered. The two different transglutaminases (TGases) could also impart a significant reduction in fabric shrinkage. The incorporation of primary amine transglutaminase substrates into wool fibres, with a view to altering wool functionality, was demonstrated using the incorporation of the fluorescent primary amine fluorescein cadaverine (FC). Incubation of wool with this fluorescent amine and transglutaminase led to high levels of incorporation into the fibres. The treatment of wool textiles with transglutaminases indicates that a number of novel and radically different finishes for wool textiles can be developed.

Increased expression of proteasome subunits in skeletal muscle of cancer patients with weight loss

Atrophy of skeletal muscle is common in patients with cancer and results in increased morbidity and mortality. In order to design effective therapy the mechanism by which this occurs needs to be elucidated. Most studies suggest that the ubiquitin-proteasome proteolytic pathway is most important in intracellular proteolysis, although there have been no reports on the activity of this pathway in patients with different extents of weight loss. In this report the expression of the ubiquitin-proteasome pathway in rectus abdominis muscle has been determined in cancer patients with weight loss of 0-34% using a competitive reverse transcriptase polymerase chain reaction to measure expression of mRNA for proteasome subunits C2 and C5, while protein expression has been determined by western blotting. Overall, both C2 and C5 gene expression was increased by about three-fold in skeletal muscle of cachectic cancer patients (average weight loss 14.5 ± 2.5%), compared with that in patients without weight loss, with or without cancer. The level of gene expression was dependent on the amount of weight loss, increasing maximally for both proteasome subunits in patients with weight loss of 12-19%. Further increases in weight loss reduced expression of mRNA for both proteasome subunits, although it was still elevated in comparison with patients with no weight loss. There was no evidence for an increase in expression at weight losses less than 10%. There was a good correlation between expression of proteasome 20Sα subunits, detected by western blotting, and C2 and C5 mRNA, showing that increased gene expression resulted in increased protein synthesis. Expression of the ubiquitin conjugating enzyme, E214k, with weight loss followed a similar pattern to that of proteasome subunits. These results suggest variations in the expression of key components of the ubiquitin-proteasome pathway with weight loss of cancer patients, and suggest that another mechanism of protein degradation must be operative for patients with weight loss less than 10%. © 2004 Elsevier Ltd. All rights reserved.

VEGF induces Tie2 shedding via a phosphoinositide 3-kinase/Akt dependent pathway to modulate Tie2 signaling

Objective— Tie2 and its ligands, the angiopoietins (Ang), are required for embryonic and postnatal angiogenesis. Previous studies have demonstrated that Tie2 is proteolytically cleaved, resulting in the production of a 75-kDa soluble receptor fragment (sTie2). We investigated mechanisms responsible for Tie2 shedding and its effects on Tie2 signaling and endothelial cellular responses. Methods and Results— sTie2 bound both Ang1 and Ang2 and inhibited angiopoietin-mediated Tie2 phosphorylation and antiapoptosis. In human umbilical vein endothelial cells, Tie2 shedding was both constitutive and induced by treatment with PMA or vascular endothelial growth factor (VEGF). Constitutive and VEGF-inducible Tie2 shedding were mediated by PI3K/Akt and p38 MAPK. Tie2 shedding was blocked by pharmacological inhibitors of either PI3K or Akt as well as by overexpression of the lipid phosphatase PTEN. In contrast, sTie2 shedding was enhanced by overexpression of either dominant negative PTEN, which increased Akt phosphorylation, or constitutively active, myristoylated Akt. Conclusions— These findings demonstrate that VEGF regulates angiopoietin-Tie2 signaling by inducing proteolytic cleavage and shedding of Tie2 via a novel PI3K/Akt-dependent pathway. These results suggest a previously unrecognized mechanism by which VEGF may inhibit vascular stabilization to promote angiogenesis and vascular remodeling.

Tissue transglutaminase and the progression of human renal scarring

ABSTRACT. Experimental renal scarring indicates that tissue transglutaminase (tTg) may be associated with the accumulation of extracellular matrix (ECM), both indirectly via TGF-β1 activation and directly by the formation of ε(γ-glutamyl) lysine dipeptide bonds within the ECM. The latter potentially accelerates deposition and confers the ECM with resistance to proteolytic digestion. Studied were 136 human renal biopsy samples from a range of chronic renal diseases (CRD) to determine changes in tTg and ε(γ-glutamyl) lysine crosslinking. Immunofluorescence for insoluble tTg showed a 14-fold increase in the kidneys of CRD patients (5.3 ± 0.5 versus 76 ± 54 mV/cm2), which was shown to be active by a similar 11-fold increase in the ε(γ-glutamyl) lysine crosslink (1.8 ± 0.2 versus 19.3 ± 14.2 mV/cm2). Correlations were obtained with renal function for tTg and crosslink. In situ hybridization for tTg mRNA showed that tubular epithelial cells were the major source of tTg; however, both mesangial and interstitial cells also contributed to elevated levels in CRD. This mRNA pattern was consistent with immunohistochemistry for soluble tTg. Changes in renal tTg and its product, the ε(γ-glutamyl) lysine crosslink, occur in progressive renal scarring in humans independently of the original etiology and in a similar manner to experimental models. tTg may therefore play a role in the pathogenesis of renal scarring and fibrosis in patients with CRD and can therefore be considered a potential therapeutic target. E-mail: T.Johnson@sheffield.ac.uk

Transglutaminases:nature's biological glues

Transglutaminases (Tgases) are a widely distributed group of enzymes that catalyse the post-translational modification of proteins by the formation of isopeptide bonds. This occurs either through protein cross-linking via epsilon-(gamma-glutamyl)lysine bonds or through incorporation of primary amines at selected peptide-bound glutamine residues. The cross-linked products, often of high molecular mass, are highly resistant to mechanical challenge and proteolytic degradation, and their accumulation is found in a number of tissues and processes where such properties are important, including skin, hair, blood clotting and wound healing. However, deregulation of enzyme activity generally associated with major disruptions in cellular homoeostatic mechanisms has resulted in these enzymes contributing to a number of human diseases, including chronic neurodegeneration, neoplastic diseases, autoimmune diseases, diseases involving progressive tissue fibrosis and diseases related to the epidermis of the skin. In the present review we detail the structural and regulatory features important in mammalian Tgases, with particular focus on the ubiquitous type 2 tissue enzyme. Physiological roles and substrates are discussed with a view to increasing and understanding the pathogenesis of the diseases associated with transglutaminases. Moreover the ability of these enzymes to modify proteins and act as biological glues has not gone unnoticed by the commercial sector. As a consequence, we have included some of the present and future biotechnological applications of this increasingly important group of enzymes.

Predicting class I major histocompatibility complex (MHC) binders using multivariate statistics:comparison of discriminant analysis and multiple linear regression

The accurate in silico identification of T-cell epitopes is a critical step in the development of peptide-based vaccines, reagents, and diagnostics. It has a direct impact on the success of subsequent experimental work. Epitopes arise as a consequence of complex proteolytic processing within the cell. Prior to being recognized by T cells, an epitope is presented on the cell surface as a complex with a major histocompatibility complex (MHC) protein. A prerequisite therefore for T-cell recognition is that an epitope is also a good MHC binder. Thus, T-cell epitope prediction overlaps strongly with the prediction of MHC binding. In the present study, we compare discriminant analysis and multiple linear regression as algorithmic engines for the definition of quantitative matrices for binding affinity prediction. We apply these methods to peptides which bind the well-studied human MHC allele HLA-A*0201. A matrix which results from combining results of the two methods proved powerfully predictive under cross-validation. The new matrix was also tested on an external set of 160 binders to HLA-A*0201; it was able to recognize 135 (84%) of them.

A proteo-liposome system for the analysis of the intracellular interactome of membrane proteins using amyloid precursor protein as a model

Transmembrane proteins play crucial roles in many important physiological processes. The intracellular domain of membrane proteins is key for their function by interacting with a wide variety of cytosolic proteins. It is therefore important to examine this interaction. A recently developed method to study these interactions, based on the use of liposomes as a model membrane, involves the covalent coupling of the cytoplasmic domains of membrane proteins to the liposome membrane. This allows for the analysis of interaction partners requiring both protein and membrane lipid binding. This thesis further establishes the liposome recruitment system and utilises it to examine the intracellular interactome of the amyloid precursor protein (APP), most well-known for its proteolytic cleavage that results in the production and accumulation of amyloid beta fragments, the main constituent of amyloid plaques in Alzheimer’s disease pathology. Despite this, the physiological function of APP remains largely unclear. Through the use of the proteo-liposome recruitment system two novel interactions of APP’s intracellular domain (AICD) are examined with a view to gaining a greater insight into APP’s physiological function. One of these novel interactions is between AICD and the mTOR complex, a serine/threonine protein kinase that integrates signals from nutrients and growth factors. The kinase domain of mTOR directly binds to AICD and the N-terminal amino acids of AICD are crucial for this interaction. The second novel interaction is between AICD and the endosomal PIKfyve complex, a lipid kinase involved in the production of phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) from phosphatidylinositol-3-phosphate, which has a role in controlling ensdosome dynamics. The scaffold protein Vac14 of the PIKfyve complex binds directly to AICD and the C-terminus of AICD is important for its interaction with the PIKfyve complex. Using a recently developed intracellular PI(3,5)P2 probe it is shown that APP controls the formation of PI(3,5)P2 positive vesicular structures and that the PIKfyve complex is involved in the trafficking and degradation of APP. Both of these novel APP interactors have important implications of both APP function and Alzheimer’s disease. The proteo-liposome recruitment method is further validated through its use to examine the recruitment and assembly of the AP-2/clathrin coat from purified components to two membrane proteins containing different sorting motifs. Taken together this thesis highlights the proteo-liposome recruitment system as a valuable tool for the study of membrane proteins intracellular interactome. It allows for the mimicking of the protein in its native configuration therefore identifying weaker interactions that are not detected by more conventional methods and also detecting interactions that are mediated by membrane phospholipids.

Redox control of protein degradation

Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies.

Secretion of tumor necrosis factor by human leukocytes stimulated with shark cartilage

To assess the role of shark cartilage as an immune modulator, acid, salt-soluble, and phosphate-buffered saline extracts were prepared from three different commercial sources (SL, TL, FDC) of cartilage and used to stimulate human leukocytes in vitro. Duplicate leukocyte cultures were set up, each containing 50 $\mu$l of endotoxin-free extract, 200 $\mu$l of cell suspension (2.4-2.5 $\times$ 10$\sp5$ cells) and 100 $\mu$l of medium and incubated at 37$\sp\circ$C. Cultures stimulated with LPS (5 $\mu$g/ml) or medium served as the positive and negative controls, respectively. Culture supernatants were assayed for TNF$\alpha$ by ELISA. Cartilage extracts stimulated cells to release significant levels of TNF$\alpha$ (p $<$.005); the highest response was obtained with the acid extract of SL cartilage. In comparison, response to corresponding extracts of bovine cartilage was lower (p $<$.05). The stimulatory activity was reduced (85%) following proteolytic digestion, and lost when extract was heated (60$\sp\circ$C, 20 min) or treated with urea (6M), suggesting that the active component(s) is a protein. ^

Stabilization of troponin I for applications in clinical chemistry and forensic medicine. Cardiac troponin I: A time of death marker

Cardiac troponin I (cTnI) is one of the most useful serum marker test for the determination of myocardial infarction (MI). The first commercial assay of cTnI was released for medical use in the United States and Europe in 1995. It is useful in determining if the source of chest pains, whose etiology may be unknown, is cardiac related. Cardiac TnI is released into the bloodstream following myocardial necrosis (cardiac cell death) as a result of an infarct (heart attack). In this research project the utility of cardiac troponin I as a potential marker for the determination of time of death is investigated. The approach of this research is not to investigate cTnI degradation in serum/plasma, but to investigate the proteolytic breakdown of this protein in heart tissue postmortem. If our hypothesis is correct, cTnI might show a distinctive temporal degradation profile after death. This temporal profile may have potential as a time of death marker in forensic medicine. The field of time of death markers has lagged behind the great advances in technology since the late 1850's. Today medical examiners are using rudimentary time of death markers that offer limited reliability in the medico-legal arena. Cardiac TnI must be stabilized in order to avoid further degradation by proteases in the extraction process. Chemically derivatized magnetic microparticles were covalently linked to anti-cTnI monoclonal antibodies. A charge capture approach was also used to eliminate the antibody from the magnetic microparticles given the negative charge on the microparticles. The magnetic microparticles were used to extract cTnI from heart tissue homogenate for further bio-analysis. Cardiac TnI was eluted from the beads with a buffer and analyzed. This technique exploits banding pattern on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a western blot transfer to polyvinylidene fluoride (PVDF) paper for probing with anti-cTnI monoclonal antibodies. Bovine hearts were used as a model to establish the relationship of time of death and concentration/band-pattern given its homology to human cardiac TnI. The final concept feasibility was tested with human heart samples from cadavers with known time of death. ^

Atividade antiofídica do decocto das folhas de jatropha gossypiifolia l. frente o veneno de bothrops jararaca

Antiophidic activity from decoct of Jatropha gossypiifolia L. leaves against Bothrops jararaca venom. Snakebites are a serious worldwide public health problem. In Latin America, about 90 % of accidents are attributed to snakes from Bothrops genus. Currently, the main available treatment is the antivenom serum therapy, which has some disadvantages such as inability to neutralize local effects, risk of immunological reactions, high cost and difficult access in some regions. In this context, the search for alternative therapies to treat snakebites is relevant. Jatropha gossypiifolia L., a medicinal plant popularly known in Brazil as “pinhão-roxo”, is very used in folk medicine as antiophidic. So, the aim of this study is to evaluate the antiophidic properties of this species against enzymatic and biological activities from Bothrops jararaca snake venom. The aqueous leaf extract of J. gossypiifolia was prepared by decoction. The inhibition studies were performed in vitro, by pre-incubation of a fixed amount of venom with different amounts of extract from J. gossypiifolia for 60 min at 37 °C, and in vivo, through oral or intraperitoneal treatment of animals, in different doses, 60 min before venom injection. The proteolytic activity upon azocasein was efficiently inhibited, indicating inhibitory action upon metalloproteinases (SVMPs) and/or serine proteases (SVSPs). The extract inhibited the fibrinogenolytic activity, which was also confirmed by zymography, where it was possible to observe that the extract preferentially inhibits fibrinogenolytic enzymes of 26 and 28 kDa. The coagulant activity upon fibrinogen and plasma were significantly inhibited, suggesting an inhibitory action upon thrombin-like enzymes (SVTLEs), as well as upon clotting factor activators toxins. The extract prolonged the activated partial thromboplastin time (aPTT), suggesting an inhibitory action toward not only to SVTLEs, but also against endogenous thrombin. The defibrinogenating activity in vivo was efficiently inhibited by the extract on oral route, confirming the previous results. The local hemorrhagic activity was also significantly inhibited by oral route, indicating an inhibitory action upon SVMPs. The phospholipase activity in vitro was not inhibited. Nevertheless, the edematogenic and myotoxic activities were efficiently inhibited, by oral and intraperitoneal route, which may indicate an inhibitory effect of the extract upon Lys49 phospholipase (PLA2) and/ or SVMPs, or also an anti-inflammatory action against endogenous chemical mediators. Regarding the possible action mechanism, was observed that the extract did not presented proteolytic activity, however, presented protein precipitating action. In addition, the extract showed significant antioxidant activity in different models, which could justify, at least partially, the antiophidic activity presented. The metal chelating action presented by extract could be correlated with SVMPs inhibition, once these enzymes are metal-dependent. The phytochemical analysis revealed the presence of sugars, alkaloids, flavonoids, tannins, terpenes and/or steroids and proteins, from which the flavonoids could be pointed as major compounds, based on chromatographic profile obtained by thin layer chromatography (TLC). In conclusion, the results demonstrate that the J. gossypiifolia leaves decoct present potential antiophidic activity, including action upon snakebite local effects, suggesting that this species may be used as a new source of bioactive molecules against bothropic venom.

Atividades biológicas de extratos salinos de sementes de plantas da caatinga contra Aedes Aegypti e investigação da participação de proteínas bioativas

Dengue fever, currently the most important arbovirus, is transmitted by the bite of the Aedes aegypti mosquito. Given the absence of a prophylactic vaccine, the disease can only be controlled by combating the vector insect. However, increasing reports of resistance and environmental damage caused by insecticides have led to the urgent search for new safer alternatives. Twenty - um plant s eed extracts from the Caatinga were prepared , tested and characterized . Sodium phosphate ( 50 mM pH 8.0) was used as extractor. All extracts showed larvicidal and ovipositional deterrence activity . Extracts of D. grandiflora, E. contortisiliquum, A. cearenses , C. ferrea and C. retusa were able to attract females for posture when in low co ncentration . In the attractive concentrations, the CE of E. contortisiliquum and A. cearenses were able to kill 52% and 100% of the larvae respectively . The extracts of A. cearenses , P. viridiflora, E. velutina, M. urundeuva and S. brasiliensis were also pupicides, while extracts of P. viridiflora, E. velutina, E. contortisiliquum , A. cearenses, A. colubrina, D. grandiflora , B. cheilantha , S. spectabilis, C. pyramidalis, M. regnelli e G. americana displayed adulticidal activity. All extracts were toxic to C. dubia zooplankton . The EB of E. velutina and E. contortisiliquum did not affect the viability of fibroblasts . In all extracts were identified at least two potential insecticidal proteins such as enzyme inhibitors, lectins and chitin - binding proteins and components of secondary metabolism . Considering all bioassays , the extracts from A. cearenses, P. viridiflora, E. contortisiliquum , S. brasiliensis, E. velutina and M. urundeuva were considered the most promising . The E. contortisiliquum extracts was the only one who did not show pupicida activity, indicating that its mechanism of action larvicide and adulticidal is related only to the ingesti on of toxic compounds by insect , so it was selected to be fragmenting. As observed for the CE , th e protein fractions of E. contortisiliquum also showed larvicidal activity, highlighting that F2 showed higher larvicidal activity and lower en vironmental toxicity than the CE source. The reduction in the proteolytic activity of larvae fed with crude extra ct and fractions of E. contortisiliquum suggest ed that the trypsin inhibitors ( ITEc) would be resp onsible for larvicidal activity . However the increase in the purification of this inhibitor resulted in loss of larvicidal activity , but the absence of trypsin inhibitor reduced the effectiveness of the fractions , indicating that the ITEC contributes to the larvicidal activity of this extract. Not been observed larvicidal activity and adulticide in rich fraction vicilin, nor evidence of the contribution o f this molecule for the larvicidal activity of the extract. The results show the potential of seeds from plant extracts of Caatinga as a source of active molecules against insects A. aegypti at different stages of its development cycle, since they are comp osed of different active compounds, including protein nature, which act on different mechanisms should result in the death of insec

Atividade antiangiogênica da forma recombinante da proteína 21 de Trypanosoma cruzi

Chagas disease, caused by the parasite Trypanosoma cruzi, is the cause of Chronic chagasic cardiomyopathy (CCC). The prospection of innovative therapeutic agents against CCC is a major task. The recombinant form of 21 (rP21), a secreted T. cruzi protein involved in host cell invasion and on progression of chronic inflammatory processes have been studied as a potential novel therapeutic target. Our present work aimed to verify and investigate the impact of rP21 in the formation of blood vessels in vitro and in vivo. First, tEnd cells were treated with different concentrations of rP21 or bacterial extract and viability and cellular adhesion were evaluated by MTT and angiogenesis inhibition by Matrigel tube formation assay and murine model. To verify the proteolytic activity of rP21 on extracellular matrix (ECM) components, fibrinogen, matrigel and fibronectin was incubated with rP21 or not. In addition, we performed proliferation assays and cell cycle analysis. Furthermore, the accumulation and distribution of F-actin was determined by Phalloidin staining using ImageJ software. Finally, tEnd cells were incubated with rP21 and the mRNA levels were analyzed by real-time PCR. Our results showed that rP21 did not alter cell viability and adhesion, but strongly inhibited vessel formation in vitro and in vivo. Tube formation assay showed that angiogenesis inhibition was dependent of the CXCR4-rP21 binding. In addition to these results, we observed that the rP21 was able to inhibit cell proliferation and promoted a significant reduction in the number of 4n cells (G2/M phase). Moreover, we found that rP21 significantly increased F-actin levels and this protein was able to modulate expression of genes related to angiogenesis and actin cytoskeleton. However, rP21 showed no significant activity on the matrix components. In this sense, we conclude that the rP21-endothelial cells (ECs) interaction via CXCR4 promotes inhibition of vessel formation through a cascade of intracellular events, such as inhibition of ECs proliferation and modulation of the expression of molecules associated with angiogenic processes and actin cytoskeleton.

Opening Basement Membrane Gaps During C. elegans Uterine-Vulval Attachment

The basement membrane (BM) is a highly conserved form of extracellular matrix that underlies or surrounds and supports most animal tissues. BMs are crossed by cells during various remodeling events in development, immune surveillance, or during cancer metastasis. Because BMs are dense and not easily penetrable, most of these cells must open a gap in order to facilitate their migration. The mechanisms by which cells execute these changes are poorly understood. A developmental event that requires the opening of a BM gap is C. elegans uterine-vulval connection. The anchor cell (AC), a specialized uterine cell, creates a de novo BM gap. Subsequent widening of the BM gap involves the underlying vulval precursor cells (VPCs) and the π cells, uterine neighbors of the AC through non-proteolytic BM sliding. Using forward and reverse genetic screening, transcriptome profiling, and live-cell imaging, I investigated how the cells in these tissues accomplish BM gap formation. In Chapter 2, I identify two potentially novel regulators of BM breaching, isolated through a large-scale forward genetic screen and characterize the invasion defect in these mutants. In Chapter 3, I describe single-cell transcriptome sequencing of the invasive AC. In Chapter 4, I describe the role of the π cells in opening the nascent BM gap. A complete developmental pathway for this process has been elucidated: the AC induces the π fate through Notch signaling, after which the π cells upregulate the Sec14 family protein CTG-1, which in turn restricts the trafficking of DGN-1 (dystroglycan), a laminin receptor, allowing the BM to slide. Chapter 5 outlines the implications of these discoveries.

Fundamental studies of the application of wheat for malting and brewing

Wheat (Triticum aestivum L.) has a long tradition as a raw material for the production of malt and beer. While breeding and cultivation efforts for barley have been highly successful in creating agronomically and brew- technical optimal specialty cultivars that have become well established as brewing barley varieties, the picture is completely different for brewing wheat. An increasing wheat beer demand results in a rising amount of raw material. Wheat has been - and still is – grown almost exclusively for the baking industry. It is this high demand that defines most of the wheat breeding objectives; and these objectives are generally not favourable in brewing industry. It is of major interest to screen wheat varieties for brewing processability and to give more focus to wheat as a brewing cereal. To obtain fast and reliable predications about the suitability of wheat cultivars a new mathematical method was developed in this work. The method allows a selection based on generally accepted quality characteristics. As selection criteria the parameters raw protein, soluble nitrogen, Kolbach index, extract and viscosity were chosen. During a triannual cultivation series, wheat varieties were evaluated on their suitability for brewing as well as stability to environmental conditions. To gain a fundamental understanding of the complex malting process, microstructural changes were evaluated and visualized by confocal laser scanning and scanning electron microscopy. Furthermore, changes observed in the micrographs were verified and endorsed by metabolic changes using established malt attributes. The degradation and formation of proteins during malting is essential for the final beer quality. To visualise fundamental protein changes taking place during malting, samples of each single process step were analysed and fractioned according their solubility. Protein fractions were analysed using a Lab-on-a-chip technique as well as OFFgel analysis. In general, a different protein distribution of wheat compared to barley or oat could be confirmed. During the malting process a degradation of proteins to small peptides and amino acids could be observed in all four Osborn fractions. Furthermore, in this study a protein profiling was performed to evaluate changes during the mashing process as well as the influence of grist composition. Differences in specific protein peaks and profile were detected for all samples during mashing. This study investigated the suitability of wheat for malting and brewing industry and closed the scientifical gap of amylolytic, cytolytic and proteolytic changes during malting and mashing.