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β-Amylase (α-1,4-glucan maltohydrolase; EC 3.2.1.2) is known to be an important enzyme in beer brewing. To identify the contribution of β-amylase in more detail, two β-amylase-deficient barley, 'Backcrossed line (BCL)-Haruna' and 'Nittakei 81', were used for the investigation of malt quality and brewing characteristics. 'BCL-Haruna' was bred by the backcross method for the introduction of the β-amylase-deficient trait from landrace barley ('Tibet Violet 1') to Japanese traditional malting barley 'Haruna Nijo'. 'Nittakei 81' was bred by crossing between Japanese malting barley 'Sukai Golden' and 'Tibet Violet 1'. With regards to the malting quality under 41.0% target cast moisture in micro-malting, the diastatic power (DP) and apparent attenuation limit (AAL) of 'BCL-Haruna' and 'Nittakei 81' were 408 oWK and 416 oWK and 12.4% and 14.8% lower than those of the control variety 'Haruna Nijo'. Oligosaccharide analysis indicated that the maltose concentration of the 'Nittakei 81' wort was 19.7 g/L lower in comparison to a standard European malting barley 'Propino' wort, although the maltotetraose and maltopentaose concentrations of the 'Nittakei 81' wort were 5.6 g/L and 2.7 g/L higher than those of the 'Propino' wort. After fermentation, the alcohol concentration of the 'Nittakei 81' beer was 0.67% lower compared with the 'Propino' beer, and the extract and concentrations of maltotetraose and maltopentaose of the 'Nittakei 81' beer were 5.0 g/L and 2.5 g/L higher than those of the 'Propino' beer. The results indicate that the β-amylase-deficient barley has a unique malting quality, especially lower DP and AAL, which might induce inadequate starch degradation or fermentation. However, this trait caused no serious issues during the brewing process, suggesting the possibility of β-amylase-deficient barley as a unique brewing material including low-alcoholic and non-alcoholic beer production with a specific saccharide composition. [ABSTRACT FROM AUTHOR]

Arabinoxylan is a major polysaccharide found in barley and wheat. As this polysaccharide has been reported to be involved in wort viscosity and filterability, as well as β-glucan, it has become increasingly important in beer brewing in recent years, despite the limited availability of a measurement methodology for arabinoxylan. In this study, we developed a simple method for determining arabinoxylan content in wort and beer products using a commercially available xylose determination kit. The results show a relationship between arabinoxylan and the viscosity of wort and beer products. These results were consistent with previous reports, suggesting that this simple method can be used to observe the behavior of arabinoxylan during malting and brewing. [ABSTRACT FROM AUTHOR]

Recent advances in high-throughput genotyping have made it easier to combine information from different mapping populations into consensus genetic maps, which provide increased marker density and genome coverage compared to individual maps. Previously, a single nucleotide polymorphism (SNP)-based genotyping platform was developed and used to genotype 373 individuals in four barley ( L.) mapping populations. This led to a 2943 SNP consensus genetic map with 975 unique positions. In this work, we add data from six additional populations and more individuals from one of the original populations to develop an improved consensus map from 1133 individuals. A stringent and systematic analysis of each of the 10 populations was performed to achieve uniformity. This involved reexamination of the four populations included in the previous map. As a consequence, we present a robust consensus genetic map that contains 2994 SNP loci mapped to 1163 unique positions. The map spans 1137.3 cM with an average density of one marker bin per 0.99 cM. A novel application of the genotyping platform for gene detection allowed the assignment of 2930 genes to flow-sorted chromosomes or arms, confirmed the position of 2545 SNP-mapped loci, added chromosome or arm allocations to an additional 370 SNP loci, and delineated pericentromeric regions for chromosomes 2H to 7H. Marker order has been improved and map resolution has been increased by almost 20%. These increased precision outcomes enable more optimized SNP selection for marker-assisted breeding and support association genetic analysis and map-based cloning. It will also improve the anchoring of DNA sequence scaffolds and the barley physical map to the genetic map.

Abstract Background Pancreatic cancer is among the most lethal malignancies worldwide. This study aimed to identify a novel prognostic biomarker, facilitating treatment selection, using mass spectrometry (MS)-based proteomic analysis with formalin-fixed paraffin-embedded (FFPE) tissue. Results The two groups with poor prognosis (n = 4) and with better prognosis (n = 4) had been carefully chosen among 96 resected cases of pancreatic cancer during 1998 to 2007 in Tohoku University Hospital. Although those 2 groups had adjusted background (UICC-Stage IIB, Grade2, R0, gemcitabine adjuvant), there was a significant difference in postoperative mean survival time (poor 21.0 months, better 58.1 months, P = 0.0067). Cancerous epithelial cells collected from FFPE tissue sections by laser micro-dissection (LMD) were processed for liquid chromatography-tandem mass spectrometry (LC-MS/MS). In total, 1099 unique proteins were identified and 6 proteins showed different expressions in the 2 groups by semi-quantitative comparison. Among these 6 proteins, we focused on Nm23/Nucleoside Diphosphate Kinase A (NDPK-A) and immunohistochemically confirmed its expression in the cohort of 96 cases. Kaplan-Meier analysis showed high Nm23/NDPK-A expression to correlate with significantly worse overall survival (P = 0.0103). Moreover, in the multivariate Cox regression model, Nm23/NDPK-A over-expression remained an independent predictor of poor survival with a hazard ratio of 1.97 (95% CI 1.16-3.56, P = 0.0110). Conclusions We identified 6 candidate prognostic markers for postoperative pancreatic cancer using FFPE tissues and immunohistochemically demonstrated high Nm23/NDPK-A expression to be a useful prognostic marker for pancreatic cancer.

The α-amylase activity of cultivated barley is critically important to the brewing industry. Here, we surveyed variation in malt α-amylase activity in 343 cultivated barley accessions from around the world. Population structure analysis based on genotype data at 1536 SNPs clustered these accessions into two groups, one comprising South-East Asian and Ethiopian accessions and one group containing the other accessions. A genome-wide association study identified significant quantitative trait loci (QTLs) for α-amylase activity on all seven chromosomes of barley. Accessions showing high and low α-amylase activity were crossed with the high-quality Japanese malting barley cv. Harun Nijo to develop F mapping populations. We identified two QTLs on chromosome 6H in a cross between Haruna Nijo (high activity) × Weal (highest activity). Single QTLs were identified each on 3H, 4H, and 5H from a cross between Haruna Nijo (high activity) × VLB-1 (low activity), indicating that the high α-amylase activity in Haruna Nijo might be derived from loci on these chromosomes. The addition of the high α-amylase activity QTL alleles from chromosome 6H in cv. Weal further increased the α-amylase activity conferred by alleles of Haruna Nijo. These results demonstrate that a target haplotype can be successfully improved using a strategy comprising diversity analysis of ex situ collections followed by introducing effective new alleles. [ABSTRACT FROM AUTHOR]

In general, barley with smoother degradation of seed storage material (modification) is known to have lower seed dormancy and a higher risk of pre-harvest sprouting (PHS). The Qsd1 and Qsd2 genes affecting seed dormancy have also been reported to affect malting characteristics. To overcome this situation, barley with an excellent balance of modification and seed dormancy was screened among the mutant population derived from 'Nittakei 68 (N68)'. In the mutant lines screened, 'N68-411' exhibited a moderate seed dormancy similar to that of 'N68', but with higher modification. It was considered that 'N68-411' was mutated in a different gene from Qsd1 and Qsd2 , resulting in no significant change in seed dormancy, only in modification. 'N68-411' also showed rapid modification during germination, suggesting a possibility of shorter germination time in malting process. Analysis of progenies from 12WY375/N68-411 and Xena/N68-411 indicated that the 'N68-411' trait was inherited and associated with a small number of genes. [Display omitted] • Sustainability of barley and malt production. • Discovery of the barley mutant line with novel traits. • Better balance between malt modification and seed dormancy. • Possibility of a shorter germination time in the malting process. [ABSTRACT FROM AUTHOR]

Background: Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of the synovial joints. Early intervention followed by early diagnosis can result in disease remission; however, both early stage diagnosis and provision of effective treatment have been impeded by the heterogeneity of RA, which details of pathological mechanism are unclear. Regardless of numerous investigations of RA by means of genomic and proteomic approaches, proteins interplaying in RA synovial tissues that contain various types of synoviocytes, are not yet sufficiently understood. Hence we have conducted an HPLC/mass spectrometry-based exploratory proteomic analysis focusing on synoviocyte lesions laser-microdissected (LMD) from formalin-fixed paraffin-embedded (FFPE) synovial tissues (RA, n = 15; OA, n = 5), where those of Osteoarthritis (OA) were used as the control. Results: A total of 508 proteins were identified from the RA and OA groups. With the semi-quantitative comparisons, the spectral index (SpI), log2 protein ratio (RSC) based on spectral counting, and statistical G-test, 98 proteins were found to be significant (pair-wise p < 0.05) to the RA synovial tissues. These include stromelysin-1 (MMP3), proteins S100-A8 and S100-A9, plastin-2, galectin-3, calreticulin, cathepsin Z, HLA-A, HLA-DRB1, ferritin, neutrophil defensin 1, CD14, MMP9 etc. Conclusions: Our results confirmed the involvement of known RA biomarkers such as stromelysin-1 (MMP3) and proteins S100-A8 and S100-A9, and also that of leukocyte antigens such as HLA-DRB1. Network analyses of protein-protein interaction for those proteins significant to RA revealed a dominant participation of ribosome pathway (p = 5.91 × 10-45), and, interestingly, the associations of the p53 signaling (p = 2.34 × 10-5). An involvement of proteins including CD14, S100-A8/S100-A9 seems to suggest an activation of the NF-kB/MAPK signaling pathway. Our strategy of laser-microdissected FFPE-tissue proteomic analysis in Rheumatoid Arthritis thus demonstrated its technical feasibility in profiling proteins expressed in synovial tissues, which may play important roles in the RA pathogenesis. [ABSTRACT FROM AUTHOR]

Foam stability is a key factor of beer quality for consumers and brewers. Recent beer proteome analyses have suggested that barley dimeric α-amylase inhibitor-1 (BDAI-1) and avenin-like protein-a (ALP) derived from barley are important for beer foam stability. In this study, BDAI-1 and ALP were purified from a Japanese commercial beer sample using salt precipitation and column chromatography. The purification level was verified using two-dimensional gel electrophoresis, mass spectrometry, and database searches. Purified BDAI-1 and ALP were added to a beer sample to compare the foam stability to that of a control beer sample. As a result, beer foam stability was significantly improved by BDAI-1 but not by ALP, thereby suggesting that BDAI-1 affects beer foam stability whereas ALP does not. [ABSTRACT FROM AUTHOR]

Background: There are no reports of proteomic analyses of inflamed islets in type 1 diabetes. Procedures: Proteins expressed in the islets of enterovirus-associated fulminant type 1 diabetes (FT1DM) with extensive insulitis were identified by laser-capture microdissection mass spectrometry using formalin-fixed paraffin-embedded pancreatic tissues. Results: Thirty-eight proteins were identified solely in FT1DM islets, most of which have not been previously linked to type 1 diabetes. Five protein-protein interacting clusters were identified, and the cellular localization of selected proteins was validated immunohistochemically. Migratory activity-related proteins, including plastin-2 (LCP1), moesin (MSN), lamin-B1 (LMNB1), Ras GTPase-activating-like protein (IQGAP1) and others, were identified in CD8+ T cells and CD68+ macrophages infiltrated to inflamed FT1DM islets. Proteins involved in successive signaling in innate/adaptive immunity were identified, including SAM domain and HD domain-containing protein 1 (SAMHD1), Ras GTPase-activating-like protein (IQGAP1), proteasome activator complex subunit 1 (PSME1), HLA class I histocompatibility antigen (HLA-C), and signal transducer and activator of transcription 1-alpha/beta (STAT1). Angiogenic (thymidine phosphorylase (TYMP)) and anti-angiogenic (tryptophan-tRNA ligase (WARS)) factors were identified in migrating CD8+ T cells and CD68+ macrophages. Proteins related to virus replication and cell proliferation, including probable ATP-dependent RNA helicase DEAD box helicase 5 (DDX5) and heterogeneous nuclear ribonucleoprotein H (HNRNPH1), were identified. The anti-apoptotic protein T-complex protein 1 subunit epsilon (CCT5), the anti-oxidative enzyme 6-phosphogluconate dehydrogenase (PDG), and the anti-viral and anti-apoptotic proteins serpin B6 (SERPINB6) and heat shock 70 kDa protein1-like (HSPA1L), were identified in FT1DM-affected islet cells. Conclusion: The identified FT1DM-characterizing proteins include those involved in aggressive beta cell destruction through massive immune cell migration and proteins involved in angiogenesis and islet vasculature bleeding, cell repair, and anti-inflammatory processes. Several target proteins for future type 1 diabetes interventions were identified. [ABSTRACT FROM AUTHOR]

An overview on targeted personalized medicine is given describing the developments in Japan of lung cancer patients. These new targeted therapies with novel personalized medicine drugs require new implementations, in order to follow and monitor drug efficacy and outcome. Examples from IRESSA (Gefitinib) and TARCEVA (Erlotinib) treatments used in medication of lung cancer patients are presented. Lung cancer is one of the most common causes of cancer mortality in the world. The importance of both the quantification of disease progression, where diagnostic-related biomarkers are being implemented, in addition to the actual measurement of disease-specific mechanisms relating to pathway signalling activation of disease-progressive protein targets is summarised. An outline is also presented, describing changes and adaptations in Japan, meeting the rising costs and challenges. Today, urgent implementation of programs to address these needs has led to a rebuilding of the entire approach of medical evaluation and clinical care. [ABSTRACT FROM AUTHOR]

The article presents a study on the oxidative stress and antioxidant capacity in space-grown barley. It supports that the space-grown barley are not significantly different from those of the ground-grown ones in their gene expression and enzyme activity of superoxide dismutase, catalase, and ascorbate peroxidase. Findings of the study recommend that the space environment does not cause oxidative stress and reduce antioxidant capacity in plants.

This study investigated whether the consumption of a diet in which high-beta-glucan barley replaced rice would reduce the visceral fat area as well as the serum low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) in hypercholesterolemic Japanese men. A randomized, double-blinded, placebo-controlled intervention study was conducted in 44 hypercholesterolemic Japanese men with a body mass index (BMI) >22 kg/m2. The subjects were randomly assigned to groups consuming either rice (placebo group) or a mixture of rice and pearl barley with a high beta-glucan content (test group, 7.0 g beta-glucan per day) for 12 weeks. Blood samples were taken, and CT scan obtained before the trial and every four weeks during the trial. The pearl barley intake significantly reduced serum concentrations of LDL-C (P = 0.041) and TC (P = 0.037) during the trial. Significant differences between the test and placebo groups were found for the visceral fat (P = 0.039), BMI (P = 0.015), and waist circumference (P = 0.011) at the end point. The consumption of pearl barley with a high beta-glucan content reduces not only LDL-C but also visceral fat area. [ABSTRACT FROM AUTHOR]

Barley intake reportedly reduces the risk of cardiovascular disease, but effects on the systemic phenotypes during healthy aging have not yet been examined. Therefore, we examined the effects of barley on the lifespan; behavioral phenotypes, such as locomotor activity, and cognitive functions, and intestinal microbiome in the senescence-accelerated mouse-prone 8 (SAMP8) mouse. We prepared two mild high-fat diets by adding lard, in which the starch components of AIN-93G were replaced by rice or barley "Motchiriboshi." SAMP8 (four weeks old, male) mice were fed AIN-93G until eight weeks old, and then rice (rice group) or barley diet (rice: barley = 1:4, barley group) until death. Changes in aging-related phenotypes, object and spatial recognition, locomotor and balancing activities, and the intestinal microbiome were recorded. Moreover, plasma cholesterol levels were analyzed at 16 weeks old. Barley intake prolonged the lifespan by approximately four weeks, delayed locomotor atrophy, and reduced balancing ability and spatial recognition. Barley intake significantly increased the medium and small particle sizes of high-density lipoprotein (HDL) cholesterol, which is associated with a reduced risk of total stroke. The Bacteroidetes to Firmicutes ratio in the barley group was significantly higher than that in the rice group during aging. Thus, lifelong barley intake may have positive effects on healthy aging. [ABSTRACT FROM AUTHOR]

Interstitial lung disease (ILD) events have been reported in Japanese non-small-cell lung cancer (NSCLC) patients receiving EGFR tyrosine kinase inhibitors. We investigated proteomic biomarkers for mechanistic insights and improved prediction of ILD. Blood plasma was collected from 43 gefitinib-treated NSCLC patients developing acute ILD (confirmed by blinded diagnostic review) and 123 randomly selected controls in a nested case-control study within a pharmacoepidemiological cohort study in Japan. We generated ∼7 million tandem mass spectrometry (MS/MS) measurements with extensive quality control and validation, producing one of the largest proteomic lung cancer datasets to date, incorporating rigorous study design, phenotype definition, and evaluation of sample processing. After alignment, scaling, and measurement batch adjustment, we identified 41 peptide peaks representing 29 proteins best predicting ILD. Multivariate peptide, protein, and pathway modeling achieved ILD prediction comparable to previously identified clinical variables; combining the two provided some improvement. The acute phase response pathway was strongly represented (17 of 29 proteins, p = 1.0×10-25), suggesting a key role with potential utility as a marker for increased risk of acute ILD events. Validation by Western blotting showed correlation for identified proteins, confirming that robust results can be generated from an MS/MS platform implementing strict quality control. [ABSTRACT FROM AUTHOR]

Abstract The lipoxygenase-1-null (LOX-less) trait has positive effects on beer quality, and in particular, improvement of flavor stability by reduction of beer deteriorating substances such as trans -2-nonenal (T2N). 'SouthernStar' was successfully developed as the first LOX-less malting barley variety in Australia by introducing LOX-less trait into 'Flagship' by successive backcross and molecular marker assisted selection (MMAS). 'Flagship' is a two-rowed malting barley variety in Australia and it was used as the recurrent parent for the successive backcrossing. The yield potential, agronomic performance, disease resistance and the general malting quality were almost identical between 'SouthernStar' and 'Flagship'. To evaluate effects of LOX-less trait on beer quality, the brewing trials were conducted using a pilot-scale brewing apparatus. The wort and beer quality were similar between 'SouthernStar' and 'Flagship'. The effect of the LOX-less trait on the reduction of beer deteriorating substances such as T2N and trihydroxyoctadecenoic acid (THOD) contents was clearly verified in this study. The scores for total staleness of the test beers were significantly lower than those of the control beers under several storage conditions in one trial. Highlights • A new lipoxygenase-1-less (LOX-less) malting barley variety 'SouthernStar' developed by backcrossing. • Similar agricultural characteristics and malting quality as a recurrent parent 'Flagship'. • Reduction of trihydroxyoctadecenoic acid (THOD) and trans -2-nonenal (T2N) contents in beer by LOX-less trait introduced. • Improvement of flavor stability evaluated by sensory analysis for stale beer. [ABSTRACT FROM AUTHOR]

Abstract: Beer proteins were analysed by two-dimensional gel electrophoresis (2DE). The protein species associated with major spots on 2DE gels were identified by mass spectrometry followed by a database search to construct a comprehensive beer proteome map. As a result, 85 out of 199 protein spots examined were positively identified and categorised into 12 protein species. A total of 11 beer samples were brewed from the malt of eight cultivars having different levels of protein modification. This experiment was designed to demonstrate the influences of barley cultivar and malt modification on beer protein composition and beer quality characters. The beers produced from these brewing trails were subsequently analysed by 2DE and their proteomes were compared. Cultivars and malt modification affected the concentration of several proteins in beer. Beer protein concentration was associated with differences in the desirable beer quality trait, foam stability. In addition, expression of yeast derived proteins were observed that may also influence beer quality. Overall, the application of a comprehensive beer proteome map provides a strong platform for detection and potential manipulation of beer quality related proteins. [Copyright &y& Elsevier]

Abstract: Colloidal haze reduces beer quality considerably. Four haze samples were analyzed by two-dimensional gel electrophoresis (2DE) in order to identify haze-active proteins. Several protein spots were observed in all of the four haze samples. Using mass spectrometry analysis followed by a database search identified these spots as barley dimeric alpha-amylase inhibitor (BDAI-1), CMb component of tetrameric alpha-amylase inhibitor (CMb) and trypsin inhibitor CMe precursor (CMe). These proteins were considered to be haze-active. Since haze-active proteins are adsorbed by silica gel in the beer filtration process, we eluted proteins adsorbed onto silica gel (PAS) and identified their species. These major PAS were identified as protein Z4, protein Z7 and trypsin/amylase inhibitor pUP13 (TAI), rather than BDAI-1, CMb and CMe. Furthermore, we analyzed proline compositions in the beer proteins, PAS and the haze proteins. Consequently, we found that the proline compositions of PAS were higher (ca. 20mol%) than those in the beer proteins (ca. 10mol%), although those of the haze-active proteins such as BDAI-1, CMb and CMe were 6.6–8.7mol%. Our results suggest that BDAI-1, CMb and CMe are not predominant haze-active proteins, but growth factors of beer colloidal haze. [Copyright &y& Elsevier]

Hokkaido-specific malting barley varieties have been developed to improve the grain yield, disease resistance, malting quality, and brewing quality. In this report we describe the breeding and evaluation of brewing quality of a hulled two-row malting barley ( Hordeum vulgare L.) variety 'Satuiku 5 go' lacking lipoxygenase-1 (LOX-1-less). 'Satuiku 5 go' was evaluated in the joint field trials for malting barley in Hokkaido from 2016 to 2018. 'Satuiku 5 go' exhibited 11% higher grain yield and 7.6 cm shorter plant height than the control variety, 'Ryohfu'. However, the disease severity of Fusarium head blight (FHB) in 'Satuiku 5 go' was higher than in 'Ryohfu'. For malting quality, 'Satuiku 5 go' exhibited higher diastatic power, soluble nitrogen content, and fine extract content, and lower wort β-glucan content than 'Ryohfu'. 100-litter pilot scale brewing trials were conducted with 'Satuiku 5 go' and 'Satuiku 2 go' as a control variety, also a LOX-1-less variety, and no clear differences were observed., (Copyright © 2024 by JAPANESE SOCIETY OF BREEDING.)

Lipoxygenase-1 (LOX-1) null 'New Sachiho Golden' is a two-row malting barley ( Hordeum vulgare L.) cultivar released in 2015 that was developed at the Tochigi Prefectural Agricultural Experimental Station by backcross breeding using the high-yield leading cultivar 'Sachiho Golden' as a recurrent parent and the LOX-1 null mutant 'Daikei LM1' as a non-recurrent parent. To develop 'New Sachiho Golden' we used a simple LOX activity assay and marker-assisted selection. This is the first LOX-1 null malting barley cultivar in Japan that is resistant to barley yellow mosaic virus (types I-III). Agronomic characteristics and malting qualities of 'New Sachiho Golden' were similar to those of 'Sachiho Golden', except that 'New Sachiho Golden' had no LOX activity in ungerminated grains and had clearly lower LOX activity during malting than 'Sachiho Golden'. The concentrations of a trans -2-nonenal (T2N) precursor in wort and beer made from 'New Sachiho Golden' were significantly lower than in those made from 'Sachiho Golden', both before and after storage.

Putative nudix hydrolase (NUDX) genes, which encode amino acid sequences showing homology with those of Arabidopsis NUDXs and conserve nudix motif, were identified from barley. The 14 deduced barley NUDXs (HvNUDX1-14) were classified into established subfamilies, except for 8-oxo-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) pyrophosphohydrolase and mRNA decapping enzyme subfamilies, and three substrate-unknown subfamilies. Drought and UV-C stresses, respectively, up-regulated 7 and 4 HvNUDX genes, but some homologs of Arabidopsis NUDXs showed different responses to abiotic stress. HvNUDX12 gene, belonging to diadenosine tetraphosphates (Ap₄A) pyrophosphohydrolase subfamily gene and up-regulated by UV-C, was expressed in Escherichia coli cells. The recombinant protein showed 8-oxo-dGTP, Ap₄A, and guanosine-3',5'-tetraphosphate (ppGpp) pyrophosphohydrolase activities, and the suppression of the lacZ amber mutation in a mutT-deficient E. coli cells caused by the incorporation of 8-oxo-GTP into mRNA was prevented to a significant degree. These results suggest that barley NUDXs have unique constitution and response of NUDX to abiotic stress.

Proteome analysis provides a way to identify proteins related to the quality traits of beer. A number of protein species in beer and wort have been identified by two-dimensional gel electrophoresis combined with enzyme digestion such as trypsin, followed by mass spectrometry analyses and/or liquid chromatography mass/mass spectrometry. In addition, low molecular weight polypeptides in beer have been identified by the combination of non-enzyme digestion and mass analyses. These data sets of various molecular weight polypeptides (i.e., proteomes) provide a platform for analyzing protein functions in beer. Several novel proteins related to beer quality traits such as foam stability and haze formation have been identified by analyzing these proteomes. Some of the proteins have been applied to the development of efficient protein or DNA markers for trait selection in malting barley breeding. In this chapter, recent proteome studies of beer and wort are reviewed, and the methods and protocols of beer and wort proteome analysis are described.

Background: Large cell neuroendocrine carcinoma (LCNEC) of the lung, a subtype of large cell carcinoma (LCC), is characterized by neuroendocrine differentiation that small cell lung carcinoma (SCLC) shares. Pre-therapeutic histological distinction between LCNEC and SCLC has so far been problematic, leading to adverse clinical outcome. We started a project establishing protein targets characteristic of LCNEC with a proteomic method using formalin fixed paraffin-embedded (FFPE) tissues, which will help make diagnosis convincing., Methods: Cancer cells were collected by laser microdissection from cancer foci in FFPE tissues of LCNEC (n = 4), SCLC (n = 5), and LCC (n = 5) with definite histological diagnosis. Proteins were extracted from the harvested sections, trypsin-digested, and subjected to HPLC/mass spectrometry. Proteins identified by database search were semi-quantified by spectral counting and statistically sorted by pair-wise G-statistics. The results were immunohistochemically verified using a total of 10 cases for each group to confirm proteomic results., Results: A total of 1981 proteins identified from the three cancer groups were subjected to pair-wise G-test under p < 0.05 and specificity of a protein's expression to LCNEC was checked using a 3D plot with the coordinates comprising G-statistic values for every two group comparisons. We identified four protein candidates preferentially expressed in LCNEC compared with SCLC with convincingly low p-values: aldehyde dehydrogenase 1 family member A1 (AL1A1) (p = 6.1 × 10-4), aldo-keto reductase family 1 members C1 (AK1C1) (p = 9.6x10-10) and C3 (AK1C3) (p = 3.9x10-10) and CD44 antigen (p = 0.021). These p-values were confirmed by non-parametric exact inference tests. Interestingly, all these candidates would belong to cancer stem cell markers. Immunohistochmistry supported proteomic results., Conclusions: These results suggest that candidate biomarkers of LCNEC were related to cancer stem cells and this proteomic approach via FFPE samples was effective to detect them.

Foam stability is an important quality trait of beer. Our previous results of two-dimensional gel electrophoresis (2DE) analyses of beer proteins implied a relationship between barley dimeric alpha-amylase inhibitor-1 (BDAI-1) and beer foam stability as judged by the NIBEM-T analyzer. To develop a novel prediction method of beer foam stability under different conditions of barley cultivar and malt modification, multiple linear regression analysis was applied. The spot intensities of major beer proteins on 2DE gel were quantified and used as explanatory variables. The foam stabilities of 25 beer samples each brewed from malt with different malt modification in one of the three cultivars (cultivars A, B, and C) were explained by the spot intensities of BDAI-1 at the 5% significance level ( r = 0.421). Furthermore, two other major protein spots (b0 and b5) were observed on the 2DE gels of Japanese commercial beer samples with different foam stability. Then, multiple regression for foam stability was calculated using these three spot intensities as explanatory variables. As a result, 72.1% of the beer foam stability in 25 beer samples was explained by a novel multiple regression equation calculated using spot b0 and BDAI-1 as positive explanatory variables and spot b5 as a negative variable. To verify the validity of the multiple regression equation and the explanatory variables, the beer foam stability in practical beer samples was analyzed. As a result, 81.5% of the beer foam stability in 10 Japanese commercial beer samples was also explained by using spot b0 and BDAI-1 as positive explanatory variables and spot b5 as a negative variable. Mass spectrometry analyses followed by database searches revealed that protein spots b0 and b5 were identified as protein Z originated from barley and thioredoxin originated from yeast, respectively. These results confirm that BDAI-1 and protein Z are foam-positive factors and identify yeast thioredoxin as a possible novel foam-negative factor.

The foam stability of beer is one of the important key factors in evaluating the quality of beer. The purpose of this study was to investigate the relationship between the level of malt modification (degradation of protein, starch, and so on) and the beer foam stability. This was achieved by examining foam-promoting proteins using two-dimensional gel electrophoresis (2DE). We found that the foam stability of beer samples brewed from the barley malts of cultivars B and C decreased as the level of malt modification increased; however, the foam stability of cultivar A did not change. To identify the property providing the increased foam stability of cultivar A, we analyzed beer proteins using 2DE. We analyzed three fractions that could contain beer foam-promoting proteins, namely, beer whole proteins, salt-precipitated proteins, and the proteins concentrated from beer foam. As a result, we found that in cultivar A, some protein spots did not change in any of these three protein fractions even when the level of malt modification increased, although the corresponding protein spots in cultivars B and C decreased. We analyzed these protein spots by peptide mass finger printing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. As a result, all of these spots were identified as barley dimeric alpha-amylase inhibitor-I (BDAI-I). These results suggest that BDAI-I is an important contributor to beer foam stability.