Proteome-Based Analysis of Colloidal Instability Enables the Detection of Haze-Active Proteins in Beer

Colloidal haze is a serious quality defect of bright beers that considerably reduces their shelf life and is thought to be triggered by hordeins, a class of proline-rich barley proteins. In this work, the proteomes of fresh and old beers were investigated in bottled pilsners and compared to the protein inventory of haze to identify specific haze-active proteins. Haze isolates dissolved in rehydration buffer contained high concentrations of proteins and sugars but provided protein gels with weak spot signals. Consequently, a treatment for the chemical deglycation with trifluoromethanesulfonic acid was applied, which resulted in the identification of protein Z4, LTP1, CMb, CMe, pUP13, 3a, and Bwiph as constituents of the haze proteome. Because only one hordein was detectable and the proline content in haze hydrolysates was lower than those of barley prolamins, our results suggest that this class of proteins is of minor importance for haze development

Unsupervised spatial segmentation of eight independent MALDI MSI analyses covering the first days of barley germination using WHIDE.

<p>A: Image scans with outlines of labeled seed compartments. B: Cluster analysis of all mass spectra of the whole grain areas. C: Cluster analysis of all mass spectra of the embryo areas as annotated in A. D: Cluster analysis of all mass spectra of the endosperm as annotated in A. E: Effect of the cluster granularity (7, 21, 56 clusters) on mapping results, exemplarily shown for four days germinated barley (G4d) from analysis B. The visualized cluster, granularity was set to 7 in A, B, and C to assign clear cluster profiles (right panels). 93 <i>m/z</i> values were selected for spatial segmentation in all analyses. Their contribution to the distinct clusters is indicated by the bar size in the right panels; for <i>m/z</i> identification see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150208#pone.0150208.t001" target="_blank">Table 1</a>. For an interactive exploration of the results in WHIDE see <a href="https://ani.cebitec.uni-bielefeld.de/barleymsi" target="_blank">https://ani.cebitec.uni-bielefeld.de/barleymsi</a>.</p

Spatio-Temporal Metabolite Profiling of the Barley Germination Process by MALDI MS Imaging

<div><p>MALDI mass spectrometry imaging was performed to localize metabolites during the first seven days of the barley germination. Up to 100 mass signals were detected of which 85 signals were identified as 48 different metabolites with highly tissue-specific localizations. Oligosaccharides were observed in the endosperm and in parts of the developed embryo. Lipids in the endosperm co-localized in dependency on their fatty acid compositions with changes in the distributions of diacyl phosphatidylcholines during germination. 26 potentially antifungal hordatines were detected in the embryo with tissue-specific localizations of their glycosylated, hydroxylated, and O-methylated derivates. In order to reveal spatio-temporal patterns in local metabolite compositions, multiple MSI data sets from a time series were analyzed in one batch. This requires a new preprocessing strategy to achieve comparability between data sets as well as a new strategy for unsupervised clustering. The resulting spatial segmentation for each time point sample is visualized in an interactive cluster map and enables simultaneous interactive exploration of all time points. Using this new analysis approach and visualization tool germination-dependent developments of metabolite patterns with single MS position accuracy were discovered. This is the first study that presents metabolite profiling of a cereals’ germination process over time by MALDI MSI with the identification of a large number of peaks of agronomically and industrially important compounds such as oligosaccharides, lipids and antifungal agents. Their detailed localization as well as the MS cluster analyses for on-tissue metabolite profile mapping revealed important information for the understanding of the germination process, which is of high scientific interest.</p></div

Localization and signal intensities of hydroxycinnamic acid derivatives and hordatines in germinating barley.

<p>Top: Localization of p-coumaroylagmatine (CA) as representative for hydroxycinnamic acid amides and hordatine B as representative for hordatines that co-localized to hordatine B when occurring in the same modification state. Intensity maps depict the non-glycosylated (<i>m/z</i> 581), glycosylated (<i>m/z</i> 743), and disaccharide-modified form (<i>m/z</i> 905) at three time points during germination (0d: non-germinated barley, G3d: three days germinated, G5d: five days germination) in longitudinal and transversal section plane. Hordatines were not detected in cross sections in non-germinated barley. The last panel shows an overlay of the three modification forms. Ion intensities were normalized to the TIC, the highest relative intensity was set to 100%. Middle panel: Average mass spectra from annotated embryo measurement regions (right) in non-germinated (green), three days (blue) and five days (red) germinated barley. Bottom: Mass spectrum with indicated peaks of hydroxycinnamic acid amides as hordatine precursors (<i>m/z</i> 250–350) and of hordatine A, B, C, and D (D not detected, grey font), hydroxylated hordatines (-OH), and hexose-modified derivates (Hex / 2 Hex) at <i>m/z</i> 550–1000. CA: coumaroylagmatine, FA: feruloylagmatine, CA-OH / FA-OH: hydroxylated CA and FA.</p

Localization of oligosaccharides in barley during germination.

<p>A) Cryo-sections of non-germinated (0d) and three day germinated (G3d) barley. B) Average mass spectrum of all MS acquired from germinated barley with [M+Na]⁺ (red) and [M+K]⁺ (blue) ions of oligosaccharides. *DP: degree of polymerization. C) Intensity heat maps of oligosaccharides with three, six, and nine hexoses in sodium [M+Na]⁺ and potassium [M+H]⁺ adducts in ungerminated barley (0d) and after three days of germination (G3d). MS intensities were normalized to the TIC of each mass spectrum; the highest relative intensity of all MS was set to 100%. The distributions of these compounds at all time points of the germination process are provided as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150208#pone.0150208.s005" target="_blank">S5 Fig</a>.</p

The barley germination process: Seedling development and sampling time points.

<p>A) Time scheme of mini malting of the <i>Optic</i> barley at 16°C for the collection of samples. Arrows indicate sampling time points with their sample name. 0d: raw barley, S: steeping, G: germination day, K: kilned malt (K not used for MSI). W: water, A: air rest, K1: kilning at 45°C (7h), K2: kilning at 65°C (17 h). B) Growth of the barley seeds during malting. Barley (0d, <i>T</i> = 1), steeped barley (S1d, <i>T</i> = 2), three of the five time points during germination (G1d, G3d, G5d (<i>T</i> = 4,6,8)) and final kilned malt (K1d) are shown as representatives. Main seed organs and compartments are indicated at the raw barley seed.</p

Detected and identified compounds from MS imaging of barley seeds.

<p>Detected and identified compounds from MS imaging of barley seeds.</p

Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium pv. campestris-3

Lar location or function. The pie charts indicate the number of proteins in each group isolated from OMVs collected from M9 medium (A) from XVM2 medium (B) and the outer membrane proteins isolated from cells grown in M9 medium (C). The percentage values within the pie chart give the amount of proteins grouped as virulence-associated, which comprise the groups of virulence factors and type III secretion proteins.<p><b>Copyright information:</b></p><p>Taken from "Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium pv. campestris"</p><p>http://www.biomedcentral.com/1471-2180/8/87</p><p>BMC Microbiology 2008;8():87-87.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2438364.</p><p></p

Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium pv. campestris-2

He brackets with numeration indicate the sections taken from the gel for tryptic digestion. (B) Protein profiles of OMVs isolated from cultures grown in M9 medium and the outer membrane (OM) fraction prepared from the same culture.<p><b>Copyright information:</b></p><p>Taken from "Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium pv. campestris"</p><p>http://www.biomedcentral.com/1471-2180/8/87</p><p>BMC Microbiology 2008;8():87-87.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2438364.</p><p></p

Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium pv. campestris-0

Ernatant of pv campestris strain B100 at a 30 k fold magnification (OMVs highlighted by arrows). (B) Immunogold electron microscopy of 1% (w/v) uranyl acetate stained cell at 14 k fold magnification after incubation with an anti-B100 antibody which was detected by a 10 nm gold particle bound to a goat anti-rabbit antibody (gold-labelled OMV highlighted by arrows). (C) Immunogold electron micrograph of 1% (w/v) uranyl acetate stained OMVs at 27 k fold magnification after incubation with an anti-B100 antibody detected with a 10 nm gold particle bound to an goat anti-rabbit antibody.<p><b>Copyright information:</b></p><p>Taken from "Analysis of outer membrane vesicle associated proteins isolated from the plant pathogenic bacterium pv. campestris"</p><p>http://www.biomedcentral.com/1471-2180/8/87</p><p>BMC Microbiology 2008;8():87-87.</p><p>Published online 2 Jun 2008</p><p>PMCID:PMC2438364.</p><p></p