Quantitative Data-Independent Acquisition Glycoproteomics of Sparkling Wine.

Sparkling wine is an alcoholic beverage enjoyed around the world. The sensory properties of sparkling wine depend on a complex interplay between the chemical and biochemical components in the final product. Glycoproteins have been linked to positive and negative qualities in sparkling wine, but the glycosylation profiles of sparkling wine have not been previously investigated in detail. We analyzed the glycoproteome of sparkling wines using protein- and glycopeptide-centric approaches. We developed an automated workflow that created ion libraries to analyze sequential window acquisition of all theoretical mass spectra data-independent acquisition mass spectrometry data based on glycopeptides identified by Byonic (Protein Metrics; version 2.13.17). We applied our workflow to three pairs of experimental sparkling wines to assess the effects of aging on lees and of different yeast strains used in the liqueur de tirage for secondary fermentation. We found that aging a cuvée on lees for 24 months compared with 8 months led to a dramatic decrease in overall protein abundance and an enrichment in large glycans at specific sites in some proteins. Secondary fermentation of a Riesling wine with Saccharomyces cerevisiae yeast strain Siha4 produced more yeast proteins and glycoproteins than with S. cerevisiae yeast strain DV10. The abundance and glycosylation profiles of grape glycoproteins were also different between grape varieties. To our knowledge, this work represents the first in-depth study into protein- and peptide-specific glycosylation in sparkling wines and describes a quantitative glycoproteomic sequential window acquisition of all theoretical mass spectra/data-independent acquisition workflow that is broadly applicable to other sample types.

Posttranslational Modifications Drive Protein Stability to Control the Dynamic Beer Brewing Proteome.

Mashing is a key step in beer brewing in which starch and proteins are solubilized from malted barley in a hot water extraction and digested to oligomaltose and free amino nitrogen. We used SWATH-MS to measure the abundance and site-specific modifications of proteins throughout a small-scale pale ale mash. Proteins extracted from the malt at low temperatures early in the mash decreased precipitously in abundance at higher temperatures late in the mash due to temperature/time-induced unfolding and aggregation. We validated these observations using experimental manipulation of time and temperature parameters in a microscale pale ale mash. Correlation analysis of temperature/time-dependent abundance showed that sequence and structure were the main features that controlled protein abundance profiles. Partial proteolysis by barley proteases was common early in the mash. The resulting proteolytically clipped proteins were particularly sensitive and were preferentially lost at high temperatures late in the mash, while intact proteins remained soluble. The beer brewing proteome is therefore driven by the interplay between protein solubilization and proteolysis, which are in turn determined by barley variety, growth conditions, and brewing process parameters.

The intrinsic and regulated proteomes of barley seeds in response to fungal infection.

Barley is an important cereal grain used for beer brewing, animal feed, and human food consumption. Fungal disease can impact barley production, as it causes substantial yield loss and lowers seed quality. We used sequential window acquisition of all theoretical ions mass spectrometry (SWATH-MS) to measure and quantify the relative abundance of proteins within seeds of different barley varieties under various fungal pathogen burdens (ProteomeXchange Datasets PXD011303 and PXD014093). Fungal burden in the leaves and stems of barley resulted in changes to the seed proteome. However, these changes were minimal and showed substantial variation among barley samples infected with different pathogens. The limited effect of intrinsic disease resistance on the seed proteome is consistent with the main mediators of disease resistance being present in the leaves and stems of the plant. The seeds of barley varieties accredited for use as malt had higher levels of proteins associated with starch synthesis and beer quality. The proteomic workflows developed and implemented here have potential application in quality control, breeding and processing of barley, and other agricultural products.

Benchtop micro-mashing: high-throughput, robust, experimental beer brewing.

Brewing science is undergoing a renaissance with the use of modern analytical chemistry and microbiology techniques. However, these modern analytical tools and techniques are not necessarily aligned with the scale and scope of brewing science. In particular, brewing processes can be time consuming, ingredient intensive, and require specialised technical equipment. These drawbacks compound with the need for appropriate numbers of replicates for adequately powered experimental design. Here, we describe a micro-scale mash method that can be performed using a common laboratory benchtop shaker/incubator, allowing for high throughput mashing and easy sample replication for statistical analysis. Proteomic profiles at both the protein and peptide levels were consistent between the 1 mL micro-mash and a 23 L Braumeister mash, and both mash scales produced wort with equivalent fermentable sugar and free amino acid profiles. The experimental flexibility offered by our micro-mash method allowed us to investigate the effects of altered mash parameters on the beer brewing proteome.

The post-translational modification landscape of commercial beers.

Beer is one of the most popular beverages worldwide. As a product of variable agricultural ingredients and processes, beer has high molecular complexity. We used DIA/SWATH-MS to investigate the proteomic complexity and diversity of 23 commercial Australian beers. While the overall complexity of the beer proteome was modest, with contributions from barley and yeast proteins, we uncovered a very high diversity of post-translational modifications (PTMs), especially proteolysis, glycation, and glycosylation. Proteolysis was widespread throughout barley proteins, but showed clear site-specificity. Oligohexose modifications were common on lysines in barley proteins, consistent with glycation by maltooligosaccharides released from starch during malting or mashing. O-glycosylation consistent with oligomannose was abundant on secreted yeast glycoproteins. We developed and used data analysis pipelines to efficiently extract and quantify site-specific PTMs from SWATH-MS data, and showed incorporating these features into proteomic analyses extended analytical precision. We found that the key differentiator of the beer glyco/proteome was the brewery, with beer from independent breweries having a distinct profile to beer from multinational breweries. Within a given brewery, beer styles also had distinct glyco/proteomes. Targeting our analyses to beers from a single brewery, Newstead Brewing Co., allowed us to identify beer style-specific features of the glyco/proteome. Specifically, we found that proteins in darker beers tended to have low glycation and high proteolysis. Finally, we objectively quantified features of foam formation and stability, and showed that these quality properties correlated with the concentration of abundant surface-active proteins from barley and yeast.