The proteome of your pint: novel ligand capture technique uncovers more proteins

The beer proteome: literary inspiration

In the 1990s, a Belgian author created a series of comic books around a family of Brewers, set over 150 years and cataloguing their growth from a brasserie to a multinational brewing company. Les Maîtres de l'Orge, translated as The Master Brewers, was conceived by Jean Van Hamme and illustrated by Francis Vallès. The series enjoyed a certain popularity before exerting an irresistible influence in an unexpected area.

It was discovered by members of an Italian research team who were inspired to carry out research into beer, specifically the beer proteome. Pier Giorgio Righetti and colleagues from the Polytechnic of Milan and the University degli Studi di Milan declared that "it was this superb saga that fired our fancy to dig deep in the beer proteome."

They were surprised to find that there were few published studies on this subject. The residual proteins in beer that survive the brewing process have an important say in the quality of the final product. They affect the properties and volume of the head foam, which are a critical factor in the acceptance of a beer by consumers.

Isolating proteins from beer: a peptide ligand library approach

The Italian team used a combinatorial peptide ligand library to isolate the proteins from beer, encouraged by its excellent performance in previous research on wine. By measuring the total sample volume, the amounts of proteins recovered (based on spiking experiments) and the volume of the harvested protein solution, they estimated the signal amplification factor of recovered protein to be 5000.

As little as 10 µg/L of residual casein could be measured in wines, with a detection limit of 1 µg/L. This gives unparalleled sensitivity.

In addition, a full bottle of wine could be extracted within three hours by the addition of just 100 µL of beads, a far more simple and rapid procedure than found in other reports.

When the methodology was transferred to beer, a preliminary step was required to remove the polyphenols because they cause smearing of the protein spots on the gels during SDS-PAGE. After degassing the beers, the best results were obtained by adding polyvinylpolypyrrolidone to precipitate the polyphenols. The resultant liquid was clear and almost colourless, indicating that the majority of the phenols had been removed.

The liquid was treated at pH 4.0, 7.0 and 9.3 with a commercial hexapeptide library or a homemade library of reduced polydispersity. The captured proteins were eluted from the library beads and subjected to SDS-PAGE. The protein spots were still smeared a little, indicating incomplete removal of the polyphenols, but they were considered to be acceptable.

The optimum pH for recovering the proteins was pH 7.0 and the homemade library captured a greater total amount of proteins than the commercial equivalent.

The beer proteome expanded: more barley, yeast and maize proteins detected

The gel spots were cut into sections and the proteins present were in-gel digested with trypsin for analysis by liquid chromatography-tandem mass spectrometry. The MS/MS spectra were searched against a database constructed from the UniProtKB/Swiss-Prot database with Hordeum vulgare (barley) Humulus lupulus (hop), Zea mays (maize) and the yeasts Saccharomyces cerevisiae, Saccharomyces pastorianus and Saccharomyces bayanus as strings. The target database had 9898 entries.

The greatest number of identified proteins was achieved using 1 L of beer and a 1:1 mixture of the two libraries. There were 20 unique barley proteins and 2 maize proteins, increasing the reported number of detected proteins in beer.

In addition, the number of known yeast proteins in beer was raised, with 40 from S. cerevisiae and one each from S. bayanus and S. pastorianus. Righetti argued that the yeast proteins must represent trace components, because only 2 had been reported in previous studies.

For several proteins, the number of peptides detected and the percentage of sequence coverage was increased, which was attributed to the larger sample volume and the greater amount of proteins captured on the ligands.

The brewer stated that only barley and corn were used to produce the beer, which was a lager beer fermented with S. carlsbergensis (pastorianus). This was confirmed by the detection of two maize proteins.

The discovery of so many extra proteins in beer will be of interest to brewers. The suppression or promotion of particular proteins could be manipulated in order to modify the flavour, aroma or foaming characteristics. The new data could also be used to confirm the types of barley or hops employed in the brewing process for particular beers.

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.