Tom Knapman
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“Bottoms Up” Proteomics

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Ahhhh beer.

It’s a ubiquitous drink found in over 90% of all countries around the world. Since the dawn of civilization, man has celebrated with beer where it can make even the most introverted person suddenly dance a little jig or belt out a top 40 song. But other than its courage-inducing alcohol, what’s really in beer? In addition to alcohol, beer is composed of a masterful mix of carbohydrates, proteins, and small molecules that all add up to one glorious glass. But the process of making that glass involves a myriad of steps where the composition of these ingredients changes drastically over time as the preparation and fermentation process progress.

Scientists at the University of Queensland in Brisbane, Australia decided to zero in on one aspect of beer production and analyze the proteome during nanoscale beer production.1 Proteins can have a profound effect on the color, viscosity, texture, and taste of beer. Therefore, a better understanding of the beer proteome and the dynamics of how it changes over the production process can lead to better quality control and manipulation of product attributes.

In their study, beer samples were produced using several different malt and grist conditions and analyzed at the sweet wort, hopped wort, and bright beer steps of production. LC-MS/MS using IDA Analysis on a TripleTOF® 5600 system and ProteinPilot™ 4.1 software identified over 200 unique quantifiable proteins from barley and yeast. ProteinPilot is particularly effective for identifying PTMs, unusual modifications, and non-specific cleavages that may happen simultaneously. The researchers then used SWATH® Acquisition to analyze how those protein abundances changed over the different stages of the beer creation process.

And their results make perfect sense. As brewing progressed, the abundance of yeast proteins increased with more yeast proteins secreted into the wort during fermentation. Changes in barley proteins correlated well with conditions in each sample, such as hydrophobic proteins showing sensitivity to organic solvent concentrations from increasing ethanol during fermentation.  In fact, major differences in the global proteomes between samples depended more upon the stage of the brewing process rather than the mill or grist conditions.

The researchers also looked for chemical and enzymatic modifications in their SWATH data and found many non-tryptic cleavages from yeast and barley enzymes. In contrast to the previous results, these, in fact, correlated strongly with the malting and mashing conditions used for the beer production. Additionally, examples of glycation of lysines, oxidation of methionines, prolines, and tryptophans were all abundant. All of these modifications can have an effect on the diverse sensory properties of beer.

To learn more about this fascinating research, grab a copy of the article from the Journal of Proteome Research, a glass of your favorite brew, and give it a read. For the backstory to the paper read our interview below with Ben Schulz one of the authors of this fascinating paper.

I’ll drink to that!

Q&A With Dr. Ben Schulz

Dr. Benjamin Schulz graduated with a degree in Chemical Engineering and Science in 2000 from The University of Queensland, after which he joined Proteome Systems, an Australian biotechnology company. In 2004 he moved to the ETH Zurich in Switzerland for his doctoral studies. He joined the School of Chemistry & Molecular Biosciences as a University of Queensland Postdoctoral Research Fellow in 2008 and NHMRC CareerDevelopment Fellow in 2012 and is now an NHMRC Career Development Fellow and Senior Lecturer.

Tell me a bit about the backstory of the paper.

Ben: The team involved in the paper was my group whose focus is using proteomics to study post-translational modifications of proteins, Claudia Vickers whose interests lie in synthetic biology, and Glen Fox who does research into barley breeding and its impact on the quality of beer. We met (over a beer) and realized there was an opportunity to take a modern systems biology approach to study the brewing process. We are working a lot with beer producers. For example, we have a collaboration with one craft brewer looking to use proteomics approaches to drive development of their production techniques. There are applications of this work in bioproduction and product development, but also in QC assurance and in product legitimacy.

Where does the historical aspect of beer brewing you mention in the paper come from?

Ben: Well we’ve been brewing beer for thousands of years using different approaches. The modern industrial approach used nowadays is a well-established and robust bioprocess, yet the fundamental biochemistry is poorly understood. There is a lot of interest in historical brewing processes and how they could be harnessed to impact modern techniques. We’ve even had people who have found beer bottles in shipwrecks or dug them up in Egypt who want to taste them and try to understand the process that would have been used to make them.

There were substantial changes to the abundances of many barley proteins at each stage of the beer brewing process. Did heat-shock proteins increase significantly from sweet wort to hopped wort (over the boil)? 

Ben: In fact, they didn’t. At the point that we are observing the proteome, the barley is no longer alive so any changes observed arise from the bioprocessing not from a cellular response. That makes it an unusual proteome to study and we are particularly interested in the unusual PTMs that can arise as a result.

Many plants don’t have their full genomes sequenced yet. For example, is hops sequenced? If not, how are you performing searches to account for any hops proteins?

Ben: We’ve had to work without data from the barley genome, which wasn’t published when we started this work. It’s since been published. We had to work with what was available in Uniprot at the time. It would be interesting to review the data again taking advantage of gene annotation and sequencing which of course is possible with data acquired by SWATH Acquisition.

Sometimes bacteria can enter the brewing process and spoil a perfectly good batch of beer! Would this technique be able to detect any pathogens that might have infiltrated along the way?

Ben: We didn’t consider this as the beer we were studying was produced using a well-controlled process, but providing you used a general library containing other proteomes this method would conceivably detect proteins from other sources, yes.

References:
1. “Process Proteomics of Beer Reveals a Dynamic Proteome with Extensive Modifications”, B.L. Schulz et. al., J. Proteome Res., February 19, 2018

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