Elsevier

Journal of Chromatography B

Volume 878, Issue 30, 15 November 2010, Pages 3143-3148
Journal of Chromatography B

2D-HPLC and MALDI-TOF/TOF analysis of barley proteins glycated during brewing

https://doi.org/10.1016/j.jchromb.2010.09.023Get rights and content

Abstract

The barley proteins have been the subject of interests of many research groups dealing with barley grains, malt and beer. The proteins which remain intact after harsh malting conditions influence the quality and flavor of beer. The characteristic feature of the proteins present in malt and beer is their extensive modification with carbohydrates, mainly glucose that comes from the starch degradation during technological processes. The degree of the protein glycation has an effect on the quality of malt and beer and on the properties of the beer foam. A combination of two-dimensional high performance liquid chromatography (2D-HPLC) and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF MS) was used for the analysis of the protein extracts that were reduced, alkylated, and degraded enzymatically without prior protein separation. This so-called “shot-gun” approach enabled us to determine glycation sites in one third of the proteins identified in the study and to propose potential glycation markers for fast and efficient monitoring during malting.

Introduction

Barley (Hordeum vulgare) is a plant commonly exploited in the brewing industry. In the course of the process of beer production, barley grains are germinated during malting to produce a large amount of α-amylase that degrades starch to sugars. The impact of the choice of a barley variety, and of the quality of grain and malt on beer properties is a major concern of the breweries worldwide. The most important components of the final product – beer – are proteins, carbohydrates, lipids, and acids from hops [1] that influence foam stability. Several proteins present in beer and foam have been characterized and found to be resistant to the malting processes [1]. The key beer and foam proteins are non-specific lipid transfer proteins (ns-LTP1 and ns-LTP2), protein Z, and hordeins that belong to the family of storage proteins. All these proteins are tolerant to high temperatures and resistant to proteolysis [2], [3], [4], [5].

Starch degradation increases the amount of monosaccharides, mainly d-glucose, during malting in the malt. A combination of high concentration of monocarbohydrates, higher temperature, and long time of the process causes glycation of the proteins [6]. This non-enzymatic glycation is a result of side chain reactions, called Maillard reactions, between d-glucose and free ɛ-amino group in lysine and guanidino group in arginine [7]. d-Glucose reacts with free amino group giving a Schiff base that rapidly rearranges to form a more stable (1-deoxy-d-fructose-1-yl)-amino acid derivative called Amadori compound [8].

There were several attempts to develop relatively fast and simple method for protein identification and determination of glycation in malt and beer. So far, most of them considered an application of gel electrophoresis (GE), in-gel degradation, and mass spectrometry (MS) [9].

In this study, we present application of a modern analytical approach, known as “shot-gun” analysis, for characterization of barley proteins undergoing glycation during brewing.

The “shot-gun” experiment usually combines two-dimensional fractionation and mass spectrometry for protein digest analysis [10]. This approach is supposed to increase dynamic range and proteome coverage. The standard analysis involves an enzymatic degradation of a complex protein mixture. Resulted peptides are then separated by HPLC and eluted fractions are analyzed by mass spectrometry. Each isolated peptide should be fragmented, and the peptide sequence and protein identity is determined based on the fragmentation (MS/MS) spectra. The general goal in the “shot-gun” approach is determination of a huge number of proteins. In our case, we were mainly interested in detection and location of non-enzymatic glycations in barley malt.

Section snippets

Chemicals

All reagents (analytical grade) were obtained from Sigma (Schnelldorf, Germany). Chymotrypsin was obtained from Roche Diagnostics GmbH (Mannheim, Germany).

Samples

Barley seeds and malt (cultivar Jersey) were provided by Research Institute of Brewing and Malting, Brno, Czech Republic. Protein extracts were obtained by the method described below. Milled barley grain and malt (50 mg) were extracted twice in deionised water (500 μl) and centrifuged at 12,000 × g. The extraction was done at the room temperature

Results and discussion

Monitoring of protein glycation during brewing was performed by comparing samples of barley seeds and grains. The choice of malt was based on a previous study of glycation of a selected protein (ns-LTP1) in barley malt, spent grains, sweet wort, boiled wort, green beer and finished beer [12]. The study revealed large amount of glycated protein in malt (presumably due to the relatively high temperature during malting and the abundance of glucose and maltose in malt), which was not altered by the

Conclusions

The 2D salt chromatographic separation of digests of barley extracts reveals the complexity of the samples and provides a sufficient separation power to deal with these complex mixtures. It is especially important in the case when there are few peptides of the same molecular weight in the sample. Comparing the chromatograms of both grain and malt extracts enabled us to observe the differences introduced during malting and to determine fractions that may contain glycated peptides. This helped to

Acknowledgments

This work was supported by grants no. 1M0570 (Research Centre for Study of Extract Compounds of Barley and Hops), from the Ministry of Education, Youth and Sports of the Czech Republic and the Institutional Research Plan AV0Z40310501 of Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, v.v.i.

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