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From malt to bright beer and beyond [Elektronische Ressource] : proteomic studies performed with HPLC and UPLC chromatography followed by (nano)ESI-QTOF-MS/MSMS identification / by Fabienne Decker

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201 pages
Publié par :
Ajouté le : 01 janvier 2010
Lecture(s) : 22
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FROM MALT TO BRIGHT BEER AND BEYOND
PROTEOMIC STUDIES PERFORMED WITH HPLC AND
UPLC CHROMATOGRAPHY FOLLOWED BY
(NANO)ESI-QTOF-MS/MSMS IDENTIFICATION

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by
Fabienne Decker


THESIS SUBMITTED IN FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR RERUM NATURALIUM
AT
THE FACULTY OF TECHNOLOGY
UNIVERSITY OF BIELEFELD, GERMANY







THIS DISSERTATION WAS CARRIED OUT AT
BRAUEREI C. & A. VELTINS GmbH Co. KG
AN DER STREUE
59872 MESCHEDE G REVENSTEIN
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THESIS PRESENTED
DECEMBER 2009












SUPERVISOR
PROF. ERWIN FLASCHEL, TECHNISCHE FAKULTÄT, UNIVERSITÄT BIELEFELD, GERMANY

SUPERVISORS AT BRAUEREI VELTINS
CIVIL ENGINEER STEFAN LOCH-AHRING, BRAUEREI VELTINS, MESCHEDE, GERMANY
JÜRGEN FARKE, BRAUEREI VELTINS, MESCHEDE, GERMANY








PROF. DR. KARL FRIEHS
GRADUATE DIRECTOR

PROF. DR. ERWIN FLASCHEL
FIRST REFEREE

PROF. DR. KARSTEN NIEHAUS
SECOND REFEREE

DR. RAIMUND HOFFROGGE
OBSERVER





DATE OF EXAMINATION
DATE OF EXAMINATION AND DEFENCE 12.03.2010


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„THE JOY OF SEEING AND UNDERSTANDING IS THE MOST PERFECT GIFT OF NATURE.
THE IMPORTANT THING IS TO NOT STOP QUESTIONING.“

ALBERT EINSTEIN



THIS IS IN MEMORY OF MY DAD








































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!STATEMENT
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Statement

The experimental work and results of this thesis are based on research carried out at the
laboratory (Instrumental Analytics, QA/QC) of Brauerei C. & A. Veltins GmbH Co. KG.
Meschede (Germany). The external doctoral thesis was performed in cooperation with the
Technische Fakultät, Universität Bielefeld (Germany).

To the best of my knowledge no part of this thesis has been submitted for any other degree or
qualification. All results presented here are my own work unless referenced to the contrary in
the text.































Printed on non aging paper according to DIN ISO 9706
iv
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Acknowledgement

I wish to thank the following persons for without whose help and expertise this thesis would
not have been possible.
I would like to thank my research advisor, civil engineer Stefan Loch Ahring, not only for the
interesting topic and his support of my doctoral thesis but also for providing the possibility of
working on it at the Veltins laboratory. Although being far away at Karlsruhe during the last
year of my thesis you unfailingly had time, helpful suggestions and interest for my work.
A special thank you also goes to Walter Bauer, Jürgen Farke and the Veltins brewing
company for their continued support of my thesis, especially in 2009.
Another very special thank you goes to my colleague Sascha Robbert for three years of
discussion, technical support and great fun. Without your knowledge, suggestions and support
I would not have never been able to complete this study.
I owe a further debt of gratitude to my colleagues, Claudia, Ricarda and Sandra for their
friendly support. A warm smile can sometimes light up the dark.
I would like to thank Reinaldo Almeida (Advion) for his support in the final stages of this
study and its advancement to new analytical levels.
A special thank you also goes to Prof. Erwin Flaschel for his regulatory support for this
external doctoral thesis and attendance for reference. Without your own and Karsten Niehaus´
support the shootings of the gushing phenomenon would not have been possible.
To Karsten Niehaus and Manfred Lissel, thank you very much for our discussions at Veltins
and at Bielefeld University. Your valuable information always provided fresh food for
thought.
To my partner, Wolli, a big thank you for all the calm and unflappable support over the whole
three years.
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!CONTENT

Content

ABSTRACT I
ZUSAMMENFASSUNG II
ABBREVIATIONS III

1 INTRODUCTION 1
1.1 “Food for thought“ 1
1.2 Proteins – a short refresher 2
1.3 From malt to beer and “beyond“ 6
1.3.1 Barley (Hordeum vulgare) 7
1.3.2 Malt production 8
1.3.3 The brewing process – wort production 9
1.3.4 The brewing process – boiling the wort and adding the hops 11
1.3.5 The brewing process – fermentation 12
1.3.6 The brewing process – filtration 13
1.3.7 Bright beer 15
1.3.8 “Going Beyond“ – colloidal stability and haze formation 17
1.4 Gushing 21
1.5 Barley protein species and classes 25
1.5.1 Barley and malt protein classification 25
1.5.2 nLTP1, nLTP2 and the class of ns LTP´s 27
1.5.3 Proteins Z4 and Z7 30
1.5.4 Hordeins 31
1.5.5 Barwin 32
1.5.6 Inhibitor protein classes 33
1.5.7 Other protein classes 35
1.5.7 Proteins with “other functions“ 36
1.6 Proteins from other origins 37
1.6.1 Hydrophobins – fungal proteins 37
1.6.2 Yeast proteins 38
1.6.3 Other proteins 38

2 INSTRUMENTAL BACKGROUND 40
!CONTENT

TM2.1 “Ultra Performance Liquid Chromatography“ – Acquity UPLC 40
2.1.1 Peptide analysis using UPLC 41
2.1.2 Protein analysis with reversed phase chromatography (RPC) 42
TM2.2 Mass spectrometer – Micromass QTOF micro 44
2.2.1 Ionization with an electrospray source 45
2.2.2 Ion separation with a quadrupole time of flight 49
2.2.3 MS strategies 50
2 2.2.4 Tandem mass spectrometry – MS mode 50
2.2.5 Bottom up approach 52
2.2.6 Top down approach 53
2.2.7 Protein sequence analysis using MassLynx and PLGS software 53

3 PROJECT OBJECTIVES 55
4 MATERIALS 56
4.1 Chemicals 56
4.2 Buffers and eluents 58
4.3 Consumables 58
4.4 Instruments 59
4.5 Sample materials 61

5 METHODOLOGY 63
5.1 Sample types and treatment 63
5.2 Preliminary investigations using HPLC and HPLC-MS 63
5.2.1 Tryptic digestion for bottom up analysis 64
5.2.2 Bottom up approaches 65
5.2.3 Sample fractionation with the Dionex HPLC 65
5.2.4 HPLC MS top down investigation 66
5.3 Protein quantification 67
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5.3.1 Biorad Roti Quant Universal assay and Nanoquant assay 67
5.3.2 2D Quant assay 67
5.4 Investigations concerning haze 68
5.4.1 Haze preparation 68
5.4.2 Solubility tests 69
!CONTENT

5.4.3 Tryptic digestion of haze 69
5.4.4 Long term storage of haze 70
5.4.5 Top down approaches 70
5.5 Gushing studies 71
5.5.1 Modified Carlsberg Test (MCT) 71
5.5.2 MCT with fractionated coarse groat 71
5.5.3 MCT with hop extract additives 72
5.5.4 MCT with enzyme additives 72
5.6 Protein precipitation 73
5.6.1 Early precipitation tests with fresh beer and haze 73
5.6.2 Phenolic extraction 74
5.6.3 Sample preparation for bottom up experiments after protein extraction 75
5.6.4 Sample preparation for top down experiments 75
5.7 UPLC MS and UPLC MS/MS studies 75
5.7.1 Bottom up approaches 75
5.7.2 Top down analysis of whole proteins 77
5.7.3 Top down and bottom up analysis in a single LCMS experiment 78

6 RESULTS AND DISCUSSION 80
6.1 Preliminary HPLC investigations 80
6.1.1 Non specific lipid transfer protein 1 (nLTP1) 80
6.1.2 Protein Z 83
6.1.3 Dionex HPLC beer sample fractionations 89
6.1.4 HPLC MS analysis of brewing process samples 90
6.2 Protein quantification 93
6.3 Haze analysis 96
6.3.1 Fundamental observations concerning haze 96
6.3.2 Haze formation – a dynamic process 99
6.3.3 Solubility tests with lyophilised and freshly prepared haze 102
6.3.4 Freshly prepared haze and basic beer turbidity 104
6.3.5 Bottom up analysis of haze 109
6.3.6 Top down analysis of haze samples 111
6.3.7 Summary 113
6.4 Gushing 113
!CONTENT

6.4.1 The “Modified Carlsberg Test“ (MCT) 115
6.4.2 MCT investigations with hop additives 118
6.4.3 The impact of enzymes on the gushing tendencies of malt and barley 119
6.4.4 First UPLC MS studies with MCT extract samples 121
6.4.5 Protein precipitation with MCT extracts 122
6.4.6 Bottom up investigations 124
6.4.7 Summary 135
6.5 From malt to beer and beyond 135
6.5.1 Top down approaches 135
6.5.2 Bottom up experiments 139
6.5.3 Top down and bottom up investigations in a single experiment 142
7 CONCLUSIONS 146
REFERENCES 148

APPENDICES 153
Appendix A, Summary of enzymatic MCT approaches 153
Appendix B, Peptide masses and sequences of MCT samples (B1 – B4) 155
Appendix C, Measurement of mass accuracy with protein standards 159
Appendix D, Precursor ion masses and charges of brewing samples (D1 – D7) 160
POSTER PUBLICATIONS 167
CURRICULUM VITAE 170









!LIST OF FIGURES

List of Figures

Figure 1 The basic structure of an α amino acid in its unionized, amphoteric form. .... 2

Figure 2 Chemical formula of an L proline (Pro/P ). ..................................................... 4

Figure 3 The three structural shapes of horse heart myoglobin (>sp: P68082, pdb:1,
WLA ), a protein standard used for mass spectrometry analyses of undigested
proteins in this thesis. ...................................................................................... 4

Figure 4 Light microscopy pictures of a Celite stabiliser on the left and PVPP on the
right. ................................................................................................................ 14

Figure 5 Haze at the bottom of a bottle: a.) chill haze after about 4 weeks of storage at
0 °C and b.) permanent haze after longterm cold storage at 0 °C. …………. 17

Figure 6 Model of haze formation. ................................................................................ 19

Figure 7 Chemical structure of polyphenols. ………………………………………… 20

Figure 8 Snapshot of a gushing beer. ………………………………………………... 22

Figure 9 Red and black grains observed with mould infection of malt material during
gushing period 2008. …………………………..…………………………… 23

Figure 10 Infection modi/symptoms following Fusarium infection of cereals!"………... 24

Figure 11 1 and 2D structural information on plant nLTPs. ... ...................................... 28

Figure 12 3D structure of barley lipid transfer protein (NMR, 4 structures). ............... . 28

TMFigure 13 Waters Acquity UPLC . ................................................................................ 40

TM
Figure 14 Micromass QTOF micro ion optics and instrument overview. ................... 44

Figure 15 Design of a normal electrospray (z-spray) source. ........................................ 46

Figure 16 Serial instrument connections used for normal and nano ESI infusion in this
thesis. .............................................................................................................. 47

Figure 17 The chip based Nanomate robot system with ESI chip. ................................. 47

Figure 18 LC coupling mode used with online LC/MS. .................................................. 48

Figure 19 Peptide fragmentation nomenclature. ............................................................ 51

Figure 20 Top down versus bottom up approaches in proteomics. ................................ 52

Figure 21 Spectrum of the tryptic digested nLTP1 standard. ......................................... 80

Figure 22 nLTP1 analysis by bottom up investigation and PLGS software identification.
.......................................................................................................................... 81

Figure 23 Top down protein spectrum of a native, lyophilised nLTP1 standard. ........... 82

Figure 24 Allocated ion series found with an nLTP1 protein standard. ......................... 82

Figure 25 MS survey scan of a tryptic digested protein Z SEC fraction. ........................ 84

Figure 26 Tuning effects observed with protein Z precursorion m/z = 1345 Da. ........... 85

Figure 27 Amino acid sequence of protein Z4 (Swissprot entry: P06293). .................... 86

Figure 28 Summary of sequence analogies determined for precursorions found in the
terminal protein Z fragment during native protein analysis. ......................... 87

Figure 29 Changes in LC separation observed for the nLTP1 peak within the brewhouse
cooking procedure. ......................................................................................... 90

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