LC-ESI-MS-MS analysis of non-enzymatic posttranslational protein modifications [Elektronische Ressource] / vorgelegt von Dima Aldiab

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LC-ESI-MS-MS Analysis of Non-enzymatic Posttranslational Protein Modifications Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Dima Aldiab aus Damaskus Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 30. 5. 2011 Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink Erstberichterstatterin: Prof. Dr. Monika. Pischetsrieder Zweitberichterstatter: Prof. Dr. Thomas. Drewello Danksagung Mein ganz herzlicher Dank gilt meiner Doktormutter Frau Prof. Dr. M. Pischetsrieder für die Betreuung der Arbeit und für die freundliche und wissenschaftliche Unterstützung während des Aufenthaltes in Deutschland. Mein Dank gilt weiterhin dem Hochschulministerium in Syrien sowie der Tishreen-Universität, mit deren Unterstützung die Durchführung dieser Abeit in Deutschland erst ermöglicht wurde. Des Weiteren möchte ich mich bei Herrn Prof. Dr. T. Drewello für die Übernahme des Zweitgutachtens bedanken, sowie Herrn Prof. Dr. M. Heinrich und Herrn PD Dr. T. Göen für die Übernahme der Doktorprüfung. Außerdem bedanke ich mich bei Herrn Dr. R. Waibel sowie dessen Mitarbeiterinnen für die Aufnahme der NMR-Spektren.
Publié le : samedi 1 janvier 2011
Lecture(s) : 67
Source : D-NB.INFO/1013086279/34
Nombre de pages : 178
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LC-ESI-MS-MS Analysis
of Non-enzymatic Posttranslational
Protein Modifications



Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.





vorgelegt von
Dima Aldiab
aus Damaskus


Als Dissertation genehmigt von der Naturwissenschaftlichen
Fakultät der Friedrich-Alexander-Universität
Erlangen-Nürnberg























Tag der mündlichen Prüfung: 30. 5. 2011

Vorsitzender
der Promotionskommission: Prof. Dr. Rainer Fink
Erstberichterstatterin: Prof. Dr. Monika. Pischetsrieder
Zweitberichterstatter: Prof. Dr. Thomas. Drewello


Danksagung
Mein ganz herzlicher Dank gilt meiner Doktormutter Frau Prof. Dr. M. Pischetsrieder
für die Betreuung der Arbeit und für die freundliche und wissenschaftliche
Unterstützung während des Aufenthaltes in Deutschland. Mein Dank gilt weiterhin
dem Hochschulministerium in Syrien sowie der Tishreen-Universität, mit deren
Unterstützung die Durchführung dieser Abeit in Deutschland erst ermöglicht wurde.
Des Weiteren möchte ich mich bei Herrn Prof. Dr. T. Drewello für die Übernahme
des Zweitgutachtens bedanken, sowie Herrn Prof. Dr. M. Heinrich und Herrn PD Dr.
T. Göen für die Übernahme der Doktorprüfung.
Außerdem bedanke ich mich bei Herrn Dr. R. Waibel sowie dessen Mitarbeiterinnen
für die Aufnahme der NMR-Spektren.
Sehr herzlich danke ich Frau C. Meißner für die freundliche Hilfe während des
Aufenthaltes in Deutschland.
Für die Freundschaft und Hilfsbereitschaft bedanke ich mich herzlich bei meinen
jetztigen und ehemaligen Kollegen im Arbeitkreis. Liebe Kollegen, ich danke euch:
Andrea Wühr, Artur Kessler, Bianca Meyer, Florian Baum, Gregor Vollmer, Ingrid
Weigel, Jasmin Meltretter, Kerstin Augner, Leonie Atzenbeck, Martin Tutsch,
Mellanie Deckert, Nina Zänglein, Nadine Schneider, Rainer Bäuerlein, Sabrina
Gensberger, Sarah Elschenbroich, Stefan Mittelmaier, Tanja Sauer, Tobias Hoch,
Ulla Müller und Viola Breyer.
Der größte Dank geht an meinen Mann Eyad und meine Eltern, sowie an meine
Freunde in Syrien.
Schließlich gilt ein besonderer Dank meiner verstorbenen Mutter. Danke Mama,
ohne deine Liebe und Unterstützung wäre diese Arbeit und viele andere wichtige
Dinge nicht möglich gewesen.


I

Table of contents
1 Introduction ........................................................................................................... 1
1.1 The Maillard reaction ........................................................................................ 1
1.2 The phases of the Maillard reaction ................................................................. 2
1.3 The effects of Maillard reaction ........................................................................ 4
1.3.1 Nutrition and food safety ........................................................................... 4
1.3.2 Advanced Glycation End-Products (AGEs) ............................................... 5
1.3.3 Physiological effects of dietary MRPs ....................................................... 6
1.3.4 Beneficial Maillard products ...................................................................... 8
1.4 Prevention of the Maillard reaction ................................................................... 9
1.5 Milk ................................................................................................................. 10
1.5.1 Milk proteins ............................................................................................ 11
1.5.1.1 Casein .............................................................................................. 12
1.5.1.2 Whey protein .................................................................................... 13
1.5.2 Heat treatment of milk ............................................................................. 14
1.5.3 Effects of heat treatment on milk ............................................................. 15
1.5.3.1 Effects of heat treatment on milk protein.......................................... 15
1.5.3.2 Effect of heat treatment on lactose .................................................. 17
1.5.3.2.1 Lactose isomerisation/degradation ........................................... 17
1.5.3.2.2 Lactose and Maillard reaction ................................................... 17
1.5.3.3 Effects of heat treatment on milk fat ................................................ 20
1.5.3.4 Effects of heat treatments on vitamins ............................................. 21
1.5.3.5 Other effects of heat treatment ........................................................ 21
1.5.3.5.1 Flavor ........................................................................................ 21
1.5.3.5.2 Allergenicity .............................................................................. 21
1.5.4 Indicators for milk heat treatment ............................................................ 22
1.6 Liquid Chromatography Electro Spray Ionization Tandem Mass Spectrometry
(LC-ESI-MS-MS) .................................................................................................. 23
1.7 Aims of the work: ............................................................................................ 26
2 Results and discussion ...................................................................................... 27
2.1 Selection of the marker compounds for protein modification .......................... 27
2.2 Standards synthesis ....................................................................................... 33
2.2.1 Synthesis of LacLys ................................................................................ 33
2.2.1.1 Affinity chromatography ................................................................... 36
2.2.1.1.1 Boronic acid affinity chromatography ........................................ 37
2.2.1.2 Hydrophilic interaction liquid chromatography (HILIC) ..................... 38
2.2.1.2.1 ZIC-HILIC column ..................................................................... 39
12.2.1.3 Identification of LacLys by H-NMR ................................................. 42
12.2.1.4 Determination of the purity of LacLys by H-NMR............................ 43
2.2.2 Synthesis of CML .................................................................................... 44
2.2.3 Synthesis of MeSO ................................................................................. 45 II
2.2.4 Trial to synthesize lysine aldehyde ......................................................... 47
2.2.5 Trial to synthesize Cys-SOH ................................................................... 48
2.2.6 OH-Trp .................................................................................................... 49
2.2.7 Discussion ............................................................................................... 49
2.3 Method development for the analysis of glycation and oxidation products in
milk by LC-ESI-MS-MS ........................................................................................ 51
2.3.1 Introduction ............................................................................................. 51
2.3.2 ESI-MS-MS conditions ............................................................................ 52
2.3.2.1 Flow-dependent MS parameters ...................................................... 53
2.3.2.2 Compound-dependent MS parameters ............................................ 53
2.3.3 MS2 fragmentation of the investigated analytes ..................................... 55
2.3.4 Chromatographic seperation of CML, LacLys, MeSO and 5-OH-Trp using
a C18 column ................................................................................................... 57
2.3.5 Glycation of α-LA in a milk model mixture ............................................... 59
2.3.6 Oxidation of α-LA in a milk model mixture ............................................... 64
2.3.7 Discussion ............................................................................................... 66
2.4 Detection of glycation and oxidation products in milk ..................................... 68
2.4.1 Analysis of MeSO and LacLys ................................................................ 69
2.4.1.1 Formation of MeSO during sample work-up .................................... 71
2.4.2 Analysis of 5-OH-Trp .............................................................................. 72
2.4.3 Analysis of CML ...................................................................................... 73
2.4.4 Separation of 5-OH-Trp, MeSO, CML and LacLys using a ZIC-HILIC
column ............................................................................................................. 74
2.4.5 Comparison of the signal intensities using C18 or ZIC-HILIC columns ... 75
2.4.6 Analysis of milk samples by LC-MS-MS using a ZIC-HILIC column ....... 76
2.4.7 Discussion ............................................................................................... 78
2.5 Analysis of ornithine in milk proteins .............................................................. 81
2.5.1 Introduction ............................................................................................. 81
2.5.2 LC-ESI-MS-MS analysis of ornithine ....................................................... 82
2.5.3 Discussion ............................................................................................... 85
2.6 Method optimization for the detection of MeSO, CML and LacLys in proteins of
processed milk ..................................................................................................... 86
2.6.1 Optimization of protein hydrolysis ........................................................... 86
2.6.2 Results .................................................................................................... 87
2.6.3 Discussion ............................................................................................... 93
2.7 Method validation for the quantification of LacLys, CML and MeSO in milk
proteins ................................................................................................................ 97
2.7.1 Introduction ............................................................................................. 97
2.7.2 Quantitative analysis by standard addition .............................................. 98
2.7.3 Results .................................................................................................. 100
2.7.3.1 Within-day repeatability .................................................................. 100
2.7.3.2 Between-day repeatability .............................................................. 101
2.7.3.3 Recovery ........................................................................................ 102
2.7.3.4 Limit of detection and limit of quantitation ...................................... 105
2.7.4 Discussion ............................................................................................. 106
2.8 Quantitation .................................................................................................. 109
2.8.1 Introduction ........................................................................................... 109
2.8.2 Results .................................................................................................. 109
2.8.3 Discussion ............................................................................................. 111
3 Materials and methods ..................................................................................... 114 III
3.1 Materials and apparatus ............................................................................... 114
3.1.1 General materials .................................................................................. 114
3.1.2 Apparatus .............................................................................................. 114
3.1.3 Materials ............................................................................................... 115
Synthesis ................................................................................................... 115
Hydrolysis .................................................................................................. 116
Materials and equipments for liquid chromatography and mass spectrometry
................................................................................................................... 116
Oxidation and glycation in milk models ...................................................... 117
3.2 Buffers and solutions .................................................................................... 117
Milk resembling Phosphate Buffer Saline (PBS), pH 6.8 ............................... 117
Solutions for enzymatic hydrolysis (Hasenkopf et al) ................................. 118
Solutions for enzymatic hydrolysis (Delatour et al) .................................... 118
Solutions for the ninhydrin assay ................................................................... 119
Solutions to isolate lysyl oxidase .................................................................... 119
Standard`s concentration to determine the fragments of LacLys, CML, MeSO
and 5-OH-Trp ................................................................................................. 120
Aqueous mobile phases for liquid chromatography ....................................... 120
Solutions for affinity chromatography ............................................................. 120
3.3 Methods ....................................................................................................... 121
3.3.1 Lactulosyllysine synthesis ..................................................................... 121
3.3.1.1. Trial to synthesize lactulosyllysine ................................................ 121
3.3.1.2 Chromatographic conditions for the separation of FMOC-LacLys . 121
3.3.1.3 Removal of the blocking group of FMOC-LacLys .......................... 122
3.3.1.4 Trial to purifiy LacLys using m-Aminoboronic acid ......................... 122
3.3.1.5 Synthesis of FMOC-LacLys ........................................................... 122
3.3.1.5.1 Fractionation of FMOC-LacLys on a ZIC-HILIC column ......... 123
13.3.1.5.2 Determination of the purity of LacLys by H-NMR .................. 123
3.3.2 Synthesis of Cbz-CML .......................................................................... 123
Removal of the blocking group of Cbz-CML .............................................. 124
3.3.3 Synthesis of methionine sulfoxide (MeSO) ........................................... 124
3.3.4 Trial to synthesize lysine aldehyde ....................................................... 124
3.3.5 Trial to synthesize cysteine sulfenic acid (Cys-SOH) ............................ 126
3.3.6 The conditions of liquid chromatography for the seperation of CML,
MeSO, LacLys and 5-OH-Trp on a C18 column ............................................ 126
3.3.7 The conditions of liquid chromatography for the seperation of CML,
MeSO, LacLys and 5-OH-Trp on a ZIC-HILIC column ................................... 127
3.3.8 Stimulation of glycation in a milk model mixture .................................... 127
3.3.9 Stimulation of oxidation in a milk model mixture ................................... 127
3.3.10 Preparation of milk samples for LC-MS-MS analysis .......................... 128
3.3.10.1 Milk defatting ................................................................................ 128
3.3.10.2 Removal of lactose and minerals from milk and milk model mixture
................................................................................................................... 128
3.3.10.3 Enzymatic protein hydrolysis (Hasenkopf et al) ........................... 128
3.3.10.4 Purification of protein hydrolyzate by ultrafiltration ....................... 128
3.3.10.5 Enzymatic protein hydrolysis (Delatour et al) ............................... 129
3.3.10.6 Acidic protein hydrolysis (Delatour et al) ...................................... 129
3.3.10.7 Purification of protein hydrolyzate by solid phase extraction ........ 130
3.3.10.7.1 The standards MeSO, LacLys and CML on a SPE column .. 131
3.3.11 Trial to detect ornithine in milk protein ................................................ 131 IV
3.3.12 Investigation the effect of sodium borohydride on MeSO .................... 131
3.3.13 Ninhydrin assay .................................................................................. 132
3.3.14 Method validation ................................................................................ 132
3.3.14.1 Within-day repeatability (intra-day variation) ................................ 132
3.3.14.2 Between-day repeatability (inter-day variation) ............................ 133
3.3.14.3 Recovery ...................................................................................... 134
3.3.14.4 LOD and LOQ .............................................................................. 135
LOD and LOQ of LacLys and MeSO ..................................................... 135
LOD and LOQ of CML ........................................................................... 136
3.3.15 Qantitation ........................................................................................... 137
3.3.15.1 Determination protein content by Kjeldahl ................................... 138
4 Summary ............................................................................................................ 139
5 Zusammenfassung ............................................................................................ 144
Bibliography ......................................................................................................... 150
List of Tables ........................................................................................................ 159
List of Figures ...................................................................................................... 161




















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