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Method establishment for the analysis of complex
biological samples using
nLC-MALDI MS/MS

DISSERTATION
ZUR ERLANGUNG DES DOKTORGRADES
der Naturwissenschaften
vorgelegt beim Fachbereich Chemie (14)
der Johann Wolfgang Goethe – Universität
in Frankfurt am Main
von
Tabiwang Ndipanquang Arrey
aus Mamfe (Cameroon)



Frankfurt (2010)
(D 30)

vom Fachbereich Chemie Biochemie, Pharmazie (14) der
Johann Wolfgang Goethe Universität als Dissertation angenommen.


Dekan: Prof. Dr. Dieter Steinhilber
Gutachter: Prof. Dr. Michael. Karas
Prof. Dr Rolf Marschalek


Datum der Disputation:


Contents
1. Proteomics .............................................................................................................. 31
1.1 Tools for proteomics research ......................................................................... 32
1.1.2 Gel-based separation methods ........................................................................ 34
1.1.2.2 Isoelectric focusing (IEF) ............................................................. 34
1.1.2.3 Two dimensional SDS-PAGE (2D-SDS-PAGE) ...................................... 35
1.1.2.4 Offgel- IEF ...........................................................5. .................... 3
1.1.3.1 Ion-exchange chromatography (IEC) ................................... .3.7. ..............
1.1.3.2 Size-exclusion chromatography (SEC) ............................... .3.7. .................
1.2.2.4 Affinity Chromatography ............................................................. 37
1.1.3.3 Normal-phase chromatography............................................... .3.8. .....
1.1.3.4 Reversed phase chromatography. ............................................ .3.8. ........
11.3 Two dimensional separation techniques ............................ .4..0 ...................
2 Introduction ..................................................................................................... 41
2.1 Mass spectrometry .......................................................................................... 41
2.1.1 Electrospray ionisation (ESI) .................................................... .4..2 .
2.1.2 Matrix assisted Laser Desorption/Ionisation (MALDI) ............................ 42
2.1.2.1 Mass Analyzers .......................................................................... 45

2.1.2.2 Time of flight (TOF) Analyzer ................................................ .4..5. ...
2.1.3 Tandem TOF Mass spectrometer............................................. .4..9. .......
2.2 Protein Identification ...................................................................................... 51
2.2.1 Bottom-up approach ...................................................................... 51
2.2.1.1 Protein identification by peptide mass fingerprinting (PMF) ................... 52
2.2.1.2 Shotgun protein/ Tandem MS identification .............................................. 52
2.2.2 Top-Down protein identification ............................................. .5.3. .......
2.2.3 HPLC; On- and Off-line coupling (ESI, MALDI) .................................... 53
3 Proteomics application ........................................................................................... 55
3.1 Lipid particle proteome ................................................................................... 55
3.2 Interaction of Mixed linage leukemia complexes ........................................... 57
3.3 Membrane Proteomics .................................................................................... 61
3.3.1 Quantification of membrane proteins ................................... .6.6. ..............
4 Objectives ............................................................................................................... 73
5 Methods and Material ............................................................................................. 74
5.1 Material ........................................................................................................... 74
5.2 Methods .......................................................................................................... 78

5.2.1 Expression plasmids, cell culture and transfection experiments
.
(AF4/ENL/AF4 MLL) .................................................................. 78
.
5.2.2 Affinity purification (AF4/ENL/AF4 MLL) .............................................. 78
.
5.2.3 Immunoprecipitation experiments (AF4/ENL/AF4 MLL) ....................... 78
5.2.4 Yeast strains and culture conditions ...................................... .7.8. ............
5.2.5 Isolation of Lipid particles from the yeast culture ..................................... 78
5.2.6 Protein quantification .................................................................... 79
5.2.7 Delipidation ........................................................... .................... 79
5.2.8 Cell culture (Human Simpson-Golabi-Behmel syndrome) ....................... 79
5.2.9 Induction of differentiation (Human Simpson-Golabi-Behmel
syndrome) ..........................................................7.9. .....................
5.2.10 Protein sample preparation (Human Simpson-Golabi-Behmel
syndrome) ..........................................................7.9. .....................
5.2.11 In-solution Proteolytic Digest ......................................................................... 80
5.2.11.1 Digestion of Purple Membrane (Elastase/Trypsin) .................................. 80
5.2.11.2 Digestion of CB membrane (Elastase/Trypsin) ........................................ 80
5.2.11.3 Tryptic digest (Lipoprotein. Adipocztes) .......................... .8.0. .....................
.
5.2.11.4 Tryptic digest (AF4, AF4 MLL and ENL protein complex) .................... 81
5.2.11.5 Tryptic/elastase digests of standard proteins............................................. 81

5.2.12 Reduction, Alkylation, Digestion and TMT-labelling of yeast plasma
membrane ..........................................................8.1. .....................
5.2.13 Construction of frit for pre-column and analytical column ...................... 82
5.2.14 Pre-column loading ....................................................................... 82
5.2.15 Biphasic pre-column loading .................................................... .8.2. .
5.2.16 Analytical column ........................................................................ 83
5.2.17 Nano-LC ......................................................................................................... 85
5.2.17.1 Agilent 1100 series ....................................................................... 85
5.2.17.2 Proxeon Easy nLC ....................................................................... 85
5.2.18 nLC gradient ............................................................................. 86
5.2.19 Matrix for manual measurement .............................................. .8.7. ......
5.2.20 Matrix for LC-MALDI spotting .............................................. .8.7. ......
5.2.21 Isoelectric focusing (IEF) fractionation (Offgel-IEF) ............................... 87
5..2.22 MALDI MS/MS ......................................................................... 88
5.2.23 Database searches ........................................................................ 88
5.2.24 Result interpretation and data analysis ................................ .9.0. ................
5.2.25 Analysis of secreted protein candidates (Human Simpson-Golabi-
Behmel syndrome). ....................................................................... 90

6 Results and discussion ............................................................................................ 92
6.1 Method Optimisation .............................................................................................. 92
6.1.1 Optimisation of spot deposition ...................................................................... 92
6.1.2 Matrix concentration for automatic spotting .................................................. 97
6.1.3 TOF/TOF method optimization ...................................................................... 99
6.1.4 Influence of mobile phase gradients on peptide identification ..................... 102
6.1.5 Improved mass accuracy ............................................................................... 106
6.1.6 Effect of temperature on LC Separation ....................................................... 109
6.1.7 Effect of temperature on column back pressure ........................................... 109
6.1.8 Effect of temperature on proteins and peptides identification ...................... 110
6.1.9 Effect of temperature on repeatability .......................................................... 113
6.1.10 Conclusion .................................................................................................... 118
6.2 Proteins from yeast lipid particles and human fat cells ................................ 119
6.2.1 Optimisation of the sample preparation procedure.................................. 119
6.2.2 Effect of carbon source on lipoprotein composition ............................... 120
6.2.3 Human fat cells ............................................................................................. 125
6.2.3.1 Protein identification ................................................................... 125
6.2.3.2 Validation of the identified secretome ............................. .1.2.8. .................

6.2.4 Summary ............................................................1.3.2. ....................
6.3 MLL Interaction partners .............................................................................. 135
6.3.1 AF4/ENL and AF4•MLLcomplex ....................................... .1.3.5. ..........
6.3.3 ENL-complex .......................................................................... 138
6.3.4 Summary ............................................................1.3.8. ....................
6.4 Result and Discussion: Membrane proteins .................................................. 141
6.4.1 Protein identification ................................................................... 141
6.4.2 Transmembrane coverage. ........................................................ .1.43
6.4.3 Fragmentation behaviour of elastase generated peptides ........................ 145
7 Two Dimensional Separation (Optimisation of the Offgel-IEF for its
use as a first dimension in a two dimensional separation plateform) .................. 148
7.1 Proteins and peptides identification................................... .1.4.8. ..............
7.2 Peptide Carryover ...................................................................... 151
7.3 Standard deviation ...................................................................... 152
7.4 pI distribution .......................................................................... 154
7.5 Using the pI of peptides to manually validate search results .................. 156
7.6 Suitability of the Offgel-IEF peptide fractionators for relative
quantification.......................................................................... 158

7.8 Protein identification ................................................................... 161
7.8 Protein quantification .................................................................. 162
7.9 Conclusion ............................................................ .................. 166
8 Method establishment for the analysis of post-translational
modifications ........................................................................................................ 168
8.1.1 Results and Discussion ................................................................................. 168
8.1.2 Crystal morphology and analyte stability ...........................6.8. ................... 1
8.1.3 Analyte sensitivity ...................................................................... 171
8.1.4 MALDI MS/MS ....................................................................... 175
8.2.1 Biphasic pre-column .................................................................... 179
8.2.2 Evaluation of non-phosphorylated acidic peptides excluders ................. 180
8.2.4 ENL protein complex .................................................................. 188
8.3 Conclusion ............................................................ .................. 193
9 General Summary ................................................................................................. 194
9.1 Zusammenfassung ................................................................................................ 197
10 References ............................................................................................................ 203
11.1 Papers in jorunals .......................................................................................... 228
11.3 ..................................................................................... Eidesstattliche Versicherung 230




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