Development and bioanalytical application of affinity-mass spectrometry for identification and

206 Pages·2014·4.92 MB·English

by  Mihaela Stumbaum

Development and bioanalytical application of affinity-
mass spectrometry for identification and structural
characterisation of protein-ligand interactions

Dissertation
zur Erlangung des akademischen Grades
des Doktors der Naturwissenschaften
(Dr. rer. nat.)

vorgelegt von
Mihaela Stumbaum

an der

Mathematisch-Naturwissenschaftliche Sektion
Fachbereich Chemie
Konstanz, 2014

Tag der mündlichen Prüfung: 29.05.2013
1. Referent: Prof. Dr. Dr. h.c. Michael Przybylski
2. Referent: Prof. Dr. Jörg Hartig
2
Science, properly understood,
heals the man of his pride;
she shows him his limits.
Albert Schweitzer

I dedicate this work to my wonderful parents Atena and Pavel Drăguşanu, and to my
loving husband Jörg.
3
The present work has been performed in the time frame from January 2007 to March
2011 in the Laboratory of Analytical Chemistry and Biopolymer Structure Analysis,
Department of Chemistry, University of Konstanz, under the supervision of Prof. Dr.
Dr. h. c. Michael Przybylski.
I would like to express my deep gratitude and appreciation to Professor Michael
Przybylski for the interesting research topic and scientific discussions, guiding my steps
throughout these years and for his continuous encouragement during my research. His
expert knowledge, enthusiasm, dedication to research, and education inspired me and
will continue to influence me in the future.
My collaborators involved in my reserach projects are acknowledged:
Prof. Dr. M. L. Gross and Dr. T. Tu for the interesting collaboration and scientific
discussions during my visits in St. Louis, Washington University, within a DAAD
Programme.
Prof. Dr. J. Hoheisel and his co-worker M. Dauber from the Functional Genome
Analysis Laboratory, DKFZ Heidelberg for providing me the Ets1 peptides library and
DNA samples, and the fascinating scientific discussions.
Prof. Dr. M. Glocker and his co-workers from the Proteome Center Rostock for
providing me with patients samples in the rheumatoid arthritis project and for
interesting scientific disscusions.
Thanks to all members of the group for the nice and sociable atmosphere, but most of
all I want to thank to Dr. Alina Petre, Dr. Marilena Manea, Dr. Camelia Vlad, Kathrin
Lindner and Adrian Moise for enlightening scientific discussions.
Special thanks to Nicole Engel for the dedicated work during her Master thesis in the
development of the online coupling of SAW biosensor with high resolution mass
spectrometry, as well as for the nice times spent together.
4
Special thanks to Dr. Lisa Jones for her support and the wonderful time spent together
in St. Louis.
Special thanks to all my friends in Konstanz for their support and the wonderful time
spent together.
Last but not least, my deepest gratitude is dedicated to my beloved family. I could never
have made it so far without the love and encouragement of my parents and my sister
LuminiŃa. Thank you with all my heart to my husband and friend, Jörg, for his
encouragement, support, help and patience.
Furthermore, I need to appreciate the funding by the German Academic Exchange
Service (DAAD, Bonn, Germany, PP 502/09).
Publications 5
This dissertation has been partially published in the following articles and presented at
the following Conferences:
Publications
1. Petre B.A., Drăguşanu M., Przybylski M. Molecular recognition specificity of
anti-3-nitrotyrosine antibodies revealed by affinity-mass spectrometry and
immunoanalytical methods in „Applications of Mass Spectrometry in Life
Safety”, Springer Science 2008, 55-67.
2. Drochioiu G., Manea M., Drăguşanu M., Murariu M., Dragan E.S., Petre B.A.,
Mezo G., Przybylski M. “Interaction of beta-amyloid (1-40) peptide with pairs
of metal ions: An electrospray ion trap mass spectrometric model study”,
Biophys. Chem. 2009, 144(1-2): 9-20.
3. Tu T., Drăguşanu M., Petre B.A., Rempel D.L., Przybylski M. and Gross M.L.
Protein-Peptide Affinity Determination Using an H/D Exchange Dilution
Strategy: Application to Antigen-Antibody Interactions, J. Am. Soc. Mass.
Spectrom. 2010, 21, 1660-1667.
4. Drăguşanu M., Petre B.A., Slamnoiu S., Vlad C., Tu T., Przybylski M., Online
bioaffinity-electrospray mass spectrometry for simultaneous detection,
identification and quantification of protein-ligand interactions, J. Am. Soc.,
Mass. Spectrom. 2010, 21, 1643-1648.
5. Drăguşanu M., Petre B.A., Przybylski M., Epitope motif of an anti-
nitrotyrosine antibody specific for nitrotyrosine-modified peptides revealed by
affinity-mass spectrometry, J. Peptide Sci. 2011, 17: 184-191.
6. Stumbaum M., Gronewold T. and Przybylski M., Biosensor-Electrospray Mass
Spectrometry, GEN 2011, 31(4).
7. Petre B.A., Ulrich M., Stumbaum M., Bernevic B., Moise A., Döring G.,
Przybylski M., When is Mass Spectrometry Combined with Affinity Approaches
Essential? A Case Study of Tyrosine Nitration in Proteins, J. Am. Soc. Mass.
Spectrom. 2012, 23 (11), 1831-1840.
Publications 6
Conference presentations
1 Oxidative Post-Translational Modifications of Proteins in Cardiovascular
Disease Conference, Boston 2008, USA, “Cysteinyl-nitrosylated peptides:
synthesis, structure and immunoanalytical characterisation”.
2 DGMS-Workshop “Affinity-Mass Spectrometry-Methods & Bioanalytical
Applications", Konstanz 2009, “Biosensor-affinity & mass spectrometry”.

Conference poster presentations
1 Drăguşanu M., Petre B.-A., Weber R. and Przybylski M. (2007) “Mass
spectrometric epitope identification of tyrosine nitrated peptides recognized by a
monoclonal 3-nitrotyrosine antibody”, Lausanne, Swiss Proteomics Society
(SPS).
2. Drăguşanu M., Petre B.-A., Döring G. and Przybylski M. (2008), “Recognition
epitope of an anti-nitrotyrosyl-antibody specific for protein nitration revealed by
affinity-mass spectrometry”, Denver, Colorado, USA, ASMS 56th Annual
Conference.
3. Drăguşanu M., Petre B.-A., Tu T., Rempel D., Gross M. L. and Przybylski M.
(2008), “Online Bioaffinity-Ion Trap mass spectrometry: combining molecular
identification and bioaffinity quantification in biopolymer interaction”, Boston
USA, "Oxidative Post-Translational Modifications of Proteins in Cardiovascular
Disease" Conference.
4. Drăguşanu M., Petre B.-A., Tu T., Gross M. L. and Przybylski M. (2009)
“Molecular recognition specificity and bioaffinity quantification in biopolymer
interaction of anti-3-nitrotyrosine antibody revealed by SAW-ESI-MS and
PLIMSTEX dilutions strategy”, Philadelphia, Pennsylvania, 57th ASMS
Conference on Mass Spectrometry.
Table of contents 7
Table of contents
1 Introduction..............................................................................................................11
1.1 Physiological and pathophysiological protein-ligand interactions..........................11
1.2 Analytical methods for characterisation of protein-ligand interactions ..................16
1.3 Bioaffinity-mass spectrometry for protein structure and interaction analysis.........23
1.4 Mass spectrometric methods for protein structure determination...........................26
1.5 Scientific goals of the dissertation...........................................................................34
2 Results and discussion.............................................................................................36
2.1 Development of online bioaffinity-mass spectrometry instrumentation.................36
2.1.1 Development of online bioaffinity-electrospray ion trap mass spectrometry..37
2.1.2 Development of online bioaffinity-high resolution mass spectrometry...........46
2.2 Application of online bioaffinity-mass spectrometry to the identification of tyrosine
nitrations in peptides and proteins...........................................................................52
2.2.1 Mass spectrometric structural characterisation of tyrosine nitrated peptides and
anti-3-nitrotyrosine antibody...................................................................................52
2.2.2 Binding affinity of the anti-3-nitrotyrosine antibody to tyrosine nitrated
peptides....................................................................................................................61
2.2.3 Identification of an epitope motif of tyrosine-nitrated peptides to the anti-3-
nitrotyrosine antibody..............................................................................................67
2.2.4 Identification and quantification of tyrosine-nitrated peptides to an anti-3-
nitrotyrosine antibody..............................................................................................76
2.2.5 Identification and quantification of tyrosine-nitrated peptides-antibody
interactions by peptide titration in combination with hydrogen/deuterium amide
exchange mass spectrometry...................................................................................88
2.3 Application of online bioaffinity-mass spectrometry to Calcium binding protein-
peptide interaction studies.....................................................................................109
2.3.1 Structure and biological function of Calmodulin...........................................109
Table of contents 8
2.3.2 Structural characterisation of Calmodulin by bioaffinity-mass spectrometry113
2.4 Interaction studies of nucleotide-transcription factor with Photo-oncogene Ets
peptides..................................................................................................................117
2.4.1 Structure and pathophysiological role of Ets1 protein in breast cancer.........117
2.4.2 Affinity of Ets1 peptides to nucleotide transcription factor...........................121
2.5 Interaction studies of autoantibodies against Rheumatoid Arthritis with nuclear
ribonucleoprotein epitope peptides........................................................................126
2.5.1 Immunopathology of Rheumatoid Arthritis...................................................126
2.5.2 Binding affinity of RA33 epitope peptides to autoantibodies........................127
3 Experimental part...................................................................................................138
3.1 Materials and reagents...........................................................................................138
3.2 Enzymes, Antibodies, Proteins and Peptides.........................................................139
3.3 Solid phase peptide synthesis................................................................................139
3.4 Chromatographic and electrophoretic separation methods....................................142
3.4.1 Reversed phase-high performance liquid chromatography............................142
3.4.2 SDS-PAGE according to Laemmli.................................................................143
3.4.2.1 Colloidal Coomassie staining.........................................................................144
3.4.3 Western Blot...................................................................................................144
3.5 Immunological methods........................................................................................146
3.5.1 Dot blot...........................................................................................................146
3.5.2 Preparation of antibody micro-column...........................................................146
3.5.3 Affinity-mass spectrometry approach ............................................................147
3.5.4 Epitope-excision and extraction mass spectrometry......................................148
3.5.5 Enzyme-linked immunosorbent assay............................................................148
3.5.6 Surface Acoustic Wave biosensor anaylsis....................................................150
Table of contents 9
3.5.6.1 Cleaning of gold surfaces...............................................................................151
3.5.6.2 Thiol monolayer formation on gold surface...................................................151
3.5.6.3 Covalent immobilisation of proteins/ peptides on self-assembled monolayer151
3.5.6.4 Experimental setup for antigen-antibody interaction studies.........................151
3.5.6.5 Determination of dissociation constants.........................................................152
3.5.7 Coupling of SAW biosensor with electrospray mass spectrometry...............153
3.5.7.1 Online coupling of SAW biosensor with ESI-Ion Trap mass spectrometry...153
3.5.7.2 Online coupling of SAW biosensor with high resolution ESI-FTICR mass
spectrometry...................................................................................................155
3.6 Peptide titration in combination with hydrogen/deuterium amide exchange mass
spectrometry...........................................................................................................156
3.7 Zip Tip pipetting procedure...................................................................................157
3.8 Mass Spectrometry methods..................................................................................158
3.8.1 MALDI-TOF-MS...........................................................................................158
3.8.2 Fourier-Transform Ion-Cyclotron Resonance mass spectrometry.................159
3.8.3 ESI-Ion Trap mass spectrometry....................................................................161
3.8.3.1 Direct infusion ESI-Ion Trap mass spectrometer...........................................161
3.8.3.2 Liquid chromatographic/Ion trap mass spectrometric analysis......................165
3.8.4 Linear trap quadrupole Orbitrap XL mass spectrometry................................165
3.9 Computer Programme............................................................................................168
3.9.1 GPMAW 5.0...................................................................................................168
3.9.2 HyperChem 6.0...............................................................................................168
3.9.3 Origin 7.5........................................................................................................168
3.9.4 Yasara.............................................................................................................168
3.9.5 Search engines for identifying proteins..........................................................169
Table of contents 10
4 Summary................................................................................................................170
5 Zusammenfassung.................................................................................................174
6 References..............................................................................................................179
7 Appendix................................................................................................................199
7.1 Appendix 1.............................................................................................................199
7.2 Appendix 2.............................................................................................................204
7.3 Appendix 3.............................................................................................................206