3 AIM OF THE THESIS
4.5 Dimethyl Sulfoxide related Effects in Protein Characterization (paper V) 33
The mechanism behind the SO3H formation was proposed as follows: The pyridazine analogues, having conjugated quinoid or semiquinoid systems, stabilize an intermediate radical via resonance delocalisation. When adding such compounds to PTP1B in the presence of reducing agent, the compounds caused catalytic oxidation of the reducing agent generating hydrogen peroxide (H2O2) that in turn efficiently oxidized the active site cysteine on PTP1B (a protein known to be sensitive to oxidation) resulting in the formation of the sulfonic derivative.
4.5 DIMETHYL SULFOXIDE RELATED EFFECTS IN PROTEIN
to a protein in the same folding state as at 0.5% DMSO concentration. The ions labelled 13+ to 11+ correspond to partly unfolded protein, whilst the cluster with ions 25+ to 21+ corresponds to fully denatured protein. Thus, at a concentration of 3%
DMSO the amount of active, native enzyme was decreased significantly.
A
B
C
1500 2000 2500 3000 3500 4000 m/z
0 100
% 0 100
% 0 100
%
2518
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1979 16351847
2518 3078
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12+
11+
10+
11+
10+
9+
8+
Denatured protein (25+ to 21+)
14+
13+
12+11+
10+
9+
8+
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0.5% DMSO
3% DMSO
1500 2000 2500 3000 3500 4000 m/z
0 100
% 0 100
% 0 100
%
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2518 3078
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0 100
% 0 100
% 0 100
%
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2518 3078
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9+
8+
Denatured protein (25+ to 21+)
14+
13+
12+11+
10+
9+
8+
No DMSO
0.5% DMSO
3% DMSO
Figure 14. DMSO effects on charge state distribution. ESI-MS spectra of the enzyme (0.7 µM) in 20 mM NH4Ac, pH 7, with 0% (A), 0.5% (B), and 3% (C) DMSO. The labels on the peaks n+, indicate the number of protons, n, attached to the protein. The pH was unchanged in the solution after the addition of DMSO.
The observed DMSO effects on proteins show the importance of thorough protein characterization. Unforeseen future assay problems may be avoided if knowledge on how proteins behave in DMSO containing solvents is established.
5 CONCLUSIONS
Pharmacological therapeutics represents the single most important commercial product of biomedical research, with a large track record of improving human health.
Nevertheless, drug discovery and development is a time-consuming, expensive, and high-risk activity.
In this thesis it is demonstrated how MS techniques can be employed in the different steps of the early drug discovery process. Ultimately these improvements together with advances in other parts of the drug discovery process could help to increase the success rate of drug discovery.
It is likely that MS will remain a primary tool for protein identification and for the analysis of post-translational modifications also in the future115, 116. New types of mass spectrometers that provide enhanced capability to discover protein identities have appeared in the field of proteomics58, 59. Top-down proteomics, i.e. deriving sequence information from intact proteins using FTICR-MS instruments with very high resolution, together with ECD fragmentation, is such an approach.
Another powerful technique, used for the identification of phenotypic markers, is the direct molecular profiling of biological samples. This technique utilizes MALDI-TOF MS to determine the spatial distribution of peptides and proteins in biological
samples in situ117,118.
In structural biology, MS plays an increasingly important role in structure based drug design due to the use of nondenaturing ESI-MS for biophysical protein characterization, and to the use of MS based methods to probe the structures of proteins (limited proteolysis and H/D exchange). Furthermore, recent papers have shown that membrane protein complexes can be studied by ESI-MS119, 120. Membrane proteins are challenging in Structural Biology. One-third of the total proteome is membrane proteins, but only ~1% of the protein structures solved so far are membrane proteins.
In recent years MS has also emerged in the lead generation phase of the drug discovery process. MS is an excellent tool in the screen to hit process for investigating the protein upon addition of ligands, e.g. to establish:
- if inhibition of the protein is a result of direct binding of the ligand.
- if the binding is reversible or covalent.
- the stoichiometry of protein-ligand complexes.
- if the stability of the protein is affected upon ligand addition.
The use of ESI-MS in a direct binding assay (paper III) has many advantages and a great potential to become a frequently used method for the direct study of binding affinities of protein-ligand interactions. Besides our own screening method, many biophysical screening techniques based on MS have emerged lately121,122, 123. Recent improvements of surface-based laser desorption/ionisation on silicon (DIOS-MS), have enabled the promising development of a rapid assay to monitor enzyme activity and enzyme inhibition124,125.
6 ACKNOWLEDGEMENTS
I started my PhD studies after 15 years of employment in the industry. During these years many people convinced me, in different ways, to move on for a PhD degree. I would like to thank everyone who has made it possible for me to bring this work to a conclusion. I am deeply grateful.
First of all I would like to thank my three supervisors:
Bill Griffiths, my main supervisor, for your great enthusiasm in my work and for sharing your huge experience and expert knowledge in mass spectrometry with me.
Janne Johansson, for all your encouragement and constant support.
Dan Hallén; without your support this thesis would not have been finished.
Hans Jörnvall, for letting me be an external student at MBB.
My co-authors: Bassam Ali, Brian Chait, Mark Field, Waltteri Hosia, Niklas Bark, Edvards Liepinsh, Bengt Persson, Dan Hallén, Johan Thyberg , Janne Johansson, Lars Tjernberg, Sofi Carnö, Frida Oliv, Kurt Benkestock, PeO Edlund, Bill Griffiths, Johan Schultz, Stephen James, Styrbjörn Byström, Johan Weigelt, and Natalia Markova.
Jan Sjövall, for taking a genuine interest in my work and for nice discussions.
Åsa Brunnström, for your never-ending support, for all good times together on trips and conferences, and most of all for your friendship.
My other (past and present) mass spec friends at KI: Johan Lengquist, Waltteri Hosia, Gunvor Alvélius, Suya Liu, Dilip Ray, Daniel Hirschberg, Yang Yang, Magnus Gustafsson, Andreas Jonsson and all the rest of you that I unfortunately never got to know properly.
The Waters company is acknowledged for their support during the years, and for helping me with my specific demands on the Q-TOF Ultima. Special thanks to Jonathan Coffey, David Varley, Cristopher Sykes, Mats Johansson, Jon Watkins, Ulf Viklund, Kennet Joelsson, Jonas Kärvin, and Thorleif Lavold.
Brian Chait, for giving me the opportunity to be a guest researcher in your lab at Rockefeller. For me this was the most fantastic year and you really made me believe that I was capable of writing a thesis.
The Chait lab members during my year at RU: Julio Cesar Padovan, Michelle Trester-Zedlitz, Andrew Krutchinsky, Jan Eriksson, Rong Wang, Wenzhu Zhang, Martine Cadene, David F, Yoshi O, Sacha U, Herbert C, and Gladys McMilleon. All of you guys made my stay in New York unforgettable.
I would like to address a special Thank You to my company Biovitrum, for giving me the opportunity to perform my PhD studies in parallel with my daily work for 3,5 years.
I also would like to render particular thanks to some people at Biovitrum who have helped me with one thing or another during my years in the industry.
Ulf Stenberg, for giving me my first job position, in the Bioanalysis group.
Bengt Norén, for saving me from the every day commuting between Uppsala and Stockholm and letting me join the mass spec. group in the BioScience Center, Stockholm.
Bo Karlén, Björn Nilsson, Johan Kördel, Martin Norin and Jan Hörling, the heads of my departments during the years. A special thanks to Johan Kördel, for your support before and during my stay in NY. Another special thanks to Martin Norin, for approving my PhD studies.
Mats Wikström, Johan Weigelt, and Joanna Chmielewska, my three section heads during my “PhD student time”, who all encouraged and supported me to go on with my thesis.
PeO Edlund for sharing your extraordinary skills and knowledge in mass spectrometry. Thank you for all your help.
Signhild Strömberg, who once taught me some important rules of survival in the research world, which I try to follow.
My friends at the 5th and 6th floor at P19, Nordenflychtsvägen 62:
Carina Norström, Kristina Zachrisson, Micael Jacobsson, Åsa Enhörning, Natalia Markova, Stefan Svensson, Dan Hallén, Jonas Uppenberg, Johan Schultz, Björn Elleby, and Catharina Sterky, my wonderful co-workers.
Anna-Lena Gustavsson and Maria Wirstam for your great support and for sharing good times with me.
Per Persson, for always being there with help and advice.
Jerk Vallgårda, Torsten Seilitz, Thomas Lundbäck, Erik Nordling, Evert Homan, Jonas Nilsson, Carmen Medina, Peter Brandt, Mats Kilén, Elisabeth Ohlsson, Thomas Wehler, Joakim Staffas, Toshi Nishida and Mats Dahlberg for sharing your specific expert knowledges and for your friendship.
Ulrica Markström for all your help whenever I need something.
My friends in the TEP group: First, Kristina Köhler Van Alstine, for your great support, Robert Svensson, Erik Holmgren, Maria Jaki-Borg, Magnus Doverskog, Lotta Berghard, Jenny Rönnmark, Karin Stefansson, Johanna Edenius, Joakim Nilsson, Susanne Gräslund, Margrit Roobol-Bóza, Martin Selander, and Annette Elmblad.
My previous P19 friends: Sofi Carnö, Kurt Benkestock, Derek Ogg, Tomas Åkerud, Frederick de Maré, Fredrik Lindqvist, Sarah Hunter, Ingela Andersson, Stina Thorell, Per Kraulis, Maria van Dongen, Yoko Rönquist-Nii, Michael Sundström, Thomas Lundquist, Maria Flocco, and Hanno Ehring.
My other colleagues in the departments of Lead Discovery, Chemistry, Pharmacology and preclinical R&D at Biovitrum.
To all my out-of-office friends: Thank you for being my friends.
To my family:
To my brother Torsten, my greatest support, his wife Åsa, and their wonderful children Peder, Ebba and Harald. Thank you for everything.
To my brave, dear mother Karin. Alzheimers; the prison of the mind.
To my late father Sven. I miss you every single day.
To my parents-in-law, Greta and Harry Tjernberg, for all your help and support during the years.
The ones who matters most:
To my children Tessie and Erik, for putting up with a hardworking mother, and for reminding me of what is valuable in life. Thank you for being my kids.
To my husband Lars, for all your love and support. Thank you for being who you are!
Last, thanks to all the people I unintentionally forgot to mention in this text.
“In vino veritas”
Plato
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