Genome-wide proteomics and quantitative analyses on halophilic archaea [Elektronische Ressource] / Konstantinos Konstantinidis

Konstantinos Konstantinidis

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 18
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Genome-Wide Proteomics and
Quantitative Analyses on
Halophilic Archaea

Konstantinos Konstantinidis









Munich 2008


Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München




Genome-Wide Proteomics and
Quantitative Analyses on
Halophilic Archaea






Konstantinos Konstantinidis
aus
Drama



2008

Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 der Promotionsordnung vom
29. Januar 1998 von Herrn Prof. Dr. Dieter Oesterhelt betreut.



Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.

München, am 21.07.2008


...................................................
(Konstantinos Konstantinidis)





Dissertation eingereicht am 21.07.2008

1. Gutachter: Herr Prof. Dr. Dieter Oesterhelt
2. Gutachter: Herr Prof. Dr. Lutz Eichacker

Mündliche Prüfung am 17.12.2008








„Ich liebe Den, dessen Seele sich verschwendet, der nicht Dank haben will und nicht
zurückgiebt: denn er schenkt immer und will sich nicht bewahren.“

Friedrich Wilhelm Nietzsche (1844-1900)


”I love him whose soul is lavish, who wanteth no thanks and doth not give back: for he
always bestoweth, and desireth not to keep for himself.”

Translated by Thomas Common (1850-1919) V

The presented thesis was prepared in the time from February 2004 to July 2008 in the
laboratory of Professor Dr. Dieter Oesterhelt at the Max Planck Institute of Biochemistry
in Martinsried near Munich.










Parts of this PhD thesis have been published:


Konstantinidis, K.; Tebbe, A.; Klein, C.; Scheffer, B.; Aivaliotis, M.; Bisle, B.; Falb,
M.; Pfeiffer, F.; Siedler, F.; Oesterhelt, D. (2007) Genome-wide proteomics of
Natronomonas pharaonis. J. Proteome Res. 6 (1),185-193

Konstantinidis, K.; Schwarz, C.; Tebbe, A; Schmidt, A; Schlesner, M.; Klein, C.;
Aivaliotis, M.; Schwaiger, R.; Pfeiffer, F.; Siedler, F.; Lottspeich, F.; Oesterhelt, D.
Quantitative analyses of heat shock response in Halobacterium salinarum. In preparation VI
Publication list

Falb, M.; Aivaliotis, M.; Garcia-Rizo, C.; Bisle, B.; Tebbe, A.; Klein, C.;
Konstantinidis, K.; Siedler, F.; Pfeiffer, F.; Oesterhelt, D. (2006) Archaeal N-terminal
protein maturation commonly involves N-terminal acetylation: a large-scale proteomics
survey. J. Mol. Biol. 362 (5), 915-924

Konstantinidis, K.; Tebbe, A.; Klein, C.; Scheffer, B.; Aivaliotis, M.; Bisle, B.; Falb,
M.; Pfeiffer, F.; Siedler, F.; Oesterhelt, D. (2007) Genome-wide proteomics of
Natronomonas pharaonis. J. Proteome Res. 6 (1),185-193

Klein, C.; Aivaliotis, M.; van Olsen, J.; Falb, M.; Besir, H.; Scheffer, B.; Bisle, B.;
Tebbe, A.; Konstantinidis, K.; Siedler, F.; Pfeiffer, F.; Mann, M.; Oesterhelt, D. (2007)
Proteome analysis of low molecular weight proteins in Halobacterium salinarum. J.
Proteome Res. 6 (4), 1510-1518

Aivaliotis, M.; Gevaert, K.; Falb, M.; Tebbe, A.; Konstantinidis, K.; Bisle, B.; Klein, C.;
Martens, L.; Staes, A.; Timmerman, E.; Van Damme, J.; Siedler, F.; Pfeiffer, F.;
Vandekerckhove, J.; Oesterhelt, D. (2007) Large scale identification of N-terminal
peptides in the halophilic archaea Halobacterium salinarum and Natronomonas
pharaonis. J. Proteome Res. 6 (6), 2195-2204

Tebbe, A.; Schmidt, A.; Konstantinidis, K.; Falb, M.; Bisle, B.; Klein, C.; Aivaliotis,
M.; Kellermann, J.; Siedler, F.; Pfeiffer, F.; Lottspeich, F.; Oesterhelt, D. Life-Style
changes of a Halophilic Archaeon analyzed by Quantitative Proteomics. In preparation

Konstantinidis, K.; Schwarz, C.; Tebbe, A; Schmidt, A; Schlesner, M.; Klein, C.;
Aivaliotis, M.; Schwaiger, R.; Pfeiffer, F.; Siedler, F.; Lottspeich, F.; Oesterhelt, D.
Quantitative analyses of heat shock response in Halobacterium salinarum. In preparation VII
Poster Presentations and Conferences

11. Arbeitstagung – Mikromethoden in der Proteinchemie, Munich (Martinsried),
Germany, June 21-24, 2004

53rd ASMS Conference on Mass Spectrometry; San Antonio, Texas, USA, June 5-9, 2005

12. Arbeitstagung – Mikromethoden in der Proteinchemie, Munich (Martinsried),
Germany, July 18-20, 2005

stHUPO 4th Annual World Congress, Munich, Germany, August 28 – September 1 , 2005

13. Arbeitstagung – Mikromethoden in der Proteinchemie, Munich (Martinsried),
Germany, June 26-29, 2006

rd3 Joint BSPR/EBI Proteomics Meeting, Hinxton, UK, July 12-14, 2006

VII European Symposium of the Protein Society, Stockholm, Sweden, May 12-16, 2007

14. Arbeitstagung – Mikromethoden in der Proteinchemie, Munich (Martinsried),
Germany, June 25-28, 2007

15. Arbeitstagung – Mikromethoden in der Proteinchemie, Munich (Martinsried),
Germany, June 23-25, 2008 VIII
Table of contents
1 Introduction __________________________________________________________1
1.1 Microorganisms in hypersaline environments ________________________________ 1
1.1.1 Characteristics of the halophilic archaea Natronomonas pharaonis and Halobacterium
salinarum _______________________________________________________________________ 2
1.2 The transcription machinery of archaea _____________________________________ 6
1.3 Molecular chaperones ____________________________________________________ 8
1.4 Mass spectrometry ______________________________________________________ 10
1.4.1 Ion Sources_________________________________________________________________ 11
1.4.1.1 Electrospray ionization (ESI)_______________________________________________ 11
1.4.1.2 Matrix-assisted laser desorption-ionization (MALDI) ___________________________ 12
1.4.2 Mass analyzers ______________________________________________________________ 14
1.4.2.1 Quadrupole mass spectrometers_____________________________________________ 14
1.4.2.2 Time of flight ___________________________________________________________ 14
1.4.2.3 Tandem mass spectrometry (MS/MS) ________________________________________ 16
1.5 Proteins and microbial proteomics_________________________________________ 16
1.5.1 Proteomic techniques for the large scale analysis of microorganisms ___________________ 20
1.5.2 Staining and destaining of proteins ______________________________________________ 22
1.5.3 Reduction and alkylation of proteins _____________________________________________ 22
1.5.4 In-gel digestion of proteins ____________________________________________________ 23
1.5.5 Extraction, concentration and desalting of peptides from polyacrylamide gels ____________ 24
1.5.6 Identification of proteins by mass spectrometry ____________________________________ 25
1.5.7 Expressional proteomics ______________________________________________________ 27
1.6 Codon adaptation index (CAI) ____________________________________________ 29
1.7 Objective ______________________________________________________________ 30
2 Materials and Methods ________________________________________________32
2.1 Materials and Devices ___________________________________________________ 32
2.1.1 Materials/Chemicals__________________________________________________________ 32
2.1.2 Devices____________________________________________________________________ 35
2.1.3 Programs __________________________________________________________________ 37
2.1.4 Oligonucleotides ____________________________________________________________ 38
2.1.5 Culture media_______________________________________________________________ 39
2.1.6 Buffers, solutions gels and standards_____________________________________________ 41
2.1.6.1 2-DE, silver staining and MS ______________________________________________ 41
2.1.6.2 SDS-PAGE and CBB staining ______________________________________________ 42
2.1.6.3Agarose gelelectrophoresis and RTqPCR______________________________________ 43
2.1.6.4 Northern blot hybridization ________________________________________________ 44
2.1.6.5 Whole genome DNA-microarrays ___________________________________________ 46
2.2 Methods_______________________________________________________________ 47
2.2.1 Growth conditions ___________________________________________________________ 47
2.2.2 Protein inventory of Natronomonas pharaonis _____________________________________ 47
2.2.2.1 Sample preparation under low ionic strength conditions (water lysis) and protein
precipitation by acetone _________________________________________________________ 47
2.2.2.2 Sample preparation under high ionic strength conditions (native lysis) with subsequent
size-exclusion chromatography and protein precipitation by TCA ________________________ 48
2.2.2.3 SDS-PAGE and in-gel digestion ____________________________________________ 50
2.2.2.4 NanoLC-MS/MS ____________________________________