FU Berlin Digitale Dissertation
Markus Kalkum : Mass Spectrometric Methods for Biochemical Proteome Research: Identity, Primary Structure and Processing of Proteins Functionally Involved in Bacterial Conjugation. Massenspektrometrische Methoden für die biochemische Proteomforschung: Identität, Primärstruktur und Prozessierung funktioneller Proteine der bakteriellen Konjugation.
|Abstract| |Table of Contents| |More Information|

Abstract For the first time, this work presents systematic investigations of micro-arrayed sample preparation on MALDI-MS targets using a robotic multi micro-dispenser system. The application of such low-volume handling techniques allow protein identification on the basis of only a few femtomoles of proteolytic peptides that are transferred into the mass spectrometer. This is ~ 1 % of the amount previously needed. In combination with the patented electro-acoustic method (HARP) described here, high density MALDI-MS sample plates can be prepared and analyzed automatically. Algorithms for the automatic interpretation of MALDI-MS spectra were invented and successfully applied. The identity of functional proteins, involved in the process of bacterial conjugation, separated by one and two-dimensional gel electrophoresis and membrane blots was determined through mass spectrometric analysis of peptides derived from enzymatic digests. Analysis of primary structure and complex chemical behavior revealed the specific complexation of iron by TraH which is part of the relaxosome. Furthermore, the existence of an N-terminal lipid modification in the entry exclusion mediating TrbK was discovered. Additionally, cleavage of the predicted signal peptide of TrbM was confirmed. In the non-induced state, Lac-I, TraL, TrbG, TrbJ and TrbM were found to be the most prominent plasmid encoded proteins. MALDI-MS analysis of samples prepared with whole cells were used to determine the mayor component of extra-cellular pilus subunits. The matrix trans-3-indolyl acrylic acid, commonly used for the analysis of synthetic polymers, enabled the sensitive detection of the trbC gene product which was found to be mayor protein component of bacterial pili. In E. coli the RP4 encoded, ribosomally synthesized and multiply processed TrbC is converted into a cyclic protein containing 78 amino acids. The head-to-tail connection of the former N- and C-terminus is realized in form of a peptide bond. At present, the resulting circular protein is the largest cyclo-peptide reported world wide. The gene products, trbC of the analogous plasmid R751 and virB2 of the Ti plasmid from Agrobacterium tumefaciens, consist of a similar circular structure. A combination of mutagenesis experiments and MALDI-MS analyses peek into the cyclization-reaction and fundamentally yield a theoretical model of it?s chemical mechanism: In the periplasmatic space the serine-protease TraF cleaves a four amino acid residue peptide from the C-terminus of TrbC. It is proposed, that the resulting acyl-enzyme reacts via aminolysis involving the a -amine group of the TrbC N-terminus, generating cyclic TrbC* and the restored TraF. >> see also: extended abstract in PDF format

Table of Contents Download the whole PhDthesis as a zip-tar file or as zip-File For download in PDF format click the chapter title Titel und detailliertes Inhaltsverzeichnis Seite 1. EINLEITUNG 1 1.1. DIE MASSENSPEKTROMETRIE IN DER PROTEOMFORSCHUNG 1 1.2. HORIZONTALER GENTRANSFER DURCH BAKTERIELLE KONJUGATION 7 2. MATERIALIEN UND METHODEN 13 2.1. VERWENDETE MATERIALIEN UND DEREN BEZUGSQUELLEN 13 2.2. MIKRODISPENSIERTECHNIK ZUR AUTOMATISCHEN MALDI-MS PROBENPRÄPARATION 15 2.3. ELEKTROAKUSTISCHES VERFAHREN ZUR EXAKTEN POSITIONIERUNG VON MEHRFACH- MIKRODISPENSEREINHEITEN (HARP) 18 2.4. BIOANALYTISCHE METHODEN 25 2.5. MASSENSPEKTROMETRIE 28 2.6. PROGRAMMIERUNG UND SOFTWARE 31 3. ERGEBNISSE 32 3.1. MINIATURISIERUNG UND AUTOMATISIERUNG DER PROBENPRÄPARATION FÜR DIE MATRIX- ASSISTIERTE LASER DESORPTIONS / IONISATIONS MASSENSPEKTROMETRIE (MALDI-MS) 32 3.2. IDENTIFIZIERUNG RP4-KODIERTER PROTEINE AUS VERGLEICHENDER ZWEIDIMENSIONALER GELELEKTROPHORETISCHER TRENNUNG 43 3.3. DER WEG ZUR AUTOMATISCHEN MALDI-MS AUSWERTUNG 52 3.4. DIE POSTTRANSLATIONALEN MODIFIZIEUNGEN DER GENPRODUKTE VON TRAH UND TRBK 56 3.5. MALDI-MS VOLLSTÄNDIGER ZELLEN UND ZELLÜBERSTÄNDE ZUR ANALYSE DER HAUPTPROTEINKOMPONENTEN KONJUGATIVER PILI. 62 4. DISKUSSION 82 4.1. AUTOMATISIERUNG PIEZOELEKTRISCHER MIKRODISPENSIERTECHNIKEN 82 4.2. NICHTINDUZIERTE EXPRESSION VON TRA2 UND TRA1 PROTEINEN 84 4.3. DIE BESTIMMUNG DER PROTEINHAUPTKOMPONENTEN KONJUGATIVER PILI VON RP4, R751 UND TI 89 5. ZUSAMMENFASSUNG 97 6. SUMMARY 98 7. BIBLIOGRAPHISCHER NACHWEIS 99 CURRICULUM VITAE 108

More Information:
Online available:	http://www.diss.fu-berlin.de/1999/44/indexe.html
Language of PhDThesis:	german
Keywords:	mass spectrometry, MALDI-MS, automation, cyclic pilin, bacterial conjugation, proteome research
DNB-Sachgruppe:	30 Chemie
Date of disputation:	01-Jun-1999
PhDThesis from:	Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee:	Prof. Dr. Hans Lehrach
Second Referee:	Prof. Dr. Walter Messer
Contact (Author):	kalkum@mpimg-berlin-dahlem.mpg.de
Contact (Advisor):	lehrach@mpimg-berlin-dahlem.mpg.de
Date created:	19-Jul-1999
Date available:	24-Aug-2000

 

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