TY  - THES
T1  - Development and application of high-performance liquid chromatography- and mass spectrometry-based two-dimensional methods for proteome analysis
A1  - Melchior,Katja
Y1  - 2008/12/03
N2  - The availability of robust, sensitive and reliable analytic methods is a highly relevant requirement for proteome analysis. In this work two gel-free two-dimensional high-performance liquid chromatography-matrix assisted laser desorption/ionization mass spectrometry (2D HPLC-MALDI-MS) platforms suitable for proteome analysis were developed and compared. 
To provide an alternative approach to the classical bottom-up method combining strong cation-exchange- and ion-pair reversed phase HPLC (SCX x IP-RP-HPLC), in which peptides are separated in both dimensions, a semi top-down method was elaborated. This new approach employed separation of proteins in the first and of peptides in the second dimension, respectively. Both separation dimensions of the new method were operated in the ion-pair reversed phase (IP-RP) chromatographic mode using poly-(styrene-divinylbenzene) (PS-DVB)-based monolithic columns and hydro-organic eluents at a pH value of 2.1. Both methods were validated using the same 10-protein mixture.
The elaborated semi top-down- and the classical bottom-up approach were applied to the proteome analysis of a Glioblastoma multiforme tissue protein extract including the comparison of the two approaches. Fewer proteins were identified with the semi top-down approach. However, the sequence coverage of the proteins identified was slightly higher. The major advantage of this new method is the elution of proteins in less fractions of the first separation dimension than for the bottom-up approach. Thus, only the fractions containing the target proteins have to be digested in further experiments. Five of 13 potential biomarkers previously identified applying the SEREX (serological identification of antigens by recombinant expression screening) approach, could be confirmed on molecular level by combining the results of both chromatographic approaches. 

KW  - Proteine
KW  - Peptide
KW  - Chromatographie
KW  - MALDI-MS
KW  - Proteomanalyse
KW  - Biomarker
CY  - Saarbrücken
PB  - Universitäts- und Landesbibliothek
AD  - Postfach 151141, 66041 Saarbrücken
UR  - http://scidok.sulb.uni-saarland.de/volltexte/2008/1888
ER  -