17th International Mass Spectrometry Conference :: Prague, 2006
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|Presentation date:||Thu, Aug 31, 2006|
|Presentation time:||14:30 – 16:00|
Andreas Schmidt1,3,4, Goran Mitulovic1, Peter Hasselblatt1, Christoph Stingl2, Gustav Ammerer3,4, Erwin F. Wagner1, Karl Mechtler11 Institute for Molecular Pathology I.M.P., Doktor-Bohr-Gasse 7, 1030, Vienna, Austria
Correspondence address: Andreas Schmidt, Christian-Doppler-Lab. /University Vienna, IMP Vienna, Dr.-Bohr-Gasse, 9, Vienna, A-1030 Austria.
Keywords: Chromatography, Liquid (LC); Isotope, Stable; MS/MS, Liquid Chromatography; Quantitative Analysis.
Novel aspect: Quantification of proteins in complex protein mixtures using multidimensional chromatography and MALDI-TOF/TOF mass spectrometry for the identification of biological pathways.
The widely expressed transcription factor c-Jun is of great importance in proliferation and survival of cells.1 However, the precise function of c-Jun seems to depend on the stage of cell differentiation. c-Jun is also a well known proto-oncogene that is up-regulated in many carcinomas. In the liver c-Jun seems to play a key role in the promotion of hepatocarcinogenesis caused by chronic hepatitis. For studies on the c-Jun mediated pathways in T-cell mediated hepatitis mice were created which were lacking c-Jun specifically in hepatocytes. This deletion caused increase in disease severity and cell death which assumes hepatoprotective functions like apoptosis initiation.
To gain more insight into c-Jun function, we applied two proteomic approaches that allowed us to identify and quantify proteins with different expression levels in the livers of genetically modified c-Jun minus mice versus unmodified mice. In the first approach liver lysates were separated by 1D-SDS gel electrophoresis. After gel staining, protein bands showing different levels in the c-Jun lacking samples were analysed by mass spectrometry. This approach allowed us to identify proteins which are associated with oxidative stress response as putative regulatory targets of c-Jun.
To avoid the low protein separation efficiency and suppression of low abundant proteins of 1D-SDS gel electrophoresis, we used multidimensional liquid chromatography in the second approach. The different liver extracts were separated on the protein level using reversed phase chromatography on monolithic column in the first dimension. After proteolytic digestion of the individual fractions, an isotopic label was introduced to allow relative quantification of proteins by mass spectrometry. For this step we used the iTRAQ (isobaric Tag for Relative and Absolute Quantification) method in which the peptides N-termini are chemically tagged with a label suitable for quantification by MS/MS.
The samples deriving from c-Jun lacking mice were labelled with the isotopes 116 and 117 while the control samples were modified with the 114 and 115 isotopes. Peptide mixtures from each sample were mixed after this step and a strong cation exchange (SCX) chromatography was applied to reduce sample complexity. Prior to mass spectrometric analysis SCX-fractions were separated by RP-HPLC using a 75 Ám ID C18 column. RP-HPLC-fractions were mixed with the MALDI matrix cyano-4-hydroxycinnamic acid (CHCA) and directly spotted onto the target. Mass spectrometry was carried out on an MALDI-TOF/TOF mass spectrometer. The Bioworks 3.2, Protein Pilot and Mascot Software were used to analyse the data from mass spectrometry. The results of this study will be discussed.
1. Eferl et al., Cell 112, 181 (2003).