Proteomics Core Unit

 

Prof. Dr. Dr. F. Grus, MD, PhD

Dr. N. Perumal, PhD

 

Mass spectrometry based proteomics

The up- and down regulation of proteins can provide important information on disease processes and can lead to a better understanding of the pathogenesis. Mass spectrometry based profiling approaches lead to the identification of important biomarkers for diseases and thus support diagnostics and predictive medical approaches.Besides conventional two-dimensional electrophoresis, our group uses state of the art technologies such as the SELDI-TOF MS (Surface Enhanced Laser Desorption/Ionization in Time-of-Flight Mass Spectrometry) ProteinChip technology or a superparamagnetic nanoparticel bead approach in a fully automated robot station. For MS analyses we use a MALDI-TOFTOF (Matrix Assisted Laser Desorption/Ionisation in Time-of-Flight; Bruker Ultraflex) mass spectrometer. Additional sample preparation, which might be necessary for biomarker identification, is performed using HPLC and nano-HPLC systems coupled with automated MALDI target spotting. Subsequently, protein microarrays are used for the validation of potential biomarkers.MS data are analysed with a specific software (protein software project, PSP), which was developed in our group. PSP searches for possible biomarkers using a combination of different algorithms such as artificial neural networks, multivariate statistics and tree algorithms.

Protein profiling by the use of magnetic beads

In a high throughput compatible approach we use superparamagnetic nanoparticels (Dynal, Bruker Daltonics) covered with different specialized chromatographic coatings for the prefractionation of the proteome of complex body fluids such as sera or tears. Sample fluids are incubated with the magnetic nanoparticels (beads) in order to capture a specific fraction of proteins, which will be eluted and spotted onto a MALDI-target. This step is necessary to allow an ultra high resolution MALDI-TOFTOF analysis, which is otherwise not feasible due to the high complexity of the samples. The processing provides much deeper insights into the proteome than common methods like 1D or 2D gelelectrophoresis. As a further benefit we decrease the day to day variability and thus increase the reproducibility of the MS spectra by use of liquid handling robots (Tecan and Beckmann robots), both for magnetic bead handling and MALDI Spotting.The wide scope on available bead coatings such as WCX, SAX, C18, C8 etc. allows a step by step proteome profiling resulting in a high increase of information. Recently beads for phospho- and glycoprotein isolation have been developed. This seems to be a very promising approach specifically for the analysis of disease correlated posttranslational protein modifications.