Core Facility Mass Spectrometry –Proteomics 

The ability to analyse the cellular proteome provides key insights into how cells function at the molecular level. The technical challenges of proteomics are protein identification and quantification in complex biological samples, identification of post translational modifications, analysis of non covalent complexes and de novo sequencing.

 

Bottom-up proteomics with high resolution (peptide mapping and sequencing of protein digests) as well as top-down protein analysis (fragmentation of whole proteins) is established on our Thermo LTQ-FT coupled to an Agilent 1200 nano-HLPC as well as on our Bruker ultrafleXtreme MALDI-TOF-TOF.

 

Consisting of a linear ion trap and a FT–ICR–MS, the LTQ-FT mass spectrometer delivers MSn spectra with mass accuracies of less than 1ppm. For fragmentation CID, IRMPD and ECD are used depending on the application.

 

The 1 kHz smartbeam II laser of the ultrafleXtreme MALDI-TOF-TOF mass spectrometer enables ultra-high data acquisition speed in both MS and MS/MS at full system performance and provides analytical and matrix flexibility while fully enabling 1-1000 Hz repetition rates.

 

LC-MALDI applications are realized by spotting (SunCollect, SunChrom) of nano-HPLC (Bruker Easy-nLC) separations on MALDI-targets.

 

Protein separation by liquid chromatographic (FPLC, HPLC) and electrophoretic methods (one and two-dimensional gelelectrophoresis) as well as protein digestion (by trypsin) and desalting are available. Differential two-dimensional gel electrophoresis (DIGE) is established. A laserscanner for highly sensitive detection of fluorescent protein stains e.g. Sypro Ruby or Krypton, as well as cyanine fluorophores (Cy2,3,5) and a spotcutter allow gel–based proteomic workflows.  Gel-free proteomic workflows utilizing multidimensional LC are currently set up.

Special emphasis is given on the development of functional proteomics technologies.
 

nano–HPLC coupled to LTQ–FT (LC–MS⁄MS)