Protein chemical analysis (Peptide Mass Spectrometry and Edman sequencing)

Along with the common methods for protein and peptide purification, our unit has advanced protein-analytical equipment:

- Peptide mass spectrometry (Dr.Peter Franke/ Dr.Mathias Dreger): MALDI-MS (Bruker Reflex) and ESI-MS (Finnigan MAT triple stage quadrupole TSQ 700). Peptide sequencing is possible with both machines (CID und PSD).

- Chemical protein micro-sequencing according to Edman (Dr.Chris Weise): ABI 473A Protein sequencer

We use these techniques mainly for samples from our own scientific projects. Additionally we offer scientific co-operations with other research groups (see list below), as long as capacities are available. However, we are no service facility and do not offer commercial services.

Contact: Dr. Chris Weise, Tel. 030-838 56424


Some fields of application:

- A standard application is protein identification by mass-fingerprinting. Here the proteins to be identified first are separated by SDS gel electrophoresis and cleaved with trypsin in the gel ("in situ"). Then, using MALDI-MS, a mass spectrum of the peptide mixture is recorded and a data-base search using these data as input is performed ("in silico-digest"). This method is very robust and reliable and particularly suited when one is dealing with organisms for which many protein sequences are known or even the entire genome has been sequenced.
If needed, the identification can be confirmed by MS-sequencing of selected peptides. -  Mass fingerprints are the basis of proteomic research. Here the complete set of proteins present in a specific subcompartent is to be analysed (1).

In many cases, partial protein sequences are needed as the basis for cloning unknown proteins. Such sequences can be determined  by N-terminal sequencing from a PVDF-type blot membrane. Blot samples for sequencing can be stained using Coomassie Blue or Ponceau Red. - N-terminally blocked proteins can be identified byinternal sequences after in-gel digestion and HPLC separation of the resulting peptides (13). In such a situation a starting amount of about 200pmol is needed.

We frequently use a ccmbination of MS techniques and chemical sequencing to generate high quality sequence data. These are particularly useful for the elucidation of post-translational (2, 6,11) or chemical modifications (7,8) or for mapping functional domains of Proteinen such as specific binding sites (3,9,12).


Selected publications: