Measurements with the Mass Spectrometer

Mass Spectrometric Analysis of the LBE Probe.

The probes were analyzed with the so-called nano-LC/MS/MS method, in which the protein bands to be analyzed are cut out of the SDS-PAGE gel (see picture 3: black striped boxes) and processed with an enzyme that cuts up proteins: the protease Trypsine. This produces short-sequenced peptides out of the long-sequenced protein. The short-sequenced peptides are later chromatographically separated (nano-LC) and their masses are determined in a first mass spectrometric step. Afterwards, there is a step in which the single peptides are further broken down into smaller fragments. The masses of these smaller fragments are then determined by a second mass spectrometric step (/MS). The amino aid sequences of the peptide can then be determined with the help of the mass measurements of the peptide fragments. With that information the sequence of the resulting protein can be assembled. In the

analysis of the insertion on artificial amino acids. that the mass of the individual peptides is changed. This is because the artificial amino acids have different masses than the natural amino acids, for example

Methionine : 149.21 Da and Ethionine: 164.24 Da.

Picture 4 shows the summary of the mass spectrometric analysis of the catalase in the LBE culture. The red stars show the positions in the protein, at which the ethionine insertion could be indentified. Admittedly, the amount of ethionine is, in comparison to the amount of methionine, still quite low (see attached analysis details). The insertion rate will hopefully be improved in the continuation of the project at the Max-Planck-Institute for Biochemistry. The first step will be for the catalase to be carried over into a plasmid-based expression system, which has not been used for research in connection with Bacillus subtilis and the use of