The exact mechanism of the proteasomal degradation of proteins is unknown. The knowledge of all cleavage products arising from a given protein would be favourable because of the fact that a number of these fragments are further loaded onto the MHC class I molecule, and become part of the antigen presentation. The successful prediction of MHC-binding peptides starting from the sequence of a virus would therefore benefit from an analysis and prediction of the cleavage patterns.
Starting from series of mass spectrometric files that contain the results of time dependent experiments of proteasomal degradations of a peptide done by different types of the proteasome, the resulting cleavage maps should be determined.
Problems arise because of
- deviants in the m/z-value between estimated and measured masses (Fig. 1)
- same or similar m/z-values of distinct peptides
- deviants in the retention time within different data files
- no clear-cut between (significant) peaks and noise (Fig. 2)
- unknown modifications of the peptides leading to mistakes between normal and modified peptides
- different quality of MS/MS data
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| Fig. 1 Typical original data near mass peaks: intensity from 0 to 12,000 (in units of 1000); mass between 1468 and 1471; retention time between 27 and 31. | Fig. 2 Definition of significance (first row: only one significant peak; second row: one significant peak and some insignificant peaks; third row: noise). |
With the developed user-friendly application it is possible to simultaneously
analyse series of MS data files (Fig. 3), including MS/MS data (Fig. 4).
The analysis results in a map of the cleavage products (Fig. 5).
Fig. 3 Series of significant peaks for different files and different z-values, most of them for z-value 2.
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| Fig. 4 Analysis of MS/MS data. | Fig. 5 Creation of degradation map - time dependent behaviour of a cleavage product. |
In a further step, all developed maps shall be analysed to extract the rules of cleavage, and to predict the cleavage map of a peptide without making experiments.