Identification of N-terminal and C-terminal peptides in proteomics

Abstract

Background: Identifying modifications made to terminal parts of proteins are very useful in understanding diseases and other out of the ordinary biological states. This thesis has focused on developing methods for enriching N-terminal and C-terminal peptides from a complex protein mixture, so that analysis of these samples can give better, more comprehensive and more reproducible results.

Materials and methods: This thesis applies a bottom-up proteomics work-flow approach to develop and compare methods for enrichment of N-terminal, using two digestion enzymes, three sample clean-up methods, and two enrichment agents in different combinations. One method for C-terminal enrichment was developed, with basis in the method that gave the best results from N-terminal enrichment.

Results and discussion: None of the N-terminal enrichment methods improved the number of terminal peptides compared to the control samples. However, the results suggest that trypsin should be the enzyme of choice when enriching for N-terminal. The method for enrichment of C-terminal peptides was developed with basis in the method that gave the best results for the N-terminal enrichment. This method yielded only one terminal peptide, which is far lower than expected based on existing literature.

Conclusion: While none of the methods developed in this thesis improves the number of terminal peptides, it would seem that trypsin should be chosen over chymotrypsin when enriching N-terminal peptides. This result, however, has not been validated and should be investigated further.