Post-translational modifications define protein functionality. The vast majority of post-translational modifications affect amino acid side-chains such as phosphorylation of serine, threonine, tyrosine residues or acetylation of lysine residues. However, post-translational modifications may also affect the N-termini of proteins. For example, in eukaryotes most proteins are co-translationally acetylated at their N-terminus following removal of the initiator methionine. New N-termini may be generated by limited proteolysis, a process that yields stable cleavage products. Limited proteolysis has important regulatory roles, e.g. in zymogen activation and proteolytic signaling.
Mass-spectrometry based proteomics has emerged as a powerful bioanalytical tool to identify and quantify post-translational modifications. For protein N-termini, so-called “N-terminomics” has been brought forward by various academic laboratories. “N-terminomics” enables mass-spectrometry based proteomics to focus on N-terminal peptides without overshadowing or interference by internal tryptic peptides. Numerous N-terminomic strategies are based on so-called “negative enrichment”, this being the enrichment of N-terminal peptides by depletion of internal (tryptic) peptides. The advantage of “negative enrichment” approaches is their ability to include natural N-terminal modifications such as acetylation. Although several robust and powerful N-terminomic workflows have been developed by different academic laboratories, their application has mostly remained the realm of specialized laboratories. In order to foster a more widespread adaption of N-terminomics, the present project has worked towards the following aims (1) integration of N-terminomics as a robust “one-pot-chemistry” workflow and (2) development of a standardized, web-based data analysis platform for N-terminomics. For the actual workflow, the originally foreseen strategy based on charge-reversal of internal peptides proved to be incompatible with a one-pot approach. A refined approach was thus implemented: amine groups of full-length proteins are chemically protected using N-Hydroxysuccinimide esters, followed by trypsination and tagging of internal peptides with compounds that introduce altered biophysical properties that enable straightforward chromatographic separation of N-terminal and internal peptides. Proof-of-concept experiments have illustrated the identification of 1000s of protein N-termini with this strategy. For the data analysis platform, we have found the Galaxy platform to be highly suitable. It enables reproducible data analysis with version control of individual tools, sharing of workflows, and their customization. Exemplary workflows have been developed and successfully tested in proof-of-concept experiments. The present project has demonstrated the feasibility of simplified N-terminomics with integrated ready-to-use data analysis. Based on these results a streamlined N-terminomic platform for broader dissemination is presently being prepared.
