The study of N-terminal protein variants, their stability and localization by means of ribosome profiling and positional proteomics

An increasing number of studies involve integrative analysis of gnee and protein expression data, taking advantage of new technologies such as next-generation transcriptome sequencing (RNA-Seq) and highly sensitive mass spectrometry (MS). Analyses of the sequence and nature of all protein amino-termini (N-termini) within proteomes (N-terminome) in eukaryotes reveal a highly understimated occurrence of database non-annotated alternative translates, thereby increasing the already high complexity of proteomes. N-terminomics provides quantitative information on the N-terminal modification states of identified proteins in addition to enabling large-scale proteome dynamics studies. Ribosome profiling, a recently developed genomics method, enables systematic monitoring of protein translation events by deep sequencing of ribosome-protected mRNA fragments and thereby enables the study of (alternative) translation (initiation)with single codon resolution. Since the global prevalence and the in vivo regulation governing the recognition of near-cognate translation intitiation codons remains poorly understood, in this project we aim at the combined usage of N-terminomics and ribosome profiling to further explore the regulatory mechanisms governing start codon selection. Moreover, proteome-wide protein turnover and subcellular localization analyses of N-terminal protein variants will be conducted, permitting the delineation of various dynamic protein parameters and protein functionality.