N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana.

Proteogenomics is an emerging research field yet lacking a standard method of analysis. In this article, we demonstrate the strength of proteogenomic analysis specific for N-terminal data that aims at the discovery of novel translational start sites. In summary, unidentified spectra were matched to a specific N-terminal peptide library encompassing all theoretical protein N-termini encoded in the genome. Gene prediction suggested 81 protein-coding models, of which several alternative proteoforms with unannotated protein starts. Next to the proteomic data, complementary ribosome footprinting data was generated from Arabidopsis thaliana cell cultures. Translation initiation site mapping by the ribosome footprinting data provided orthogonal evidence for 14 novel peptides identified by our proteogenomics pipeline. Overall design: Ribosome profiling sequencing of Arabidopsis thaliana wild-type ecotype Landsberg erecta cells treated with lactimidomycin or cycloheximide.