Parallel reaction monitoring reveals N-terminal acetylation of plastid precursor proteins

The N-terminus of a protein has an important regulatory impact on its in vivo stability and half-life. Proteins destined to chloroplasts and mitochondria are synthesized as precursor proteins in the cytosol with an N-terminal peptide sequence that ensures their correct targeting. During their cytosolic passage, precursor proteins are exposed to the cytosolic protein degradation machinery, hence, their N-termini must comply with regulatory processes for proteolytic degradation in the cytosol. We present here a method to determine the identity and modification state of plastid precursor protein N-termini in the cytosol by combining protoplast protein import assays with targeted mass spectrometry by means of parallel reaction monitoring (PRM). This method requires a hypothesis on potential modifications at the protein N-terminus such as methionine removal or N-terminal acetylation, that is decoded into an inclusion mass list to guide mass spectrometric data acquisition to specific peptides. This type of approach largely eliminates the stochastic nature of MS acquisition allowing different modifications to be tested as alternative hypotheses. Using Skyline, a quantitative assessment of different N-terminal modifications can be performed. We have used this method to determine the modification state of a model precursor protein RNP29 in two different genotypic backgrounds, but our workflow is easily expandable to different precursors.