Mass spectrometry of co-immunoprecipitation using tobacco Maturase K as bait

Chloroplast group IIA introns derive from bacterial ribozymes. Their splicing likely requires Maturase K (MatK), which has been largely inaccessible to functional analyses being itself a chloroplast intron-encoded protein. Here we used co-immunoprecipitation coupled with proteomics to identify proteins bound to MatK in tobacco (Nicotiana tabacum) seedlings. We used homoplastomic tobacco lines modified to express NtMatK-HA (line NtMatK C+) or control lines carrying only the selection marker (line NtMatK C-). Proteins co-precipitating with NtMatK-HA were purified via anti-HA beads and identified via shotgun proteomics (n=3 seedling pools). The mass spectrometry data were further processed using Philosopher followed by label-free quantification based on precursor intensities using IonQuant and statistical analysis using Amica.