Analysis of sporamin forms expressed in different subcellular compartments of transgenic tobacco plants by IMAC and ESI-MS

Various forms of sporamin (spo, Δpro, ΔproHDEL and spoHDEL) corresponding to different targetings within the plant cell, respectively, to the vacuole, the extracellular compartment and the endoplasmic reticulum (ER) for the last two were used as model proteins to study further structural modifications. These proteins were expressed in tobacco plants (Nicotiana tabacum L. cv. PB D6), purified by immobilized metal ion affinity chromatography (IMAC) and then analyzed by electrospray ionization mass spectrometry (ESI-MS). Conformational and post-translational modifications were observed between these different forms of sporamin, extracted from leaves. By the combination of these two techniques, detection of discrete intermediate species was achieved. Three distinct entities were detected for the vacuolar form of sporamin (spo) indicating a two-step processing of the protein, while only one entity, shorter by two amino-acids at its N-terminus, was detected for the extracellular form of sporamin (Δpro). The analysis of sporamin forms with an HDEL amino-acid extension can not be deduced easily from MS data and may reflect post-translational modifications distinct from proteolytic processing. Thus post-translational modifications appear to be closely related to targeting within the plant cell.