CORESTA Meeting, Smoke Science/Product Technology, 2015, Jeju, ST 36

Comprehensive analysis of proteins in tobacco leaf

MASUGI E.(1); ORTIZ A.(2); YOSHIMURA Y.(1); MITSUI K.(1); DAVID F.(2); SANDRA P.(2); SANDRA K.(2)
(1) Japan Tobacco Inc., Tobacco Science Research Center, Yokohama, Kanagawa, Japan; (2) Research Institute for Chromatography, Kortrijk, Belgium

In the last two decades, proteomics has been a vastly expanding field of research. Proteomics aims at the comprehensive and comparative measurement of the proteins present in a sample, e.g. plants. Major developments in separation and analytical sciences have substantially extended protein identification rates and protein quantification accuracies. Proteins, which constitute about 10% of the dry weight of tobacco leaf, are known to affect the flavour of cigarettes. Understanding tobacco protein profiles and the changes in the functions of curing, storage and aging might broaden our understanding in the tobacco flavour.

The purpose of this study was to develop a comprehensive analytical method for proteins in tobacco leaf.

Following protein extraction, solubilisation and in-solution proteolysis, the resulting peptides mixtures were analysed by two-dimensional reversed-phase liquid chromatography coupled to high resolution quadrupole time-of-flight mass spectrometry (Q-TOF). Fifteen peptide fractions, sampled in an off-line manner from a reversed-phase column operated at high pH, were subsequently analysed by nano-LC Q-TOF MS/MS under acidic conditions. Proteins were identified by the Spectrum Mill platform.

About three hundred different tobacco specific proteins were identified representing a four-fold increase over the one dimensional approach.

Differentiating protein profiles were observed depending on tobacco curing type. As an example, proteins related to photosynthesis were particularly abundant in Oriental leaf types, a direct result of sun exposure.