The effect of enzymatic removal of proteins from plant leaf material as studied by pyrolysis-mass spectrometry: detection of additional protein marker fragment ions

This analytical pyrolysis study investigates the effects of ethanol extraction and of enzymatic protein removal from leaf material of two grass species, Poa annua and Pou pratensis. After pyrolysis the leaf fragments were analysed by low-voltage electron impact (El) and by ammonia chemical ionization (NH₃-Cl). Pronase-treated and untreated material were compared using multivariate analysis of the PyMS data. The resulting discriminant function spectra among others show a kind of 'negative image' of the PyMS spectrum of the plant proteins originally present in Poa leaves. In addition to known protein marker fragment ions several hithertoo unrecognised ones were apparent as well. The nitrogen concentration was determined for several plant fractions. Quantitative comparison of relative intensities of masses found by discriminant analysis led to selection of an additional group of protein marker fragments (m/z 54, 70, 84, 107, 130, 209, 225 and 243 for El and 70, 72, 75, 84, 86, 89, 125, 131, 136, 146, 165, 201, 226, 229, 244, 262, 281 and 295 for NH₃-Cl) which showed a significant correlation (r² > 0.5) with the total nitrogen content in the Poa leaves. The origin of grass protein marker fragments was discussed in comparison with reference spectra of two new plant cell-wall proteins, a synthetic polyamine and of albumin. Enzymatic digestion, in addition, yielded a better exposure of the plant cell-wall skeleton, and, therefore, also of the biomacromolecule lignin. Using previously obtained wet chemical data response values were calculated for marker fragments comparative to the analysed content of proteins, polysaccharides and different lignins.