Plant Physiol, December 2002, Vol. 130, pp. 1938-1950

Proteomics of Light-Harvesting Proteins in Different Plant Species. Analysis and Comparison by Liquid Chromatography-Electrospray Ionization Mass Spectrometry. Photosystem I¹

Department of Environmental Sciences, University of Tuscia, Viterbo, Italy (L.Z., S.R., A.M.T.); and Instrumental Analysis and Bioanalysis, Saarland University, Saarbrücken, Germany (C.G.H.)

The light-harvesting proteins (Lhca) of photosystem I (PSI) from four monocot and five dicot species were extracted from plant material, separated by reversed-phase high-performance liquid chromatography (HPLC) and subsequently identified on the basis of their intact molecular masses upon on-line hyphenation with electrospray ionization mass spectrometry. Although their migration behavior in gel electrophoresis was very similar, the elution times among the four antenna types in reversed-phase-HPLC differed significantly, even more than those observed for the light-harvesting proteins of photosystem II. Identification of proteins is based on the good agreement between the measured intact molecular masses and the values calculated on the basis of their nucleotide-derived amino acid sequences, which makes the intact molecular masses applicable as intact mass tags. These values match excellently for Arabidopsis, most probably because of the availability of high-quality DNA sequence data. In all species examined, the four antennae eluted in the same order, namely Lhca1 > Lhca3 > Lhca4 > Lhca2. These characteristic patterns enabled an unequivocal assignment of the proteins in preparations from different species. Interestingly, in all species examined, Lhca1 and Lhca2 were present in two or three isoforms. A fifth antenna protein, corresponding to the Lhca6 gene, was found in tomato (Lycopersicon esculentum). However PSI showed a lower heterogeneity than photosystem II. In most plant species, Lhca2 and Lhca4 proteins are the most abundant PSI antenna proteins. The HPLC method used in this study was found to be highly reproducible, and the chromatograms may serve as a highly confident fingerprint for comparison within a single and among different species for future studies of the PSI antenna.