Characterization of a variant of the spinach PSII type I light-harvesting protein using kinetically controlled digestion and RP-HPLC-ESI-MS

A previously unknown isoform of the type I major antenna protein of photosystem II of spinach was identified, and its amino-terminal sequence was characterized by a novel kinetic digestion approach, in which sequential tryptic digestion was followed by analysis of both released peptides and truncated proteins by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry. Using nonpolar, monolithic, 200-microm-i.d. separation columns based on poly(styrene/divinylbenzene) copolymer and applying gradients of acetonitrile in 0.05% aqueous trifluoroacetic acid, released peptides and truncated proteins could be separated and mass analyzed in a single chromatographic run. This enabled a straightforward identification of the fragments removed from the amino-terminal ends of the protein, which was essential for the characterization of the antenna isomers showing the most significant sequence variation in the amino-terminal region. The sequences of the amino termini were derived from the differences in molecular mass between intact and truncated proteins and were corroborated by sequencing using tandem mass spectrometry and database searching. The sequence of the 23 amino-terminal residues of the previously unknown isoform differed from that of the other two known isoforms only in one and three amino acids, respectively. Such subtle changes in amino acid sequence are supposed to play an important role in the supramolecular organization of photosynthetic antenna proteins.