Novel protein phosphorylation site identification in spinach stroma membranes by titanium dioxide microcolumns and tandem mass spectrometry

In this work, spinach stroma membrane, instead of thylakoid, has been investigated for the presence of phosphorylated proteins. We identified seven previously unknown phosphorylation sites by taking advantage of TiO2 phosphopeptides enrichment coupled to mass spectrometric analysis. Upon illumination at 100 mu mol m(-2) s(-1), two novel phosphopeptides belonging to the N-terminal region of Lhcb1 light-harvesting protein were detected: NVSSGS(p)PWYGPDR and T(p)VQSSSPWYGPDR. Moreover, three new threonine residues in CP43 (Thr-6, Thr-8, and Thr-346) and, for the first time, two amino acid residues of the N-terminus of Rieske Fe-S protein of the cytochrome b(6)f complex (Thr-2 and Ser-3) were revealed to be phosphorylated. Since Lhcb1 and CP43 have been reported as mobile proteins, it may be suggested that illumination derived phosphorylation, and consequently the addition of negatively charged groups to the protein, is a necessary condition to induce a significant protein structural change.