PARENT SESSION
Posters P3D Genomic, proteomic and related technologies. Abstracts (720-730)

Identification of in vivo phosphorylated proteins in thylakoid membranes of Arabidopsis thaliana by electrospray ionization mass spectrometry. Maria Hansson^*,1, Alexander Vener¹, ¹ Division of Cell Biology, Linköping University, Linköping, Sweden

ABSTRACT- Protein phosphorylation plays a major regulatory role in all cellular functions including photosynthesis. Identification of the sites of protein phosphorylation is experimentally challenging and we have developed dedicated mass spectrometric techniques for characterization of in vivo protein phosphorylation in photosynthetic membranes, which exclude the use of radioactive phosphate and antibodies against phosphoproteins. We isolate thylakoid membranes from leaves of Arabidopsis thaliana. The surface exposed parts of the membrane proteins are released by trypsin treatment and then hydrophobic segments of the proteins are removed with the membranes by centrifugation. The released hydrophilic peptides, including phosphorylated regions, are directly analysed by nanospray-quadropole-TOF mass spectrometry (QSTAR, Applied Biosystems). The negative ion mode precursor of -79 and -97 ions scanning allows for selective and sensitive detection of just phosphopeptides present in the complex peptide mixture. Further detection of diagnostic product phosphoryl ions -79 and -97 during fragmenting of the negatively charged peptide ions allows determination of exact masses of the phosphopeptides. Then we use high resolution positive mode MS/MS to identify the sequences of the phosphopeptides. The phosphorylation sites in these peptides are identified due to the prominent loss of phosphoric acid (H₃PO₄, 98 Da) from the fragment ions containing phosphorylated residue. Despite of the possibility for direct mass spectrometric characterization of the phosphopeptides, the complexity of the peptide mixture often requires enrichment for the phosphopeptides present in low amounts to allow de novo sequencing. Thus, we also use immobilized metal-affinity chromatography (IMAC) after methylation of the carboxylic groups in the peptides (Ficarro et al., 2002, Nat Biotechnol. 20, 301-305) to get enrichment of the phosphopeptides. At present we have identified three novel phosphorylation sites in thylakoid proteins from Arabidopsis thaliana. Further improvement in mass spectrometric analysis and phosphopeptide enrichment is on the way to identify the minor phosphorylated proteins in the photosynthetic membranes.

KEY WORDS: mass spectrometry, protein phosphorylation, thylakoid membrane