Beta-elimination coupled with tandem mass spectrometry for the identification of in vivo and in vitro phosphorylation sites in maize dehydrin DHN1 protein.
Dehydrins are a group of proteins that are accumulated during environmental stress such as drought and low temperature or during late embryogenesis. In the present study, we isolated dehydrin DHN1, also known as Rab17 protein, from maize kernel by an acid extraction method, removed the phosphoric acid groups from phosphorylated residues by beta-elimination via treating the protein with barium hydroxide, and identified the sites of phosphorylation by tandem mass spectrometry. Our results showed that each of the seven contiguous serine residues (Ser78-Ser84) in the serine tract could be phosphorylated. The beta-elimination procedure was shown to be essential for the detection and subsequent site mapping of the heavily phosphorylated peptide by mass spectrometry. We also found that protein kinase CK2 could catalyze the phosphorylation of the DHN1 protein in vitro and the level of phosphorylation was comparable to that of the DHN1 isolated from maize seeds. Moreover, the in vitro phosphorylation also occurred on the serine residues in the serine tract region, suggesting that CK2 might be involved in the phosphorylation of the serine track region in maize kernel in vivo.
Jiang
Xinzhao
X
Department of Chemistry, Mail Drop 027, University of California, Riverside, California 92521-0403, USA.
Wang
Yinsheng
Y
D000595:Amino Acid Sequence; D017609:Barium Compounds / Q000494:pharmacology*; D047390:Casein Kinase II / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012639:Seeds / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D003313:Zea mays
D017609:Barium Compounds; D010940:Plant Proteins; C070556:dehydrin proteins, plant; D010768:Phosphoserine; C012766:barium hydroxide; D047390:Casein Kinase II