Isolation of the Arabidopsis phosphoproteome using a biotin-tagging approach.
Protein phosphorylation plays a key role in signal transduction in cells. Since phosphoproteins are present in low abundance, enrichment methods are required for their purification and analysis. Chemical derivatization strategies have been devised for enriching phosphoproteins and phosphopeptides. In this report, we employed a strategy that replaces the phosphate moieties on serine and threonine residues with a biotin-containing tag via a series of chemical reactions. Ribulose 1,5-bis-phosphate carboxylase/oxygenase (RUBISCO)-depleted protein extracts prepared from Arabidopsis seedlings were chemically modified for 'biotin-tagging'. The biotinylated (previously phosphorylated) proteins were then selectively isolated by avidin-biotin affinity chromatography, followed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). This led to the identification of 31 protein spots, representing 18 different proteins, which are implicated in a variety of cellular processes. Despite its current technical limitations, with further improvements in tools and techniques this strategy may be developed into a useful approach.
Kwon
Sun Jae
SJ
School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea.
Choi
Eun Young
EY
Seo
Jong Bok
JB
Park
Ohkmae K
OK
D017360:Arabidopsis / Q000737:chemistry* / Q000378:metabolism; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation; D005591:Chemical Fractionation; D015180:Electrophoresis, Gel, Two-Dimensional; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D010940:Plant Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D020543:Proteome / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D012273:Ribulose-Bisphosphate Carboxylase / Q000172:deficiency; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010750:Phosphoproteins; D010940:Plant Proteins; D020543:Proteome; D011092:Polyethylene Glycols; D001710:Biotin; D012273:Ribulose-Bisphosphate Carboxylase