Plant protein phosphorylation monitored by capillary liquid chromatography--element mass spectrometry.
Many essential cellular functions such as growth rate, motility, and metabolic activity are linked to reversible protein phosphorylation, since they are controlled by signaling cascades based mainly on phosphorylation/dephosphorylation events. Quantification of global or site-specific protein phosphorylation is not straightforward with standard proteomic techniques. The coupling of capillary liquid chromatography (microLC) with ICP-MS (inductively coupled plasma-mass spectrometry) is a method which allows a quantitative screening of protein extracts for their phosphorus and sulfur content, and thus provides access to the protein phosphorylation degree. In extension of a recent pilot study, we analyzed protein extracts from the model organisms Arabidopsis thaliana and Chlamydomonas reinhardtii as representatives for multicellular and unicellular green photosynthetically active organisms. The results indicate that the average protein phosphorylation level of the algae C. reinhardtii is higher than that of A. thaliana. Both the average phosphorylation levels were found to be between the extreme values determined so far for prokaryotes (C. glutamicum, lowest levels) and eukaryotes (Mus musculus, highest levels). Tissue samples of A. thaliana representing different stages of plant development showed varying levels of protein phosphorylation indicating a different adjustment of the kinase/phosphatase system. We also utilized the microLC-ICP-MS technology to estimate the efficiency of a novel phosphoprotein enrichment method based on aluminum hydroxide, since the enrichment of phosphorylated species is often an essential step for their molecular characterization.
Krüger
Ralf
R
Central Spectroscopy, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. krueger@zentrallabor.klinik.uni-mainz.de
Wolschin
Florian
F
Weckwerth
Wolfram
W
Bettmer
Jörg
J
Lehmann
Wolf D
WD
D000818:Animals; D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000302:isolation & purification / Q000378:metabolism; D002478:Cells, Cultured; D016825:Chlamydomonas reinhardtii / Q000302:isolation & purification / Q000378:metabolism; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D051379:Mice; D010446:Peptide Fragments / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010758:Phosphorus / Q000032:analysis; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism*; D040901:Proteomics; D015800:Protozoan Proteins / Q000302:isolation & purification / Q000378:metabolism; D013455:Sulfur / Q000032:analysis
D029681:Arabidopsis Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D010940:Plant Proteins; D015800:Protozoan Proteins; D010758:Phosphorus; D013455:Sulfur