Stable isotope labeling of Arabidopsis thaliana cells and quantitative proteomics by mass spectrometry.
Quantitative analysis of protein expression is an important tool for the examination of complex biological systems. Albeit its importance, quantitative proteomics is still a challenging task because of the high dynamic range of protein amounts in the cell and the variation in the physical properties of proteins. Stable isotope labeling by amino acids in cell culture (SILAC) has been successfully used in yeast and mammalian cells to measure relative protein abundance by mass spectrometry. Here we show for the first time that proteins from Arabidopsis thaliana cell cultures can be selectively isotope-labeled in vivo by growing cells in the presence of a single stable isotope-labeled amino acid. Among the tested amino acids ([2H3]-leucine, [13C6]arginine, and [2H4]lysine), [13C6]arginine proved to be the most suitable. Incorporation of [13C6]arginine into the proteome was homogeneous and reached efficiencies of about 80%. [13C6]Arginine-labeled A. thaliana suspension cells were used to study the regulation of glutathione S-transferase expression in response to abiotic stress caused by salicylic acid and to identify proteins that bind specifically to phosphorylated 14-3-3 binding motifs on synthesized bait peptides in affinity purification experiments. In conclusion, the combination of stable isotope labeling of plant cells and mass spectrometry is a powerful technology that can be applied to study complex biological processes that involve changes in protein expression such as cellular responses to various kinds of stress or activation of cell signaling.
Gruhler
Albrecht
A
Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Schulze
Waltraud X
WX
Matthiesen
Rune
R
Mann
Matthias
M
Jensen
Ole N
ON
D000595:Amino Acid Sequence; D000596:Amino Acids; D017360:Arabidopsis / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000032:analysis* / Q000737:chemistry; D002478:Cells, Cultured; D008401:Gas Chromatography-Mass Spectrometry; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins / Q000032:analysis / Q000737:chemistry; D007525:Isoelectric Focusing; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012273:Ribulose-Bisphosphate Carboxylase / Q000032:analysis / Q000737:chemistry; D020156:Salicylic Acid / Q000494:pharmacology
D000596:Amino Acids; D029681:Arabidopsis Proteins; D050883:HSC70 Heat-Shock Proteins; D020543:Proteome; D005982:Glutathione Transferase; D012273:Ribulose-Bisphosphate Carboxylase; D020156:Salicylic Acid