Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC).
We have recently described a method, stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of relative protein abundances. Cells were metabolically labeled with deuterated leucine, leading to complete incorporation within about five cell doublings. Here, we investigate fully substituted 13C-labeled arginine in the SILAC method. After tryptic digestion, there is a single label at the C-terminal position in half of the peptides. Labeled and unlabeled peptides coelute in liquid chromatography-mass spectrometric analysis, eliminating quantitation error due to unequal sampling of ion profiles. Tandem mass spectrum interpretation and database identification are aided by the predictable shift of the y-ions in the labeled form. The quantitation of mixtures of total cell lysates in known ratios resolved on a one-dimensional SDS-PAGE gel produced consistent and reproducible results with relative standard deviations better than five percent under optimal conditions.
Ong
Shao-En
SE
Center for Experimental Bioinformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Kratchmarova
Irina
I
Mann
Matthias
M
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D002247:Carbon Isotopes; D018929:Cell Culture Techniques; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D041681:NIH 3T3 Cells; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods; D020539:Sequence Analysis, Protein
D000596:Amino Acids; D002247:Carbon Isotopes; D011506:Proteins; D001120:Arginine