Comparison of full versus partial metabolic labeling for quantitative proteomics analysis in Arabidopsis thaliana.
In recent years a variety of quantitative proteomics techniques have been developed, allowing characterization of changes in protein abundance in a variety of organisms under various biological conditions. Because it allows excellent control for error at all steps in sample preparation and analysis, full metabolic labeling using (15)N has emerged as an important strategy for quantitative proteomics, having been applied in a variety of organisms from yeast to Arabidopsis and even rats. However, challenges associated with complete replacement of (14)N with (15)N can make its application in many complex eukaryotic systems impractical on a routine basis. Extending a concept proposed by Whitelegge et al. (Whitelegge, J. P., Katz, J. E., Pihakari, K. A., Hale, R., Aguilera, R., Gomez, S. M., Faull, K. F., Vavilin, D., and Vermaas, W. (2004) Subtle modification of isotope ratio proteomics; an integrated strategy for expression proteomics. Phytochemistry 65, 1507-1515), we investigate an alternative strategy for quantitative proteomics that relies upon the subtle changes in isotopic envelope shape that result from partial metabolic labeling to compare relative abundances of labeled and unlabeled peptides in complex mixtures. We present a novel algorithm for the automated quantitative analysis of partial incorporation samples via LC-MS. We then compare the performance of partial metabolic labeling with traditional full metabolic labeling for quantification of controlled mixtures of labeled and unlabeled Arabidopsis peptides. Finally we evaluate the performance of each technique for comparison of light- versus dark-grown Arabidopsis with respect to reproducibility and numbers of peptide and protein identifications under more realistic experimental conditions. Overall full metabolic labeling and partial metabolic labeling prove to be comparable with respect to dynamic range, accuracy, and reproducibility, although partial metabolic labeling consistently allows quantification of a higher percentage of peptide observations across the dynamic range. This difference is especially pronounced at extreme ratios. Ultimately both full metabolic labeling and partial metabolic labeling prove to be well suited for quantitative proteomics characterization.
Huttlin
Edward L
EL
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA. elhuttin@wisc.edu
Hegeman
Adrian D
AD
Harms
Amy C
AC
Sussman
Michael R
MR
D000465:Algorithms; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry* / Q000254:growth & development / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000032:analysis*; D002853:Chromatography, Liquid; D003624:Darkness; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000378:metabolism; D010455:Peptides / Q000032:analysis; D040901:Proteomics / Q000379:methods*
D029681:Arabidopsis Proteins; D009587:Nitrogen Isotopes; D010455:Peptides