Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics.
A crucial issue in comparative proteomics is the accurate quantification of differences in protein expression levels. To achieve this, several methods have been developed in which proteins are labeled with stable isotopes either in vivo via metabolic labeling or in vitro by protein derivatization. Although metabolic labeling is the only way to obtain labeling of all proteins, it has thus far only been applied to single- celled organisms and cells in culture. Here we describe quantitative 15N metabolic labeling of the multicellular organisms Caenorhabditis elegans, a nematode, and Drosophila melanogaster, the common fruit fly, achieved by feeding them on 15N-labeled Escherichia coli and yeast, respectively. The relative abundance of individual proteins obtained from different samples can then be determined by mass spectrometry (MS). The applicability of the method is exemplified by the comparison of protein expression levels in two C. elegans strains, one with and one without a germ line. The methodology described provides tools for accurate quantitative proteomic studies in these model organisms.
Krijgsveld
Jeroen
J
Center for Biomedical Genetics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands. j.krijgsveld@chem.uu.nl
Ketting
René F
RF
Mahmoudi
Tokameh
T
Johansen
Janik
J
Artal-Sanz
Marta
M
Verrijzer
C Peter
CP
Plasterk
Ronald H A
RH
Heck
Albert J R
AJ
D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000032:analysis / Q000378:metabolism*; D029721:Drosophila Proteins / Q000032:analysis / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D009587:Nitrogen Isotopes; D020543:Proteome / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013045:Species Specificity
D029742:Caenorhabditis elegans Proteins; D029721:Drosophila Proteins; D009587:Nitrogen Isotopes; D020543:Proteome