Abstract
Stable isotope labeling by amino acids combined with mass spectrometry is a widely used methodology for measuring relative changes in protein and phosphorylation levels at a global level. We have applied this method to the model organism Caenorhabditis elegans in combination with RNAi-mediated gene knockdown by feeding the nematode on pre-labeled lysine auxotroph Escherichia coli. In this chapter, we describe in details the generation of the E. coli strain, incorporation of heavy isotope-labeled lysine in C. elegans, and the procedure for a comprehensive global phosphoproteomic experiment.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Caenorhabditis elegans / cytology*
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Caenorhabditis elegans / genetics
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Caenorhabditis elegans / metabolism*
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Caenorhabditis elegans Proteins / chemistry
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Caenorhabditis elegans Proteins / genetics
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Caenorhabditis elegans Proteins / isolation & purification
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Caenorhabditis elegans Proteins / metabolism
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Escherichia coli / genetics
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Gene Knockdown Techniques*
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Isotope Labeling
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Mass Spectrometry
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Phosphopeptides / chemistry
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Phosphopeptides / metabolism
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Phosphoproteins / chemistry
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Phosphoproteins / genetics*
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Phosphoproteins / isolation & purification
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Phosphoproteins / metabolism*
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Phosphorylation
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Proteolysis
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Proteomics / methods*
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RNA Interference
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Signal Transduction*
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Titanium / chemistry
Substances
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Caenorhabditis elegans Proteins
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Phosphopeptides
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Phosphoproteins
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titanium dioxide
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Titanium