Metabolic Labeling Approaches

Metabolic labeling is an in situ method which employs or isotope-encoded ( C or H) amino acids. Organisms are grown in controlled media under defined conditions in order to ensure translation of mRNA entities to uniformly isotopically encoded proteins. Consequently, the approach is generally restricted to cell culture systems and is not applicable to quantitative MS analysis of proteins derived from 

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Hang HC, Yu C, Kato DL, Bertozzi CR. A metabolic labeling approach toward proteomic analysis of mucin-type O-linked gly-cosylation. Proc. Natl. Acad. Sci. U.S.A. 2003 100 14846-14851. 

Using this isotope labeling approach, metabolites in human plasma have been quantified (38) and altered metabolites have been detected in urine that are due to metabolic disorders such as tyrosinemia type II, argininosuccinic aciduria, homocystinuria, and phenylketonuria (20). Most recently, a smart isotope tag, 15N-cholamine, has been developed for effective detection of the same metabolites using both NMR and MS methods. This approach maximizes the combined strengths of two powerful analytical techniques for a variety of metabolomics applications. 15N-cholamine possesses dual characteristics an NMR-sensitive heteronuclear isotope with good chemical shift dispersion and a permanent charge that improves MS sensitivity (48). 

Luxen A, Guillaume M, Melega WP et al (1992). Production of 6-[18P]fluoro-L-dopa and its metabolism in vivo - a critical review. Nucl Med Biol 19 149 Machula HJ, Blocher A, Kuntzch M et al (2000). Simplified labeling approach for synthesizing 3[Pg.149]

Not only do mRNAs move more freely to equilibrate through the cytoplasm if they are injected into the vegetal pole, but this also reduces the chance of injection into the nucleus, which is more likely to be toxic to the oocyte. Although the distribution of mRNA is not important for metabolic labeling studies, some analytical approaches such as electrophysiological measurements will be affected by an uneven distribution of the protein product. In these cases, oocytes should be incubated for 2-3 days before analysis. 

To identify cell surface ligand-binding proteins, two different approaches to cross-linking may be employed. Figure 1 illustrates these two strategies which we have used successfully to identify the tissue-type plasminogen activator (tPA)-binding protein on hepatoma cells (Bu et al., 1992) either radiolabeled tPA crosslinked to unlabeled cells, or unlabeled tPA crosslinked to [ Sjmethionine metabolically labeled cells. Each of these two approaches is separately described in detail. 

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