Abstract
Despite progress in the characterization of their genomes, proteomes of several model organisms are often only poorly characterized. This problem is aggravated by the presence of large numbers of expressed sequence tag clones that lack homologues in other species, which makes it difficult to identify new proteins irrespective of whether such molecules are involved in species-specific biological processes. We have used a pulsed stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry method, which is based on the detection of paired peptides after [(13)C(6)]lysine incorporation into proteins in vivo, to greatly increase the confidence of protein identification in cross-species database searches. The method was applied to identify nearly 3000 proteins in regenerating tails of the urodele amphibian Notophthalmus viridescens, which possesses outstanding capabilities in the regeneration of complex tissues. We reason that pulsed in vivo SILAC represents a versatile tool to identify new proteins in species for which only limited sequence information exists.
Highlights
Despite progress in the characterization of their genomes, proteomes of several model organisms are often only poorly characterized
Reliable Identification of Newt Proteins by Pulsed in Vivo SILAC—Labeling of newt tissue with [13C6]lysine was achieved by maintaining adult N. viridescens for 20 days on a diet consisting of mouse liver derived from fully labeled SILAC mice [21]
To accelerate labeling and to mark proteins that were newly synthesized during regeneration, we divided the animals into two groups after completion of the initial incorporation phase
Summary
Despite progress in the characterization of their genomes, proteomes of several model organisms are often only poorly characterized. In Vivo SILAC of Uncharacterized Proteomes needs in this unique process To identify such proteins, which might lack counterparts in other organisms as well as to increase the confidence level for the detection of potential homologues in other species we have developed a new approach. This approach is based on labeling of proteins in vivo that permits reliable peptide verification by mass spectrometry (MS) for organisms with little or no available sequence information
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