Advanced Identification of Proteins in Uncharacterized Proteomes by Pulsed in Vivo Stable Isotope Labeling-based Mass Spectrometry

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. Molecular & Cellular Proteomics 9:1157-1166, 2010.Lack of sequence information has greatly impeded analysis of biological processes and identification of proteins in several nonstandard model organisms for which no comprehensive genome characterization is available. Even if nucleotide sequence data (i.e. EST libraries) are available, it is often difficult to use these data for identification of new proteins, particularly if EST clones lack obvious homologues in other species. Furthermore, it is often difficult to distinguish open reading frames in EST clones lacking obvious homologues from 3Ј-untranslated regions, intermediate splice products, or cloning artifacts. In addition, several other problems aggravate identification of peptides in partially characterized genomes, which spurred the development of a number of alternative approaches (reviewed in Refs. 1 and 2). Traditional database searches usually allow only identification of peptides that are conserved in newly detected proteins and putative homologous proteins from closely related species (3, 4). Unfortunately, such an approach is not efficient to recognize proteins that are phylogenetically distant from available reference organisms, or belong to poorly conserved protein families. The reliable identification of unknown proteins in "isolated" model organisms currently remains unsolved, although new software tools based on MS BLAST sequencesimilarity searches that use multiple redundant and partially accurate candidate peptide sequences have been developed to cope with this difficulty. One potential solution for this problem is de novo protein sequencing, which, however, remains a challenging problem (reviewed in Refs. 5 and 6).The urodele amphibian Notophthalmus viridescens, vulgo newts, which is one of the best-characterized organisms for the regeneration of complex tissues, is an example of an "isolated" model organism. Newts are able to completely regenerate limbs (7) and ...

The SILAC Fly Allows for Accurate Protein Quantification in Vivo

“…This zoo is likely to grow further and will probably contain all popular animal models in the future. In fact, partial labeling of newt and chicken using a pulsed approach has already been reported (21,23). Our data show that the SILAC fly can provide new insights into biological processes in vivo.…”

“…Three years later, Krü ger et al (22) achieved essentially complete labeling of the laboratory mouse. Until now, this so-called "SILAC mouse" was the only multicellular organism that has been completely labeled with the SILAC approach, and partial labeling was recently achieved in newts (21,23).…”

In Vivo Quantitative Proteome Profiling: Planning and Evaluation of SILAC Experiments

“…Similarly, labeled worms are obtained by feeding Caenorhabditis elegans with heavy SILAC-labeled E. coli ( 12 ) . Finally, partial labeling of newts was achieved by feeding them with tissue derived from SILAC mice ( 13 ) . If none of the strategies is applicable or unreasonable (e.g., human tissue, animal models with long generation time), the use of a heavy labeled reference sample, known as Super-SILAC, should be considered ( 10 ) .…”

Quantitative proteomic strategies for the identification of microRNA targets

“…These two reports have provided a wealth of data on potential miRNA targets and clues for future research directions. Furthermore, pSILAC has also been applied to determine translation rates [63] and identify new proteins in species for which only limited sequence information exists [65]. However, this method is relatively expensive, time consuming and not practical for analyzing biological samples that cannot be grown in culture, such as tissues or body fluids.…”