Stable Isotope Labeling with Amino Acids in Drosophila for Quantifying Proteins and Modifications
- 10 August 2012
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of Proteome Research
- Vol. 11 (9), 4403-4412
- https://doi.org/10.1021/pr300613c
Abstract
Drosophila melanogaster is a common animal model for genetics studies, and quantitative proteomics studies of the fly are emerging. Here, we present in detail the development of a procedure to incorporate stable isotope-labeled amino acids into the fly proteome. In the method of stable isotope labeling with amino acids in Drosophila melanogaster (SILAC fly), flies were fed with SILAC-labeled yeast grown with modified media, enabling near complete labeling in a single generation. Biological variation in the proteome among individual flies was evaluated in a series of null experiments. We further applied the SILAC fly method to profile proteins from a model of fragile X syndrome, the most common cause of inherited mental retardation in human. The analysis identified a number of altered proteins in the disease model, including actin-binding protein profilin and microtubulin-associated protein futsch. The change of both proteins was validated by immunoblotting analysis. Moreover, we extended the SILAC fly strategy to study the dynamics of protein ubiquitination during the fly life span (from day 1 to day 30), by measuring the level of ubiquitin along with two major polyubiquitin chains (K48 and K63 linkages). The results show that the abundance of protein ubiquitination and the two major linkages do not change significantly within the measured age range. Together, the data demonstrate the application of the SILAC principle in D. melanogaster, facilitating the integration of powerful fly genomics with emerging proteomics.Keywords
This publication has 49 references indexed in Scilit:
- Quantitative Proteomic Analysis of Protein ComplexesMolecular & Cellular Proteomics, 2008
- The biological impact of mass-spectrometry-based proteomicsNature, 2007
- Highly Robust, Automated, and Sensitive Online TiO2-Based Phosphoproteomics Applied To Study Endogenous Phosphorylation in Drosophila melanogasterJournal of Proteome Research, 2007
- A high-quality catalog of the Drosophila melanogaster proteomeNature Biotechnology, 2007
- An integrated chemical, mass spectrometric and computational strategy for (quantitative) phosphoproteomics: application to Drosophila melanogaster Kc167 cellsMolecular BioSystems, 2007
- One hundred years of high-throughput Drosophila researchChromosome Research, 2006
- Proteomic analysis of the wing imaginal discs ofDrosophila melanogasterProteomics, 2004
- Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomicsNature Biotechnology, 2003
- Mass spectrometry-based proteomicsNature, 2003
- Homophila: human disease gene cognates in DrosophilaNucleic Acids Research, 2002