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Author Notes:

E-mail: junmin.peng@stjude.org.

Subjects:

Research Funding:

This work was supported by a grant (RR025822) from National Institutes of Health and a Research Scholar Grant RSG-09-181 from the American Cancer Society.

P.X. is supported by Chinese National Basic Research Program (2011CB910602), National Natural Science Foundation of China 31070673 and 31170780.

H.T. is supported by China Scholarship Council. H.L. is supported by National Nature Science Foundation of China 30430260 and 30225024.

P.J. is supported by NIH grants NS051630 and MH076090.

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Biochemical Research Methods
  • Biochemistry & Molecular Biology
  • SILAC
  • Drosophila melanogaster
  • proteomics
  • mass spectrometry
  • fragile X syndrome
  • ubiquitin
  • SPECTROMETRY-BASED PROTEOMICS
  • FRAGILE-X-SYNDROME
  • MASS-SPECTROMETRY
  • QUANTITATIVE PROTEOMICS
  • IN-VIVO
  • CELL-CULTURE
  • MELANOGASTER
  • REVEALS
  • PHOSPHOPROTEOMICS

Stable Isotope Labeling with Amino Acids in Drosophila for Quantifying Proteins and Modifications

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Journal Title:

Journal of Proteome Research

Volume:

Volume 11, Number 9

Publisher:

, Pages 4403-4412

Type of Work:

Article | Post-print: After Peer Review

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.

Copyright information:

© 2012 American Chemical Society.

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