Proteomic SILAC Labeling in Drosophila melanogaster

Drosophila melanogaster has been used as a model organism in numerous developmental studies, and its use in proteomic studies is on the rise.¹ Quantitative proteomics using in vivo stable isotope-labeled amino acids (SILAC) has also been extended to fruit flies.² Aimed at evaluating the accuracy and versatility of SILAC in fruit flies, Chang et al.³ performed a comprehensive analysis of in vivo labeling strategies in Drosophila.

Instead of purchasing expensive SILAC-labeled yeast as used previously,² homemade labeled yeast with 5% SILAC food was developed. This strategy helped to lessen costs and preserved the growth rate of the fruit flies. Embryos were transferred to juice plates labeled with light or heavy food. The homemade yeast was able to achieve nearly complete labeling. For light labeling, yeast was supplemented with 40 mg/L of [¹²C₆¹⁴N₂]-lysine (Lys0) and [¹²C₆¹⁴N₄]-arginine (Arg0). For heavy lysine labeling, media was supplemented with 40 mg/L of [¹³C₆¹⁵N₂]-lysine (Lys8) and 40 mg/L of [¹²C₆ ¹⁴N₄]-arginine (Arg0). For heavy arginine labeling, media were supplemented with 40 mg/L of [¹²C₆¹⁴N₂]-lysine (Lys0) and 40 mg/L of [¹³C₆¹⁵N₄]-arginine(Arg10). For labeling with both heavy lysine and heavy arginine, the YNB media were supplemented with 40 mg/L of [¹³C₆]-lysine (Lys6) and 40 mg/L of [¹³C₆¹⁵N₄]-arginine (Arg10).

Adult and embryonic aged flies were dechorionated, and proteins were extracted. Proteins were next separated via SDS-PAGE, and gels were stained with Coomassie Blue. Gel slices were digested with trypsin in preparation for high-resolution mass spectrometry. Peptides were diluted with 1% formic acid and analyzed by LC-MS/MS on an LTQ-Orbitrap (XL or Velos) mass spectrometer (Thermo Scientific) coupled to a nanoACQUITY UPLC system (Waters) via a PicoView nanospray interface (New Objective).

Allowing up to two missed cleavages and an FDR of 1%, spectra were searched in three sequence databases, including the Drosophila database, Andromeda, FlyBase (version 5.17; comprising 21,753 entries), and the online Saccharomyces cerevisiae sequence (download from NCBI on March 30, 2009; comprising 5880 entries).

Results of these experiments demonstrated the simplicity of using SILAC in Drosophila. For future studies, Chang et al.³ recommended this method be used along with a normalization peptide that can be applied to avoid any misrepresentation of protein levels.

References

1. Brunner, E., et al. (2007) ‘A high-quality catalog of the Drosophila melanogaster proteome‘, Nature Biotechnology, 25 (5), (pp. 576−583)

2. Sury, M.D., et al. (2010) ‘The SILAC fly allows for accurate protein quantification in vivo‘, Molecular and Cellular Proteomics, 9 (10), (pp. 2173−2183)

3. Chang, Y.C., et al. (2013) ‘Evaluation of Drosophila metabolic labeling strategies for in vivo quantitative proteomic analyses with applications to early pupa formation and amino acid starvation‘, Journal of Proteome Research, 12 (5), (pp. 2138–2150)