The SILAC fly allows for accurate protein quantification in vivo

Abstract

Stable isotope labeling by amino acids in cell culture (SILAC) is widely used to quantify protein abundance in tissue culture cells. Until now, the only multicellular organism completely labeled at the amino acid level was the laboratory mouse. The fruit fly Drosophila melanogaster is one of the most widely used small animal models in biology. Here, we show that feeding flies with SILAC-labeled yeast leads to almost complete labeling in the first filial generation. We used these "SILAC flies" to investigate sexual dimorphism of protein abundance in D. melanogaster. Quantitative proteome comparison of adult male and female flies revealed distinct biological processes specific for each sex. Using a tudor mutant that is defective for germ cell generation allowed us to differentiate between sex-specific protein expression in the germ line and somatic tissue. We identified many proteins with known sex-specific expression bias. In addition, several new proteins with a potential role in sexual dimorphism were identified. Collectively, our data show that the SILAC fly can be used to accurately quantify protein abundance in vivo. The approach is simple, fast, and cost-effective, making SILAC flies an attractive model system for the emerging field of in vivo quantitative proteomics.

Fig. 1.

Work flow for labeling of D. melanogaster with heavy isotope-containing amino acids. Embryos are collected, and hatched larvae are fed with “light” l-[¹²C₆,¹⁴N₂]lysine (L) or “heavy” l-[¹³C₆,¹⁵N₂]lysine (H) labeled S. cerevisiae. Different adult F₁ subpopulations (male and female) are mixed and analyzed by LC-MS/MS. Pairs of identical peptides with different stable isotope compositions can be distinguished in the mass spectrometer based on their mass difference (8 Da). Consistently, the ratio of peak intensities of heavy versus light peptides reflects differences in protein abundance in vivo. rel., relative.

Fig. 2.

Effective labeling of D. melanogaster with heavy lysine. A, labeling efficiency of S. cerevisiae and adult D. melanogaster. Total protein extracts of heavy labeled yeast and adult flies from the F₁ or F₂ generation were analyzed by LC-MS/MS. H/L ratios of the 100 most intense proteins were calculated and expressed as median of log₂ -fold changes. Whiskers indicate 2.5 and 97.5 percentiles. Labeling efficiencies were 96.7% (S. cerevisiae), 96.2% (D. melanogaster F₁), and 96.9% (D. melanogaster F₂). B, representative mass spectrum of an alcohol dehydrogenase (Adh)-derived peptide from heavy adult flies in the F₁ generation. The mass shift between the light and the heavy forms of the peptide is 8 Da due to one heavy lysine. Dots mark light and heavy monoisotopic peaks (log₂ -fold change is 4.4, corresponding to an incorporation rate of 95.6%).

Fig. 3.

Precision of label-free versus SILAC-based protein quantification. Heavy and light flies were mixed and analyzed by two different LC-MS/MS runs (n = 1578 quantified proteins). A, for label-free quantification, the ratios of light protein intensities between both replicates were calculated and plotted in a histogram. B, SILAC-based quantification of light proteins between both replicates was performed using the heavy peptides as an internal standard (ratio of ratios). Comparison of standard deviations demonstrates that SILAC is ∼4-fold more precise than label-free quantification.

Fig. 4.

Identification of sex-specific protein clusters by GO analysis. Total protein extracts of mixed heavy female and light male flies were analyzed by LC-MS/MS, and log₂ -fold changes of H/L protein ratios were calculated (n = 1913). Proteins were divided into five bins according to their H/L ratio. p values of GO terms that were significantly enriched (p < 0.01) in at least in one bin were log-transformed, z-transformed, hierarchically clustered, and plotted as a heat map.

Fig. 5.

Identification of sex-specific germ line and somatic protein expression in adult D. melanogaster. The histogram on the right shows male versus female protein ratios in the w¹¹¹⁸ strain (n = 2106). The histogram on top shows ratios of male versus female protein ratios in tud¹ progeny (n = 2079). Proteins quantified in all experiments are shown in the central scatter plot. The numbered regions in the scatter plot contain different sets of proteins (supplemental Table 1). Known male accessory gland proteins in region 1 are shown in blue. Selected proteins that are mentioned in the main text have labels and are shown in red. Dashed lines indicate log₂ -fold change of 1.5 and −1.5, respectively.

Fig. 6.

Correlation between male versus female protein -fold change and previously published mRNA data (n = 1816). The trend line indicates a good overall correlation between mRNA and protein -fold changes (slope, 0.9; R² = 0.5). Strongly regulated genes tend to change more at the protein level than at the mRNA level.