23Drosophila melanogaster has been a working horse of genetics and cell biology for more than a 24 century. However, proteomic-based methods have been limited due to technical obstacles, 25 especially the lack of reliable labelling methods. Here, we advanced a chemically defined food 26 source into stable-isotope labelling of amino acids in flies (SILAF). It allows for the rapid generation 27 of a large number of flies with full incorporation of lysine-6. SILAF followed by fractionation and 28 enrichment gave proteomic insights at a depth of 5,966 proteins and 7,496 phosphorylation sites, 29 which substantiated metabolic regulation on enzymatic level. Furthermore, the label can be traced 30 and predicts protein turnover rates during different developmental stages. The ease and versatility 31 of the method actuates the fruit fly as an appealing model in proteomic and post-translational 32 modification studies and it enlarges potential metabolic applications based on heavy amino acid 33 diets. 34Schober et al., 3 of 32 relying on accurate quantification of peptides, e.g. post-translational modification scans have not 50 been feasible in Drosophila yet. Furthermore, the somewhat unpredictable yeast metabolism does 51 not allow precise tracing of amino acid labels in order to reliably predict protein turnover rates and 52 complicates the introduction of several labels or additive substrates at the same time. Here, we 53 present an alternative, direct labelling approach by employing a fully defined chemical food source 54 that contains heavy amino acids isotopes, which addresses several key concerns of stable-isotope 55 labelling in fruit flies. 56 RESULTS 57To overcome the difficulties associated with traditional yeast-based food in SILAC, we used a 58 holidic food source in this study containing 47 ingredients (9, 10), including defined amounts of 59 eight essential amino acids. Flies grown on this holidic food are viable and fertile with a comparable 60 lifespan, although they do present with a prolonged developmental larvae stage, pupating after 61 approximately nine instead of five days after egg laying (dae) at 25°C (9, 10). 62 SILAF enables full amino acid labelling 63In its more popular application in cell culture, SILAC relies on labelling with both "heavy" 13 C 6 -lysine 64 and 13 C 6 15 N 4 -arginine followed by digestion with trypsin, which cuts C-terminally of both amino 65 acids. However, previous results in both cells and the fly suggested that arginine can be 66 metabolised into other amino acids (8, 11). In agreement with this, growing larvae on holidic food 67 containing heavy-lysine and -arginine, followed by protein identification using liquid 68 chromatography-tandem mass spectrometry (LC-MS/MS), identified a number of heavy [ 13 C 5 15 N 1 ] 69 proline-containing peptides ( Supplementary Fig. 1a, b). In addition, we observed a decrease in the 70 average intensity of the heavy fraction in a peptide mixture from heavy and light holidic food 71 indicating a "loss" of the heavy label ( Supplementary ...